Ahlers I¹, Ahlersová E¹ , Bojková B¹ , Kubatka P¹ , Alberty R² , Adámeková E¹ , Marková M¹

Nimesulide (NIM) represents the non steroidal antiinflammatory drug, inhibitor of prostanoid synthesis, with marked influence on cyclooxygenase-2 activity. The aim of recent study was to determine the metabolic effect of this compound in rats, in the nycthemeral arrangement of the experiment. Male Wistar: han rats were kept under standard conditions, with light : dark regimen 12 : 12 h (light on at 7 a.m.). Nimesulide (Helsinn Healthcare SA, Lugan, Switzerland) was administered 1 hour after light onset (i.e. at 8 a.m.), or 1 hour after onset of darkness (i.e. at 8 p.m.) in the dose of 10 mg/kg b.w. subcutaneously. The control group was injected by the same volume of the vehicle, used for NIM dissolution (i.e. at 11 a.m. and 11 p.m.) the rats were sacrified and selected parameters of lipid and carbohydrate metabolism in the serum, liver, bone marrow and heart muscle were determined.

A marked increase in the serum glucose concentration, a moderate increase in serum cholesterol, liver triacylglycerol and bone marrow phospholipid concentration and a decrease in lipid peroxide concentration in the bone marrow was observed after NIM administration in the darkness. A decrease in liver glycogen and an increase in liver cholesterol and triacylglycerol concentration occured after NIM administration in the ligth part of day. Chronopharmacological studies for the determination of NIM effects are required in human subjects too.

Ahlersová E, Kassayová M , _majda B , Ahlers I

In the laboratory rat - exhibiting low photoperiodic activity - moderate response to various photoperiods on circadian oscillations of thyroid hormones were noted. In the present study we examined the effect of short artificial photoperiod LD 6 : 18 applied in a single season, on circadian variations of serum thyrotropin (TSH), thyroxine (T₄), triiodothyronine (T₃) and reverse T₃ (rT₃). The control group was kept in LD 12 : 12 regimen.

Male Wistar rats were adapted for five weeks to artificial light regimens LD 6 : 18 and 12 : 12. Cold light was switched on at 07.00 h in both regimens. The rats were examined at 3-hour intervals within 24 h, the serum concentration of THS, T₄, T₃ and rT₃ were determined radioimmunochemically. The results were evaluated by the cosinor analysis with a chosen 24 h and 12 h rhythm periods. In the short day the circadian oscillations of TSH, T₄ and rT₃ did not show rhythmicity except of T₃ with rhythm detection. Circadian oscillations of TSH and thyroid hormones examined in LD 12 : 12 regimen were rhythmic, T₄ rhythm was of borderline significance. The mesor values of TSH, T₄, T₃ resp. rT₃ were significantly higher in the LD 6 : 18 regimen in comparison with values of control group (LD 12 : 12 regimen).

The effect of short artificial photoperiod applied in spring influenced circadian oscillations of serum TSH and thyroid hormones: the mesors were remarkably higher in comparison with LD 12 : 12 regimen values, the rhythmicity was not present with the exception of T₃.

Babinská K¹, Klvanová J², Béderová A³

The fatty acid intake and plasma fatty acid composition were assessed in a group of iron deficient subjects (n=25, mean age 42,8±8,3y) and in a control group of matched healthy subjects (n=25, mean age 44,5±12y). Serum iron levels were assessed spectrophotometrically, plasma fatty acids were determined by gas chromatography. Fatty acid consumption was estimated from 24-hour dietary recalls.

The intake of essential fatty acids (18:2n6 and 18:3n3) did not differ significantly between both groups. Intake of 18:3n3 (1,5 g per day) was lower than the recommendation. Similarly, extremely low consumption of LCPUFA n-3 (20:5n3 and 22:6n3) was observed. Lower (p<0,04) fatty acid index (calculated as product to substrate ratio) for the formation of g-linolenic acid (18:3n6/18:2n6) and dihomo-g-linolenic acid (20:3n6/18:2n6) was found in the iron deficient group. Serum iron level significantly correlated with the ratio 18:3n6/18:2n6 (r=0,33; p<0,03), as well with 20:3n6/18:2n6 (r=0,33; p<0,03). Significantly lower plasma level of 20:5n3 was observed in the iron deficient group.

Bahníková M, Matějovič P, Pásek M, _imurdová M, _imurda J

Ajmaline, a derivative of rauwolfia, is known as a potent antiarrhythmic drug currently used in the treatment of a variety of both ventricular and atrial tachyarrhythmias. It is regarded as a representative of class Ia according to Vaughan Williams classification. Numerous clinical studies have shown that ajmalin slows conduction of excitation, prolongs the refractory period, QT and QRS interval. Surprisingly, only sporadic cellular electrophysiological data are available.

The whole cell patch clamp technique was applied to rat ventricular myocytes at room temperature. Ajmaline in the range 3.10^-7 - 5.10^-3 M was found to suppress three currents: the fast sodium current (I_Na), 4-aminopyridine sensitive transient outward current (I_to) and current measured at the end of the imposed 0.1 s rectangular pulses (I_K) in a dose-dependent manner with EC₅₀ of 8.2, 27.5, and 166 mM and the Hill coefficient of 0.62, 0.67, and 0.44, respectively. Although the block of ajmaline on I_Na and I_to was affected by depolarization considerably, it did not show significant frequency dependency (0.33 - 3.3 Hz). The effect was reversible in all concentrations applied. The half times of the onset of the block action was less than 150 s for all the explored current components. A significant voltage dependence of I_K block was not observed.

This study demonstrates low blocking specificity and frequency independent effect of ajmalin in rat ventricular myocytes. The latter corresponds well with the observed fast equilibration of block level following repolarization.

Barancik M^1,2, Strohm C², Bruehl v.ML², Schaper W^2 
1Inst Heart Res, Slovak Acad Sci, Bratislava, Slovak Republic, and ²Dept Experim Cardiol, Max-Planck-Instit, Bad Nauheim, Germany

Evidence accumulates suggesting that mitogen-activated protein kinases (MAPKs) could play an important role in the stress responses and for survival and cell death. We investigated the role of MAPK cascades in the ischemic preconditioning (IP) mediated cardioprotection in pig myocardium. Methods: The hearts (open chest model) were subjected to regional ischemia. IP was induced by two cycles of 10 min LAD-occlusion and 10 min reperfusion, prior to the onset of prolonged index ischemia. "Stress" kinase activator (anisomycin- AN), specific inhibitors (PD98059, UO126 for ERKs; SB203580 for p38-MAPK) and actinomycin-D (Act-D) were infused systemically or intramyocardially. The infarct size (IS) measurements, kinase and Western blot assays were performed as described previously (1). Results: IP significantly reduced the IS. During the IP procedure an activation of ERKs, JNKs and down-regulation of p38-MAPK activities was observed. Infusions of PD, UO and Act-D significantly reduced the IP-induced cardioprotection and this was connected with inhibition of ERK activities. Inhibition of p38-MAPK cascade by SB significantly reduced IS after index ischemia but did not influence the IS reduction mediated by IP. AN reduced IS when infused prior to index ischemia and this was associated with a specific activation of JNKs. Conclusion: The results point to the positive role of ERKs and SAPK/JNKs in adaptive responses of myocardium to ischemia. On the other hand, p38-MAPK activation is believed to accelerate the death pathway, the inhibition of which shifts the balance toward cell survival.

1. Barancik et al., J Cardiovasc Pharmacol 35; 2000:474-483

Barcal J, Jelínková D, _tenglová V, Vožeh F, Žalud V

Lurcher mutant mice as a model of cerebellar degeneration were used. Because our previous studies have shown important changes not only in the cognitive functions but also in the level of excitability, this work is focused on the hippocampal activity (electrically evoked, LTP). Healthy mice ("wild-type") were used as a control group. Hippocampal potentiation was performed in acute experiments under urethane anesthesia (20%, 2g/kg). For stimulation (perforant path: lambda—3.0-1,9) and registration (ipsilateral dentate gyrus 2.0 -1.7-2.0) stainless steel electrodes were used. Biphasic pulses 3V, basal low frequency 0.1 Hz, duration 0.1 ms, high-frequency stimulation (HFS) 100Hz, 10 bursts each 10 s were applied. On the contralateral side L-arginine (substrate for NO synthesis) and nitro-L-arginine (blockator of NO-synthase) were administered intracerebroventricularly (1ml, 30 min after HFS). In the selected animals NADPH-diaphorase positivity has been used as an NOS marker. The block of NOS impaired evoked responses in both groups of animals (wild and Lurcher) whereas increase of NO bioavailability (after L-arginine administration) caused an enhancement of responses, but only in healthy animals with statistically significant effect. Taken together, the cognitive deficit in this cerebellar degeneration is characterized by the change of NOS activity in hippocampal region. Results after influencing the nitric oxide bioavailability suggest that NO plays an important role (retrograde messenger?) in various types of the hippocampal activity in our cerebellar degeneration model.

Béder I¹, Kittová M¹, Maťa_eje A¹, Čársky J², Országhová Z², Babinská K ¹

Aminoguanidine (AG) and its adducts with aldehydes (Schiff bases) are membranal active substances.

The aim of this work was to observe the effect of AG, its adduct with pyridoxal-pyridoxylidenaminoguanidine (PAG) and pyridoxal on erythrocyte elasticity with expected differentiated protective influence. In 7 non-diabetic and 16 diabetic patients with insulin dependent diabetes mellitus erythrocyte elasticity was estimated by determination of their deformability using filtration method with centrifugation. Other hematological variables including erythrocyte and reticulocyte counts, hematocrit values, hemoglobin concentration and the mean cell volume were determined. AG improved erythrocyte filterability by 4%, aminoguanidine derivate PAG by 11% and pyridoxal by 13% in healthy subjects. In diabetic patients the AG effect on erythrocyte filtrability was improved by 7%, PAG effect by 9 % and pyridoxal effect by 15% in comparison to the control group. The other investigated hematological variables in both groups were within the range of the physiological standard.

Beňačka R ¹, Tomori Z ¹

Introduction: The present work was designed to compare the resuscitation effects of mechanical, air-puff and/or electrical stimulation of epipharyngeal (EPS), inferior nasal (INS) & nasal philtre areas (NPS) during respiro-circulatory failure induced by peracute systemic anoxia, asphyxia and/or short brain ischaemia.

Methods: In 14 anaesthetised nonparalyzed cats we analysed diaphragmal EMG, heart rate (HR), systemic blood pressure (BP), native & spectral EEGs, brainstem auditory/ median or trigeminal sensory evoked potentials (EP), trachel air-flows and ABS.

Results: EPS could revive respiration within 90s of apnoea Recovery was indicated by instantaneous series of aspiration reflexes (ARs) (316±12 ms, M±SE). This was followed by immediate rise in both BP (2-3x) and HR (4x) and sudden restore of pontine EP. EEG revived within next 20-40s. Rhythmic breathing reappeared 40-60 s after EPS, while thalamocortical EP recovered with 80-120s delay. Bilateral carotic occlusion (BCO) abolished renewal of brain activity in spite of present AR and sustained BP. INS evoked similar effect to EPS. although less effective (only in 63% at 30-60 s of apnoea). NPS could reactivate cardiovascular, respiratory and brain responses even 120-150 s after apnoea onset Recovery started with series of 4-10 AR-like bursts (216±12ms) followed by 2-4x increase in HR and BP and periodic gasps resuming into normal rhythm. When BP>30-40 mmHg repetitive NPS could preserve automatic breathing even after cortical death induced by BCO.

Benick_ J¹, Najvirtová M¹, Baqi L¹, Križanová O², _trbák V¹

Introduction: Neonatal streptozotocin (nSTZ) treatment in rats leads to parallel depletion of pancreatic insulin and TRH followed by spontaneous regeneration of insulin- but not TRH-containing cells. An impaired insulin response to glucose stimulation persists in nSTZ rats for the rest of life, which is the similar effect as that observed in mice with prepro-TRH gene knock-out. We therefore used the model of nSTZ rats to determine the possible role of perinatal absence of TRH in the development of insulin responsiveness dysfunction.

Methods: At the day of birth the rats were injected with 90 mg/g BW STZ i.p. TRH (10 ng/g BW/day s.c.) was injected daily during the first week of life and function of isolated pancreatic islets was studied at the age 12-14 weeks. Plasma glucose, content of insulin in plasma and in pancreatic islets, and insulin release in vitro were measured by radioimmunoassay.

Results: The nSTZ rats had normal glycemia and insulinemia. Insulin content in islets was lowered and we observed no insulin response to glucose and high KCl stimulation in vitro in nSTZ animals. Perinatal TRH treatment deeply decreased protein and insulin content in islets of both control and nSTZ rats, and enhanced basal insulin secretion in nSTZ animals in vitro. Insulin response to glucose stimulation reappeared only in TRH-treated nSTZ females.

Beręsewicz A, Mączewski M, Duda M

Cardiac ischemia/reperfusion (IR) causes damage to cardiomyocytes and coronary endothelium, the changes preventable by ischemic preconditioning (IPC). Adverse consequences of endothelial dysfunction (ED) include increased granulocyte accumulation within IR myocardium. In this study the hypothesis was tested that IPC and diazoxide, an activator of mitochondrial K_ATP channel, confer protection against ED via the mechanism that involves attenuation of myocardial peroxynitrite formation (a product of the reaction: superoxide (O₂^-) + NO). This may involve decreased O₂^- production, increased O₂^- removal, and/or decreased NO production. Perfused guinea-pig hearts were subjected either to (i) 30 min/30 min IR or to (ii) 3x5 min IPC prior to IR; (iii) 3x5 min diazoxide infusion/washout prior to IR or (iv) the hearts were infused for 10 min before the ischemia and during the initial 10 min of the reperfusion with either SOD or L-NMMA. Coronary flow responses to acetylcholine served as measures of endothelial function. IR impaired acetylocholine response by 62%, an effect prevented by IPC, diazoxide, SOD, and L-NMMA. The reperfusion-induced outflow of O₂^- was reduced, that of .OH increased, and that of NO not affected by IPC, diazoxide and SOD. Thus, it is enhanced O₂^- removal rather then decreased O₂^- production that accounts for the reduction in O₂^- outflow in IPC and diazoxide groups (perhaps via increased activation of mitochondrial K_ATP and then activation of SOD). Reperfusion-induced outflow of NO was reduced and that of O₂^- and .OH not affected by L-NMMA. These results implicate that it was the product of the reaction O₂^- + NO, but not O₂^-, per se, that mediated ED in IR guinea-pig heart, and that IPC and diazoxide afforded endothelial protection because they attenuated the biological availability of O₂^-, and thereby of peroxynitrite.

Biernat J, Sendur R, Pawlik M, Obuchowicz R, Zejc-Bajsarowicz M, Pawlik WW

The aim of our study was to evaluate the role of sensory neurons and sensory peptides in the protective action against CCl₄ induced liver injury.

Experiments were performed on anesthetized rats, weighing 220-240 g. Hepatic blood flow (HBF) was determined by Laser Doppler flowmetry and arterial pressure (AP) was measured. AspAT and AlAT serum levels were measured 24 hours after CCl₄ 

The first group: placebo, in the second sensory denervation was made by administration of Capsaicin (CAP) in increasing doses. In the third group carbon tetrachloride (CCl₄) alone (0,3 ml/kg s.c.) was given. In the fourth group CCl₄ was given 14 days after capsaicin (CAP) s.c. In the fifth and the sixth group CCl₄ was administered after CGRP (0,16 mg/kg i.p.) and Substance P (0,1mg/kg i.p.)respectively.

CCl₄ (0,3 ml/kg s.c.) produced liver damage in rats which was manifested at 24 hr in serum rise of AspAT and ALAT to 485±120 U/L and 368±83 U/L respectively. AP and HBF were decreased by 15±7% and 25±6% respectively. In chronically CAP denervated rats: CCl₄ produced significantly less hepatic damage as occurred in a rise of AspAT and ALAT to 358±45 and 255±38 U/L respectively, while HBF was increased by 58±8%. Pretreatment with CGRP and substance P did not significantly changed AspAT¢s and ALAT¢s values but HBF increased by 19±4% and 24±6% respectively in comparison to CCl₄ alone.

Biliczki P¹, L. Virág¹, N. Iost², Papp JGy^1,2, Varró A¹:

The aim of this study was to investigate the possible role of the repolarization reserve and the interaction of different potassium channels in the dog ventricular muscle preparations, by applying the conventional microelectrode and whole cell patch-clamp techniques at 37 °C. Complete block of I_Kr by 1 µM dofetilide (DOF) lengthened action potential duration (APD) by 45.6±3.6 %, n=13 (at cycle length CL=5000 ms). Chromanol 293B (CRO) at 10 µM (concentration which selectively blocks I_Ks current) applied alone did not markedly lengthen APD (< 7 %), but when repolarization was already prolonged by complete I_Kr block (1 µM DOF), inhibition of I_Ks with 10 µM CRO substantially delayed repolarization (38.5±8.2 %, n=6, at CL=5000 ms). BaCl₂ (BA) applied alone in the concentration of 10 µM (which selectively blocks I_k1 current) lengthened APD by 33.0±3.1 % (n=11), but when I_Kr was blocked (1 µM DOF) 10 µM BA produced an excessive frequency dependent lengthening in APD by 104.3±22.1 % (n=7), frequently initiating early afterdepolarizations (EAD). We have concluded that in the dog ventricular muscle when only one type of potassium channels is inhibited, excessive APD lengthening is not likely to occur. Dog ventricular myocytes seem to repolarize with a strong safety margin ("repolarization reserve"). However, when this normal "repolarization reserve" is attenuated (due to drugs, remodelling or genetic disorders), the otherwise minimal or moderate potassium current inhibition can result in excessive and potentially proarrhythmic prolongation of the ventricular action potential duration. Therefore, application of drugs which are able to block more than one type of potassium channels is probably more hazardous than the use of specific inhibitors of a given potassium channel.

CIRCULATORY, RESPIRATORY AND OTHER MARKERS AT HEMORRHAGIC HYPOVOLEMIA.

Bračoková I, _vorc P, Dorko E, Kassayová K, _timmelová J

Breier A¹, Barančík M², Kvačkajová J², Boháčová V²

Mouse leukemic cell line L1210/VCR used in our studies were prepared by adaptation of parental sensitive L1210 cell line to vincristine. In L1210/VCR cells was observed also increased cross-resistance to other cytostatics such as vinblastine, doxorubicin, mitomycin C, and actinomycin D. Important factors influencing the realization of MDR in L1210/VCR cells are flexibility of structure, lipophilicity and molecular weight of used cytostatics. Resistance to vincristine and doxorubicin was found to be associated with decreased intracellular accumulation of these drugs. Multidrug resistant cell line L1210/VCR is characterized by overexpression of PGP (determined at protein and mRNA levels) but not by an increase of activities of glutathione S-transferase. Moreover, the exposure of resistant cells to several cytostatics (vincristine, doxorubicin, mitomycin C) did not influence the activities of GST. Several drugs (chemosensitisers, like: calcium entry blockers, inhibitors of calmodulin, local anesthetics and some xantine derivatives) were found to depress the P-glycoprotein mediated MDR. The primary structure of PGP contains sequences with consensus for phosphorylation sites of some protein kinases and the linker region of MDR1-PGP was found to be phosphorylated in vivo within the acidic domain. Some protein kinase activators (phorbol myristate: PKC) and inhibitors (bisindolylmaleimid: PKC; SB203580: p38-MAPK; PD098059: ERKs) on the resistance of L1210/VCR cells were found to influence the MDR of our cells. Development of MDR in L1210/VCR cell line was associated with significant changes in content, phosphorylation and activity of p38-MAPK. These facts indicate that the phosphorylation of PGP represents important mechanism involved in the regulation of its transport activity.

Brezová A¹, Uhrík B¹, Heizmann CW²

S100A1 protein is a small acidic protein with two EF-hand type Ca²⁺-binding motifs. S100A1 can form homodimers (slow skeletal and heart muscles, kidney) or heterodimers with S100B (brain). Function and localization of S100A1 has not been clearly revealed yet, but it is assumed to have regulatory effects along the Ca²⁺-signal transmission pathway. The aim of our work was to study the subcellular localization of S100A1 protein in normal rat heart.

Rat hearts were fixed by coronary perfusion with low aldehyde concentration solution. Samples from left and right ventricles and left and right atriums were dissected and processed according to Tokuyasu technique. Ultrathin cryo-sections were immunolabelled and examined in fluorescent and electron microscope. Immunolabelling was performed with polyclonal rabbit IgG against human recombinant S100A1 diluted 1:100. The specifity of primary antibody was tested by saturated primary antibody and by rabbit normal serum.

In sections from all types of heart tissue the most pronounced immunolabelling was seen in mitochondria. Much lower labelling could be detected in myofibrils, at different levels of sarcomere. Up to the present no target proteins for S100A1 are known in mitochondria. However, the functional significance of S100A1 for cardiac muscle may be assumed from results of studies on patologically changed heart: a lowered expression of S100A1 in human cardiomyopathy and its right ventricular upregulation in chronic pulmonary hypertension.

Brtko J¹, Podoba J², Schmutzler C³, Köhrle J³

The majority of thyroid adenomas are of clonal origin. The present study was undertaken to investigate the status of functional thyroid hormone receptors, major thyroid hormone signal mediators, in both the human TAs and CNs in comparison with a normal thyroid tissue from the same patient. Electrophoretic mobility shift assays using a DR4 („direct repeats" 4), a thyroid hormone responsive element (TRE) derived from the human type I iodothyronine 5´-deiodinase promoter demonstrated the DNA-binding of thyroid hormone receptors (TRs) in thyroid tissue nuclear extracts. A significant increase (p ‹ 0.05) in the functional binding properties of TRs to the DR4 TRE was found in TAs when compared to normal thyroid tissue. Contrary, a marked diminution in the TR-TRE complex formation was found in CNs in comparison with normal thyroid tissue. In addition, functional activity of the iodothyronine 5´-deiodinase (5´-DI) was analyzed in benign tumours, thyroid TAs and CNs in comparison with that of normal thyroid tissue. A significantly increased (p ‹ 0.01) activity of 5´-DI was demonstrated in TAs, and in contrast, decreased values of the enzyme activity were found in CNs when compared to a normal tissue.

From the data it is suggested that both the status of TR-TRE complex formation and the activity of the 5´-DI may be altered in benign tumours of human thyroid gland.

Cagalinec M^1,3, Matea_ik A³, Chorvátová A^2,3, Chorvát D³

In recent decades we could see evident progress in biological optical microscopy methods, caused by wide availability of lasers, CCD detectors and digital data processing. Meanwhile, there is an apparent paradigm shift from applications using simple measurement of light intensity to the utilisation of derived, more complex fluorescence parameters like excited state lifetime, anisotropy etc.

Here, we present an extension of mentioned philosophy towards characterisation of spatially resolved calcium waves in isolated left ventricular rat cardiomyocytes, labelled with fluorescence calcium indicator Fluo-3. In addition to CCD imaging providing series of fluorescence images, we have constructed normalised temporal gradient of fluorescence intensity in each pixel and find it’s extremes describing local dynamic characteristics of the calcium wave (rise and decay constant). Using this methodology it is possible to obtain spatially resolved images of cell function parameters instead of simple calcium concentration mapping. We describe a simplified kinetic model that is possible to use for analysis of fluorescence data practically in real time, what is essential regarding possible future use of this setup in regular experiments for cellular physiology or cellular pharmacology.

The translocation of GLUT4 glucose transporter protein to the plasma membrane (PM) 30 min after oral application of glucose (3g/kg b.wt.) after overnight fasting was studied in insulin resistant hereditary hypertriglyceridemic rats (HHTg) in epidydimal fat pad, diaphragm, m. gastrocnemius and heart. In addition, the glycogen content in muscles was examined as one of the indicators of glucose utilisation. The adult HHTg males and control (C) males were kept one week on high sucrose diet. After the diet the HHTg group had worse glucose tolerance compared with control group and significantly higher content of serum triglycerides (3,7 ± 0,1 vs 1,6 ± 0,2 mmol/l).

The major differences in the GLUT4 content in PM between HHTg and control groups after glucose load were found in adipose tissue (the increase of 29% vs 90%). In m. gastrocnemius of control group the GLUT4 content increased by 50%, in diaphragm by 37% whereas in HHTg group we found no changes. GLUT4 content in myocardium was not different between HHTg and control rats. The changes in glycogen content in m. gastrocnemius reflected the changes in PM GLUT4 content.

We conclude that the impaired sensitivity of tissues to insulin in HHTg rats is associated with impaired ability to translocate GLUT4 transporters to the plasma membrane and the decreased synthesis of glycogen in skeletal muscle.

Pulmonary surfactant is a film of surface active agents that coats the alveolar surface and the small conducting airways. The physiological advantages of lung surfactant are that (i) it reduces the work of breathing and thereby reduces the muscular effort needed to expand the lungs; (ii) it lowers the surface tension at low lung volume, thus preventing the alveoli to collapse at the end of expiration; (iii) it prevents the deflation of individual alveoli at different rates.

Natural surfactant is a mixture of lipids, proteins (about 10%), and a small portion (less than 1%) of carbohydrates. The major lipid component is dipalmitoylphosphatidylcholine (DPPC). Adequate surface activity in vivo is only found in surfactant preparations that contain phospholipids and specific, surfactant associated proteins (SP). Four surfactant proteins have been described to date (SP-A, SP-B, SP-C, SP-D).

Surfactant is synthesised, stored, and recycled or catabolized by alveolar type II epithelial cells. It is packed in dense lamellar bodies, after exocytosis converted to tubular myelin in alveolar space and spread into a film at air/liquid interface. Control of metabolism of surfactant takes part at local, humoral and neural levels.

Camborova P¹,Pechan J², Ferak I³, Hubka P⁴, Jakubovsky J⁵, Hulin I¹.

The relation between gastric electrical activity measured at the surface of the skin and contractile activity is one of the complications of electrogastrography (EGG). Dominant frequency of the gastric slow wave in the EGG determins the maximum frequency of the contractions and could reflect the activity of the ICC network. The aim of our study was to detect the changes in stomach electrical activity in a patient with gastric outlet obstruction lasting 2 years. The EGG recordings were performed before the surgical correction and 10 monthsafter the operation.Before the surgical correction stable electrical activity was recorded (2.5cpm) and ultrasound revealed absence of gastric contractions. This electrical activity was slightly lower in comparison with mean values in our group of healthy subjects. 10 months following the surgery activity of 2 cpm was observed..The surgery confirmed gastric dilatation with major curvature length of 80 cm. Histology revealed mild dysplasia, chronic gastritis and Helicobacter pylori.

Even enormous gastric dilatation and structural changes haven’t resulted in a loss of electrical activity. The question is, why has the synchronized electrical activity not initiated gastric contractions.

Cendelín J¹, Žalud V¹, Jelínková D¹, _tenglová V¹, Vožeh F¹, Vrba J²

We studied the effect of high frequency electromagnetic field (HF EMF) on healthy wild type (+/+) and Lurcher mutant (+/Lc) mice (C3H strain). Lurcher mutants served as a model of olivocerebellar degeneration.

Spatial learning ability of adult animals was tested immediately after acute exposure to HF EMF or control conditions. Young mice were exposed to long-term influence (postnatal days 2 — 21) of HF EMF that involved the rapid brain growth spurt. Spatial learning was tested using Morris water maze. Brains of selected animals were processed histochemically and immunohistochemically to detect NADPH diaphorase and c-Fos.

Both +/+ and +/Lc adult animals exhibited no changes of spatial learning ability after acute exposure to HF EMF. The long-term exposure caused deterioration of spatial learning ability in +/+ and it slightly improved results in +/Lc. These differences were statistically insignificant. Detection of NADPH diaphorase showed no differences between exposed and control adult mice. In young control +/Lc was higher NADPH diaphorase activity in hippocampus and in some other brain structures as compared with exposed mice. Certain changes of c-Fos immunoreactivity in dependence on HF EMF exposition also were found.

We found no significant effect of HF EMF. However, some changes were discovered in mice exposed to HF EMF during their early ontogenetic development.

Cernayova A, Pancza D, Ravingerova T

Myocardial adaptation to ischemia has been recently shown to trigger multiple signaling pathways including activation of receptor tyrosine kinase and subsequent stimulation of p38-MAP kinase pathway. The aims of the study were to investigate their role in the protective effect of ischemic preconditioning (IP).

Isolated Langendorff-perfused rat hearts were subjected to test ischemic challenge (TI) induced by 25 min global ischemia (GI) followed by 35 min reperfusion (R). Recovery of function at the end of reperfusion (left ventricular developed pressure, LVDP) expressed as % of the initial value served as the end-point of injury. The hearts were adapted to ischemia by 2 episodes of 5 min GI and 5 min R before TI. To block receptor tyrosine kinase pathway, the hearts were pretreated with genistein (50 and 100 µM), and SB 203580 (5 µM) was used to inhibit p38-MAP kinase. Both drugs were applied 10 min before the onset of ischemia.

After TI/R, LVDP in the control group was 22.6 ± 3.5%, whereas preconditioned hearts showed a significantly better recovery of function (LVDP 63.0 ± 5.1%; P<0.05). 100 µM genistein partially blocked protective effect of IP, whereas SB 203580 completely abolished protection afforded by IP (LVDP 27.7 ± 7.8%).

IP protects rat hearts against myocardial dysfunction caused by ischemia/reperfusion. Blockade of cardioprotection with SB 203580 suggests a potential role of the activation of p38-MAP kinase pathway in the protective effect of IP. The activation of the upstream signaling mechanisms cannot be excluded.

Červenáková Ž¹, Penesová A¹, Ježová D¹, Kvetňansk_ R¹, Hinghofer-Szalkay H², Macho L¹, Ko_ka J¹

Changes in body fluid distribution are known to influence endocrine gland function. We studied the effects of altered plasma volume (PV), achieved by different body positions, on neuroendocrine response to insulin-induced hypoglycaemia.

Tests were performed in 12 subjects on 2 occasions: "head-up" (+60º head-up tilt standing for 30 min and hypoglycaemia in sitting position afterwards) and "leg-up" (leg-up position for 30 min and hypoglycaemia in leg-up position afterwards) in a random order. Controlled hypoglycaemia was induced by insulin infusion and adjusted to 2.7 mmol/l for 15 min by glucose infusion.

PV was significantly greater in leg-up (p<0.001) and lower in head-up (p<0.001) position than basal value. Head-up position caused increases in ACTH, aldosterone, noradrenaline (NA) and PRA (p<0.01). Leg-up position resulted in decreases in GH and adrenaline (A) (p<0.05). Contraregulatory response to controlled hypoglycaemia was mild. The significant increase in GH and A responses to hypoglycaemia used did not differ between the positions. Hypoglycaemia failed to activate ACTH release in head-up position. As to the gender differences, A response to hypoglycaemia was greater in males than in females (p<0.001).

&#These results show that reduced PV is associated with elevated plasma NA, ACTH, aldosterone and PRA while augmented PV with decreased A and GH concentrations. In the case of ACTH, the first stimulus is consequential for the subsequent response. Rise in A levels during hypoglycaemia depends on gender and is decreased in women.

Červinková Z, Radvaková D

&#The aim of our study was to evaluate impact of bile duct ligation on the functional and morphological parameters of small intestine.

Material and methods: The experiments were performed on 30 male albino Wistar rats with an initial body mass of 210-225 g. In the first group secondary biliary cirrhosis (BC) was induced by ligation of bile duct, the second control group (C) underwent median laparotomy. The rats were sacrificed three weeks after the operations. The extent of liver damage and liver regeneration was determined by assessment of serum activities of AST, ALT, GMP and AP, serum concentrations of albumin and CRP, liver DNA synthesis, and mitotic activity of hepatocytes. Intestine permeability was measured by lactulose-mannitol test, morphological changes were evaluated using histological estimation.

Results: Significant increase (p<0.05) of biochemical parameters together with liver morphological changes clearly documented development of BC in rats with bile duct ligation. Lactulose-mannitol test and intestine DNA synthesis were significantly increased (p<0.05) in BC rats to compare with controls.

Conclusion: Secondary biliary cirrhosis induced injury of small intestine shown by histological (confluence and irregularity of villi) and functional changes (impairment of intestinal barrier). These changes are probably to some extent related to the presence of portal hypertension. Intestine injury was followed by induction of reparative process judging by increased DNA synthesis in the small intestine.

Chvátal A, Anděrová M, Syková E

In conclusion, our experimental data and mathematical analysis reveal a more compact ECS volume around oligodendrocytes than around astrocytes. Such heterogeneities in the gray matter may selectively affect the diffusion of neuroactive substances in specific areas and directions and facilitate spatial K⁺ buffering in the nervous tissue.

Czakóová G¹, Kasalová L², Novotná J², Hermochová D¹, Korynta J³, Herget J¹

We have shown in the rat lungs that chronic hypoxia initiates increase in lung collagenolytic activity which results in the presence of low molecule weight collagen type I cleavages . The oxygenation of cornea is provided mainly directly from the athmosferic air. Therefore the minimal participation of the blood born substances may be anticipated in hypoxic corneal tissue. The aim of our study was to find the low molecular collagen peptides in the corneas of rats exposed to hypoxia. The rats were exposed to isobaric chronic hypoxia 10%O₂ for 4 and 14 days. Gel electrophoretic separations (SDS-PAGE) of the collagenous proteins isolated by limited pepsin digestion from corneas of 6 rats exposed to 4 days hypoxia, 6 rats exposed to 14 days hypoxia and 6 and 6 respective normoxic controls were performed. We found no differences in gel electrophoretic prophile of collagenous proteins between the normoxic rats and groups of rats exposed to hypoxia. There were no signs of vascularization of corneal tissue in rats exposed to hypoxia. We conclude that it is unlikely that the presence of collagenous cleavages in chronic hypoxia is a direct effect of lack of oxygen on collagenous matric proteins.

1.

Insuline-like growth factor-1 (IGF) overexpression was reported in acute pancreatitis. The aim of our studies was to determine the effect of IGF administration on the development of caerulein-induced pancreatitis (CIP).

Acute pancreatitis was induced by s.c. infusion of caerulein (10 mg/kg/h) for 5 h. IGF was administrated twice (30 min prior to caerulein or saline infusion and 3 h later) at the doses: 2, 10 or 50 mg/kg s.c. Immediately after cessation of caerulein or saline infusion the pancreatic blood flow (PBF), plasma amylase and lipase activity, plasma interleukin-1b (IL-1b) and interleukin 10 (IL-10) concentration, cell proliferation, and morphological signs of pancreatitis were examined.

Administration of IGF without induction of CIP increased plasma IL-10. Treatment with IGF, during induction of pancreatitis, increased plasma IL-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in DNA synthesis and PBF, and the reduction in pancreatitis evoked increase in plasma amylase, lipase and IL-1b level. Protective effect of IGF administration was dose-dependent, the dose 2 x 50 mg/kg caused the maximal reduction in pancreatic damage.

Conclusions: (1) Administration of IGF attenuates pancreatic damage in CIP; (2) This effect seems to be related to the increase in production of IL-10, reduction in release of IL-1β, the improvement of PBF and the stimulation of pancreatic tissue growth.

Donič V, Doničová V, Le_ko R, Tomori Z

Many sleep laboratories reported elevated blood pressure (BP) and changes in heart rate variability (HRV) of patients with sleep related breathing disorders (SRBD). These changes are not always clearly visible by using standard polysomnographic recordings. Sleep apnoea patients create usually not a homogenous group, but may have obstructive sleep apnoea syndrome (OSAS), OSAS treated with CPAP, upper airway resistance syndrome, simple snoring, etc. Therefore 24-h ambulatory (BP) devices, which offer blood pressure and HRV assessment during sleep, but also during wake period of the day, could be useful for study and better understanding of this phenomena.

Methods: Heart rate and BP were monitored using 24-h ambulatory blood pressure device (Cardiotens, Meditech Budapest) in adults and power spectral analysis of HRV was performed. Low frequency/high frequency ratio LF/HF, which reflects the sympatho-vagal balance, was calculated for the period of sleep vs. wakefulness (S/W). Our aim was 1) to detect the sensitivity - utility of our parameter in a group of patients with differentiation of OSAS against a group treated by CPAP, with respect to sympatho-vagal tone, 2) to select patients with high probability of SRBD

Results: Increased S/W ratio reflecting sympathetic activation was observed in patients with OSAS having (RDI 43.8) (0.9), compared to patients treated with CPAP (0.68), and people without SRBD.

Distribution of GABAergic neurons in the auditory cortex and in subcortical auditory structures.

Druga R, Ouda L, Syka J

Gamma-aminobutyric acid (GABA) is considered the major inhibitory transmitter in the CNS. The distribution of GABA-producing neurons in the auditory cortex (AC), the medial geniculate body (MGB) and the inferior colliculus (IC) of the rat was investigated by using an antibody to GAD and GABA in effort to obtain normative data for future experiments.

The IC contained a large population of GABA immunoreactive neurons distributed throughout all this subdivisions. A higher density of GABAergic neurons was evident in the basal part of the central nucleus and in the basal part of the external cortex of the IC.

The density of GABAergic neurons within the MGB was lower than in the IC and GABAergic neurons prevailed in the ventral division of the MGB.

In the auditory cortex GABAegic neurons were found in all fields (Te 1, Te 3 and Te 2 ) and were distributed in all cortical layers, particularly in layers IV and VI.

Dvorakova M^1,2, Kummer W¹

1Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany, and

Eckhardt A^1,4, Mik_ík I^1,4, Cserháti T², Forgács E², Zicha J¹, Deyl Z^1,3

Depository effects in collagen (and slowly metabolized proteins in general), are very difficult to assess owing to extremely low concentration of the modifying (deposited) entities in the protein matrix. Typical of these nonenzymatic changes is glycation (Maillard reaction). In order to reveal such alterations we applied deep enzymatic fragmentation by bacterial collagenase resulting in a set of small peptides which, if modified, are likely to change their chromatographic and electrophoretic properties and can be visualized on the resulting peptide profile. Bacterial collagenase cleaves collagen mainly to tripeptides (some of them we successfuly identified).

Collagen from tail tendons of four groups of rats was studied. The tissue (a mixture of type I and III collagens) was digested by bacterial collagenase, the arising peptides were separated by HPLC and divided into five fractions. Further characterization of the profiles was done by capillary electrophoresis (CE).

It was demonstrated that the combination of HPLC and CE peptide mapping with subsequent statistical evaluation (principal component analysis and t-test) represents a reliable approach for revealing posttranslational modifications in slowly metabolized model protein in vivo.

Ekmekcioglu C¹, Haslmayer P², Philipp C¹, Mehrabi MR³, Glogar HD³, Grimm M⁴, Thalhammer T², Marktl W¹

El-Saggan AHH, Uhrík B

Polycationic dye Ruthenium red (RR) stains negative binding sites in external coats of cells, related to transport, barrier, or receptive functions, and has been used as a marker of glycoconjugates in cells of different origin. However, RR penetration into deeper parts of tissue or cells is limited due to repulsive forces of those RR⁶⁺ polycations already bound to cellular matrix. This problem may severely limit the use of RR in studies of staining properties both of tissues and of isolated cells concentrated by centrifugation. In the present study a new method of RR staining of negative binding sites on cryosections of glutaraldehyde-fixed frozen cells has been introduced with the aim to expose simultaneously all the cells and their components to the cationic dye treatment.

The mouse leukemic cell line L1210 and rat heart muscle cells from a left ventricle were used. Cells were fixed with 2% glutaraldehyde in cacodylate buffer (CB), soaked in 2.2 mol/l sucrose and frozen by plunging into liquid nitrogen. Ultrathin cryosections were cut at a temperature of -90°C, transferred with a wire-loop to Formvar coated copper grids, postfixed with 1% OsO₄ and stained with 0.05% RR in CB for 60-120 min. After removing RR solution with filter the grids were dried and examined electron microscopically.

The resulting staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat).

Ficková M, ¹Kotyzová D, ¹Mičková V, ¹Eybl V, Brtko J

Cadmium (Cd) and mercury (Hg) are inorganic toxicants of great environmental and occupational concern. Moreover, chronic exposure of rats to oral Cd exerts diabetogenic effect characterized by hyperglycemia and lower glucose stimulated insulin release. This study was performed to investigate the influence of toxic environmental contaminates — Cd and Hg on insulin receptors in isolated fat cells.

Male Wistar rats were drinking ad libitum either CdCl₂ (9,7 mg/l) or HgCl₂ (11,5 mg/l) in tap water for 6 weeks. This intake did not influenced weight gain (C=+237 g, Cd+255 g, Hg=+239 g). Cd treatment induced slight increase in glycemia (6,0±0,2 mmol/l) as compared to control rats (5,4±0,2 mmol/l), p<0,05. Elevated insulinemia in Cd group (0,678±0,1 ng/ml) was not statistically different either to control (0,47±0,05) or Hg (0,45±0,09) group. The number of insulin receptors (IR) in adipocytes was significantly lowered in both treated groups (C=22,3±3,6x10³/cell, Cd=9,4±1,1x10³, p<0,01; Hg = 11,1±0,9x10³, p<0.05). Despite the similar fat cell size in all groups, significantly smaller density of IR in both, Cd and Hg rats was observed. Less insulin receptors (Cd) displayed higher apparent affinity binding constant K_a (p<0,05 vs. C) and increased ED₅₀ values for insulin. A significant negative correlation (r=-0,89, p<0.001) between insulinemia and insulin receptors in isolated adipocytes of Cd group was present.

The results demonstrate the presence of IR disturbances in adipocytes as the effect of long-term intake of toxic elements.

Fi_árková B, Vytá_ek R, Vízek M,

Turanlahti et al. recently showed that hyperoxia attenuated free radical-mediated effects of NO inhalation. To test whether hyperoxia could similarly attenuate effects of endogenously produced NO, we measured nitrotyrosine concentrations in serum of rats exposed to hyperoxia during experimental pneumonia. 34 Wistar male rats were assigned to one of 4 groups. In groups 1 and 2 0,5 ml of 0,7 % carragheenan was applied intratracheally (carragheenan groups). Groups 1 and 3 were then placed for 48 hours into hyperoxia (F_IO₂ 0,75 — 0, 83) (hyperoxic groups). Rats of groups 2 and 4 breathed air (normoxic groups). 48 hours later were all rats anesthetized, intubated, their ventilation measured, their expired air collected for NO concentration measurement and 1 ml of blood taken to examine serum nitrotyrosine concentration (ELISA). ANOVA was used for statistical evaluation.

Carragheenan installation combined with hyperoxia increased NO production (159,8 ± 36,9 ppb/min compared to 22,6 ± 6,6 in controls, p< 0,05). Nitrotyrosine concentrations were in both hyperoxic groups lower than in normoxic (carragheenan-hyperoxic group 5,1 ± 0,3 uM, caragheenan-normoxic 8,3 ± 0,7; p< 0,05, control-hyperoxic 4,5 ± 0,9 and control-normoxic 7,5 ± 0,7; p< 0,05). The results suggest that hyperoxia prevents nitration of plasmatic proteins.

Turanlahti et al.: Nitric oxide and hyperoxia in oxidative lung injury. Acta Paediatr., 89, 996 -70, 2000.

Franěk M¹, Vaculín _¹, Hess L², Rokyta R¹

Medetomidin (ME), a₂ agonist, has been used in veterinary medicine for a long time to evoke sedation or as a component of combined anaesthesia. In present paper the effects of intramuscular ME alone and its combination with ketamine (KE) on pain threshold in rats are described.

ME was administered to Wistar male rats weighing 200- 230g in the doses: 100-1500 mg/kg, its effect was compared with two control groups (saline; morphine 5 mg/kg). KE (25 mg/kg) was administered alone and in combination with ME (100 and 200 mg/kg). Plantar test and tail flick were used to measure pain thresholds before, 10 and 20 min after the administrations. These two methods partially enable to distinguish spinal and supraspinal antinociceptive effects of ME.

In plantar test me evoked a shortening of response latencies for the doses 100-500 mg/kg and had no effects for the doses 750-1500 mg/kg. In tail-flick test, me evoked dose dependent antinociception for the doses over 500 mg/kg. The combination of me 100 mg/kg and ke 25 mg/kg evoked strong antinociception, 200 mg/kg me and 25 mg/kg ke evoked total anaesthesia. Ke alone (25 mg/kg) had no effect on nociception.

The obtained results indicate, that me affects nociception at two levels and in different-opposite manner: supraspinally facilitates and spinally inhibits. It is suggested that the supraspinal facilitation results from the inhibition of a tonic antinociceptive effect of bulbar nuclei (i.e. Disinhibition) while the spinal inhibition is caused by a decrease of the activity of the dorsal horn neurons participating in the nociceptive pathways.

Friedl R¹, Moeslinger T¹, Kopp B² and Spieckermann PG¹

1Institute of Physiology, Vienna, Austria

The human retina offers the unique possibility to study erythrocyte and leukocyte movement in vivo with two independent methods. The blue field entoptic technique assesses leukocyte movement in the perifoveal retinal capillaries. Using laser Doppler velocimetry and fundus photography erythrocyte movement in larger retinal vessels can be quantified. Little is, however, known about possible interactions between erythrocytes and leukocytes in the human retina. We investigated erythrocyte and leukocyte flow in the retina during administration of granulocyte colony-stimulating factor (G-CSF) under basal conditions and during states of pronounced vasoconstriction induced by systemic hyperoxia. After 8 hours G-CSF increased total leukocyte counts four-fold. This increase in circulating leukocytes was reflected by an increase in retinal white blood cell density (110 ± 48%). By contrast, neither retinal vessel diameters nor red blood cell velocity were altered by administration of G-CSF. During systemic hyperoxia a pronounced decrease in red and white blood cell flux was observed. 8 hours after G-CSF administration the reduction in white blood cell flux during systemic hyperoxia was comparable to that under baseline conditions. By contrast, the response in erythrocyte flow during systemic hyperoxia was more pronounced when G-CSF was pre-administered. These observations appear to be related to the "train" effect, a phenomenon which describes the complex interactions of leukocytes and erythrocytes in capillaries and smaller arterioles and venules. Our observation also indicates that leukocytes play an important role in retinal vascular resistance.

Gaba_ová E¹, Béder I¹, Babinská K¹, Béderová A³, Tureck_ L², Uhlíková E²

1Instute of Physiology and ²Institute of Medical Chemistry and Biochemistry, Medical School, Comenius University, ³State Health Institute, Bratislava, Slovakia

This study was designed to identify the beliefs, motivation and personal and enviromental influences shaping eating habits of a group of college students in Slovakia. We studied 167 students of medical faculty- 43 men and 124 women, average age 21.5years, who provided information on demographic and socio-economic variables, responded to an interviewer-administered, food-frequency questionnaire that assessed the consumption of more than 100 food items and 24-hours recalls of food intake. Study included measuring of blood pressure, anthropometric and lipid parameters as well as lipid peroxidation levels- conjugated diens. The prevalence of overweight was only 4% in this study, more often in women. Higher level of total cholesterol has been found in 26% of students, high levels of LDL cholesterol in 13,6%. Almost a half of students did not take food regularly, in most of the cases they replaced the main meal with the fast food. One third of the investigated group, takes vitamin and mineral supplements. However, there were variations between individuals, with specific practices being influenced by personal food preferences, time availability, health beliefs and concern, food availability, and the physical and social enviroment. Results indicate that, in general, the study group was reasonably well nourished. However, fat consumption was 30% higher than the recommended intake, for both males and females. The percentage of energy derived from carbohydrates was below the guideline value in both sexes. Relatively low iron and fiber intakes were found for females. Even though all students participated in some physical activity, less females participated in high activity sport. Based on these results, some concern about the dietary habits and the related health consequences in medical students appears justified.

The coupling between increased work and metabolism, which has been postulated for the brain more than 100 years ago, has been demonstrated now for many different tissues. In the retina, this coupling between augmented neuronal activity and increased blood flow exists as well. Using diffuse luminance flicker for photic stimulation this has been verified in several animal and human studies. A variety of techniques have been employed to show that diffuse luminance flicker induces retinal and optic nerve head vasodilatation. However, despite may efforts, the mechanism behind the coupling between increased neural activity and augmented blood as well as possible mediators involved in this process are still a matter of controversy. New knowledge on this coupling is now being accumulating thanks to the development of different techniques as laser Doppler flowmetry and new methods for exact determination of retinal vessel size, which allow the accurate assessment of the activity induced blood flow response. This presentation will provide an update on our current understanding of the coupling between increased neural activity and ocular blood flow and the factors which may potentially interfere with it. Furthermore a short overview of the techniques currently available for the assessment of neurovascular coupling will be given.

Gerová M, Smie_ko V, Kristek F

The huge body of data dealing with the role of arginine metabolism and/or NO production in genetic hypertensive rats (SHR) provide 3 possibilities: (i) NO production increase, (ii) decrease and (iii) no change. The aim of the study was to compare the response of systemic blood pressure (BP) to activators of NO synthase (acetylcholine - Ach and bradykinin - BK) in SHR and NO deficient rats (NODH). BP in age matched, anaesthetized Wistar control rats (n=8), SHR (n=7) and NODH (n=8) was 137.0 ± 2.7 mmHg, 177.1 ± 5.4 mmHg (P<0.05, vs controls), and 166.0 ± 2.7 mmHg (P<0.05, vs controls), respectively. BP decrease induced by Ach i.v. (10 µg/0.1 ml physiol. sol./10sec) amounted in controls 63.8 ± 3.2 mmHg, in SHR 108.1 ± 6.4 mmHg (P<0.05, vs controls), and in NODH 107.8±4.9 mmHg (P<0.05, vs controls). BP decrease induced by BK (100µg/0.1ml/10sec) amounted in controls 53.3 ± 5.2 mmHg, SHR 106.6 ± 8.3 mmHg (P<0.05, vs controls), and NODH 105.2 ± 4.5 mmHg (P<0.05, vs controls).

Significant increase in hypotensive response of SHR and NODH to both Ach and BK vs controls was found. No difference in rate of hypotension was found in two models of hypertension. The later finding might indicate either (i) an increased NOS activity in SHR, or (ii) a common mechanism, however different from arginine ® NO pathway, in both SHR and NODH.

Hamar J,Turchányi B, Tóth B, Vendégh Z

Aims: Ad and C fibers are responsible for antidromic vasodilatation and neurogenic inflammation (Szolcsányi J. Agents and Actions 23:4-11. 1988.). In the present studies we wanted to see whether the inactivation of these fibers could influence reperfusion injury of the skeletal muscle.

Methods: Sensory fibers of the left sciatic nerve were selectively damaged with capsaicin treatment in the anesthetized rat. 7 days later the effected hind limb underwent tourniquet ischemia for 30, 60, and 120 min. Then the limb was allowed to be reperfused for 1, 24, 72, and 168 hours. The isometric tetanic contraction force was measured of the extensor digitorum longus muscle (EDL) on both sides (damaged vs. control). Contractions were elicited with direct and indirect electric stimulations of the EDL and the sciatic nerve, respectively.

Main results: Capsaicin alone does not effect the EDL contraction force. Selective denervation did not influence the contractions after 30 min ischemia. After 60 min ischemia the selectively denervated muscle contracted slightly weaker at 168 hours of reperfusion. Contraction force of the capsaicin treated side was significantly stronger at all reperfusion times and stimulation modes.

Conclusions: A 2-hour ischemia produces qualitatively more serious damages. Exclusion of the peptidergic sensory afferents can reduce the inflammatory reaction, a major component of reperfusion injury.

Herget J¹ , Novotná J, Bláhová L¹

Hypercapnia attenuates the hypoxic pulmonary hypertension (HPH) and oxygen radical injury induced by exposure to chronic hypoxia . HPH results from remodeling of peripheral pulmonary arteries. We hypothesize that the remodeling is induced by matrix collagen cleavage in the vascular wall . Collagen composition of peripheral pulmonary arteries was studied in male adult rats exposed to 4 days chronic isobaric hypoxia (F_iO2 = 0.1, F_iCO2 = 0, n = 7), hypoxia and hypercapnia (F_iO2 = 0.1, F_iCO2 = 0.045, n = 7) and in normoxic controls (n = 7). Collagenous proteins were extracted from the isolated peripheral pulmonary arteries and analyzed by SDS electrophoresis . Similarly to our previous study in all hypoxic rats, we detected the 3/4 and 1/4 collagen type I cleavages. Cleavages were not present in rats exposed to normoxia and in rats exposed to hypoxia combined with hypercapnia.

We conclude that hypercapnia attenuates HPH by inhibition of collagen cleavage in the walls of peripheral pulmonary arteries. The mechanism is probably related to the CO₂ inhibition of hypoxia induced radical tissue injury.

Hijová E¹, Ni_tiar F², Petrá_ová D¹

Mercury is a serious ubiquitous pollutant (environmental and occupational), which have been reported as potent toxic and/or carcinogenic agens in humans and animals. Mercury enters an organism in a variety of chemical forms, causes the production of reactive ogygen species leading to the formation of an oxidative stress.

Methods: Adult male Wistar albino rats (n=60, 10 per group) were for 30d. fed on a normal laboratory diet and supplied with drinking water containing mercury (as mercuric chloride) in different doses of LD₅₀. The LD₅₀ for HgCl₂ is 37 mg.kg^-1. The rats in control group received drinking water without mercuric chloride. Plasma specimens were used for determination of vitamin C as antioxidant and malondialdehyde as product of lipid peroxidation and stress indicator.

Results: In the group receiving dose LD₅₀ of mercuric chloride the concentration of ascorbic acid and malondialdehyde were increased by 22.19% and by 6.88% respectively, compared with control group. Statistically significant was comparison dose of LD₅₀ of HgCl₂ with 1/4 of LD₅₀ for ascorbic acid (p<0.01), and for malondialdehyde (p<0.001).

Hinghofer-Szalkay H, ¹Jezova D, Loder I, Roessler A

Purpose of our study was to identify stimulus combinations of lower body negative pressure (LBNP) and head-down tilt (HDT) which compensate each other in their effect on cardiovascular variables, electrical thoracic impedance, hematocrit and plasma mass density, and hormone concentrations in blood plasma. We hypothetized that „neutral points" (NP’s) can be demonstrated (hypothesis 1); NP’s may vary with variables (hypothesis 2); and NP’s might change as a function of time (hypothesis 3). Tests were performed in 10 healthy young subjects. Normalized values were used to compare with supine control conditions SC to get rid of time-dependent shifts unrelated to the stimulus. The effect of a certain stimulus pair X at time t was defined as the normalized value at(X,t) minus the normalized value at (SC,t). Data were plotted as a function of tilt angle, data points connected by a best-fit line or curve, and the NP determined as inter- or extrapolated NP’s, defined as the intersection with the respective supine control line. Preliminary findings suggest that with LBNP-15, the NP is 13-16° HDT for thoracic impedance (irrespective of stimulus duration), and plasma renin activity (PRA), and >24° for aldosterone; no clear NP was found for heart rate, blood pressure, plasma density, and hematocrit. With LBNP-35, the NP is about 16-30° for heart rate (increasing with stimulus duration), 26-28° HDT for PRA and aldosterone, and Ò30° HDT for thoracic impedance, plasma density, and hematocrit. It might be concluded that NP’s do not always clearly show (refutes H1), that NP’s differ for different variables (confirms H2), and there is a dependency on stimulus duration in some instances (partially confirms H3).

Hitka P, Vízek M

Analyses of expired air became an important resource of clinical information (1). Such analyses require relatively large volumes of expired air therefore, in studies using small experimental animals, long lasting collection of expired air is necessary. This paper describes a system exhausting expired air of spontaneously breathing anesthetized, intubated rats.

Tidal volume of anesthetized, intubated rat was recorded by classical body plethysmography. The volume signal was used to trigger pump, sucking air from the side port of tracheal cannula. Onset of either inspiration or expiration activated an electronic controller regulating the time delay with which the pump was switched on and intensity of the sucking. The flow sucked by the pump resembled the expiratory flow of the animal.

The system was tested by comparing CO₂ output measured by collecting the expired air by our system with classical collection of expired air. The differences between the methods ranged from +0,2 to —0,1 ml/min. The respective CO₂ outputs (1,6 ± 0,4 and 1,5 ± 0,4 ml/min (mean ± SD)) did not significantly differ.

References:

1. Mutlu, G.M. et al.: Collection and analysis of exhaled breath condensate in humans. Am J Respir Crit Care Med, 2001, 164, 731-737

Hlavacka F, Polonyova A

Postural instability in cosmonauts returning from spaceflight probably results from in-flight adaptation of central nervous system processing of sensory inputs from vestibular, somatosensory and visual systems. We tested how post-spaceflight postural reactions to unilateral vibration of lower leg muscles (somatosensory input) were changed. For comparison the postural responses to soleus and tibialis anterior muscles vibration with frequencies (40-100 Hz) were measured in healthy subjects.

Vibration (duration 8s, amplitude 1 mm and frequency 80 Hz) was applied to the belly of tibialis anterior (TA) or soleus (SOL) muscle. The force platform measurements (center of foot pressure - CoP) were performed before spaceflight and on the first day (10 hours) after landing, the second day and the fifth day.

Unilateral vibration of the TA muscle induces tilt of body forwards and in the direction of the stimulated leg. While unilateral vibration of SOL muscle causes a backward tilt and in the direction opposite to the stimulated leg. In the first days after spaceflight an increased postural responses to soleus vibration were observed. Influence of microgravity on postural response to TA vibration was negligible. Comparison of body responses to the left and to the right leg vibration showed changes in proprio-postural asymmetry. The balance recovery was successfully finished after 5 days.

The results showed that the magnitude of postural responses (body tilt) to leg muscle vibration was modulated by the frequency of the vibratory stimulus. The frequency of vibration does not influence direction of body tilt, which was determined only by the muscle stimulated.

Impairment of the ocular perfusion represents a major pathophysiological mechanism for the development of several ocular diseases including open-angle glaucoma, which is one of the leading causes of blindness. Endothelin-1 has an important role in the regulation of the optic nerve head and retinal blood supply, systemic blood pressure as well as aqueous humour outflow and consequently regulates the intraocular pressure. These factors in combination determine the ocular perfusion pressure and its diurnal fluctuation, which seem to be key mechanisms in the development and progression of the retinal ganglion cell apoptosis in open-angle glaucoma. Animal models for the endothelin induced damage of the optic nerve head perfusion, decrease of the aqueous humour outflow and elevation of the intraocular pressure as well as low systemic blood pressure as a consequence of low plasma endothelin-1 concentration in the human will be shown in the lecture. The clinical consequences for glaucoma will be also discussed.

Honzíková N¹, Hrstková H², Hak J³, Balcárková P¹, Závodná E¹, Nováková Z¹ 

Balance between the reflex and tonic autonomic control of the heart in children with bronchial asthma was studied.

Blood pressure was recorded in 15 children (mean age±SD: 12.9±1.5 years, range 11-15 years) with diagnosis of bronchial asthma at the beginning (group A1) and after 6 weeks of balneal therapy (group A2) and in 45 healthy controls (C), age and sex matched (spectral method, Finapres blood pressure monitoring for 5 minutes, metronome controlled breathing 0.33 Hz). Baroreflex sensitivity was determined by spectral method and it was expressed in ms/mmHg (BRS) and in Hz/mmHg (BRSf). Relationships between age and height, mean pulse interval (PI), systolic blood pressure (SBP), BRS and BRSf were tested.

In both groups, height correlated with age (C: r=0.7560, p<0.01, A1, A2: r=0.6324, p<0.05). An age-dependent increase of SBP (r=0.4872, p<0.01) and PI (r=0.4578, p<0.01) and a decrease of BRSf (r=-0.4685, p<0.01) were found in C. None of these parameters correlated with age in A1. In C, age-dependent development of SBP and PI was not accompanied by any significant change of BRS, but BRS correlated with mean PI (r=0.4214, p<0.01). Correlation between BRS and mean PI was not present in A1, but it was normalised in A2 (r=0.6571, p<0.01). In A2, SBP correlated with age as well (r=0.5577, p<0.05).

RECEPTOR LOCALIZATION WITH FLUORESCENT COUPLED SELECTIVE D1 LIGANDS IN RETINA
Huemer K-H¹, Simader C², Barisani T²

Dopamine is known to have different roles within retinal circuitry. Unlike most transmitters its action is not restricted to local synaptic effects; it also has neuromodulatory functions in the outer retina. In this study we used the selective D1 receptor type antagonist SCH23390 linked to the fluorochrome BODIPY FL (Molecular Probes, USA).

Rabbit retinae were isolated and transferred to cooled culture medium. The fluorescent coupled D1 antagonist SCH23390-BODIPY-FL (Molecular Probes) was added to the medium (25 — 100 nM) and retinae incubated for 15 to 120 min. Retinae were examined as whole mounts or cut into 50µm slices. Images were taken with an inverted laser scanning confocal microscope (Argon Laser 488nm, EM 505nm).

We found two different types of labeling. The first kind is restricted to cell surfaces and displays a diffuse fluorescence of most cells of the outer plexiform layer and a specific cell population of the inner nuclear layer. In inner plexiform layer a distinct focal pattern of nerve terminals was labeled. This fluorescence can be inhibited by unlabeled SCH23390. The other kind of labeling, found chiefly in Müller and ganglion cells is not restricted to the surface, but also includes the cytosol excluding only the nuclei. This fluorescence can not be blocked by SCH23390 and only partly by desipramine.

Imrich R¹, Rovensk_ J², Červenáková Ž¹, K_inantová L¹, Kvetňansk_ R¹, Ko_ka J¹ 

The aim of our study was to prove the potential inhibitory effect of hyperprolactinemia on response of HPA. It has been suggested that hyperprolactinemia may contribute to relative HPA insufficiency in rheumatic diseases.

Insuline-induced hypoglycemia (0,1 IU/kg) was performed in 10 female volunteers in fertile age during their follicular phase twice, 60 minutes after either domperidone (10mg orally,COSTI) or placebo administration. Blood samples were collected from an indwelling catheter inserted into cubital vein at -60, 0, 30, 45, 60 and 90 minutes. The concentration of prolactin (PRL), ACTH and cortisol were measured in plasma.

The plasma levels of PRL 1 hour (0 min) after domperidon (dpd) administration were significantly higher than in controls (71±11 ng/ml vs 14 ±6 ng/ml; p<0,01). Insulin-induced hypoglycemia resulted in significant rises in the mean plasma ACTH level from 10±1 pg/ml (dpd) and 11±1 pg/ml (controls) to 148±19 pg/ml (dpd) and 139±12 pg/ml (controls) at 45 min and in plasma cortisol from 407±62nmol/l (dpd) and 391±42 nmol/l (controls) to 925±60 nmol/l (dpd) and 810±52 nmol/l (controls) at 60 min. The significant increase in ACTH and cortisol responses to hypoglycemia did not differ between dpd and controls.

Pharmacologicaly induced hyperprolactinemia did not induced significant reduction of HPA function in healthy young women.

Iost N¹, Virág L², Opincariu M³, Bogáts G³, Szécsi J³, Varró A², Papp JGy^1,2

Properties of the rapid (I_Kr) and the slow (I_Ks) components of the delayed rectifier potassium currents were compared in undiseased human (HM), dog (DM) and guinea pig (GM) ventricular myocytes by using the patch-clamp technique at 37 °C.

Ivanics T¹, van Riel N², op de Buijs J², Miklos Zs¹, Szekeres M¹, Toth A¹, Dezsi L¹, van der Vusse² and Ligeti L¹

Entry and removal of intracellular free calcium ions are responsible for the rhythmic contractile activity of the myocardium. The sum of these processes result in the Ca²⁺_i transient. According to our present knowledge a flux of small amount of calcium ions across the L-type channels of the sarcolemma induces the release of a large amount of calcium ions from the sarcoplasmic reticulum (SR) via the RyR2 type Ca²⁺ release channels. Following the peak of the Ca²⁺_i transient removing processes including the Na⁺/Ca²⁺ (NCX) exchanger and Ca²⁺-ATPase of the SR (SERCA2a) will be turned on. In this study we provide a quantitative analysis of the Ca²⁺_i transient of the beating rat heart. Ca²⁺_i transients were collected from Indo-PE3 AM loaded rat hearts. A simple model was developed to shed light on the cellular structures and processes involved in Ca²⁺_i handling. Flux of Ca²⁺ ions through RyR2 was calculated as the first derivative of the upstroke of the Ca²⁺_i transient. The removal processes of Ca²⁺_i from the cytosol were calculated from a set of differential equations. In these equations the following parameters were used: free Ca²⁺ concentration in the interstitium, the cytosol and SR; fluxes generated by various Ca²⁺ pumps; conductances of the L-type Ca²⁺ channel and RyR2; the buffer capacities of the cytosol and the SR. The removal processes were modeled by a simple Michaelis-Menten kinetics yielding the kinetic parameters, V_max, K_m and Hill-coefficient for SERCA2a and NCX.

A possible brain stem imbalance due to receptor hyperexcitability proposed in panic disorder patients was studied by means of recording the changes in saccadic eye movements (SEMs), saccadic eye movement related potentials (SEMRPs) and optokinetic nystagmus (OKN) as well. The above electrophysiological correlates were compared in groups of 10 healthy undergraduates and 10 panic disorder outpatients.

The accuracy of SEMs was evaluated by means of number of over- and undershootings, the SEMRP were analyzed by a special PC programme (Jagla et al.,1994) as also the angular velocity of OKN slow phase (AV-OKN).

No differences were found in number of over- and under- shootings of SEMs as well as in their directions (centrifugal vs. centripetal). On the other hand, the magnitude of SEMs inaccuracy was markedly increased in patients (p<0.001). The latencies of the premovement components of parietal SEMRPs were higher (p<0.02) and the overall duration of frontal SEMRPs was longer (p<0.01) in panic disorder patients. The AV-OKN in patients was significantly slower with stimuli of higher velocities (60º and 90ºs‾_) as compared to healthy subjects (p<0.01) and its intraindividual fluctuations and interindividual variability were markedly expressed (p<0.001).

The changes in the visual-oculomotor integrations due to brain stem oculomotor circuits imbalance can be proposed.

Jagla, F. et al.,: Physiol.Res., 43, 1994, 229-232

Janovská A, Gregor M, Mejsnar J, _tefl B

&#To achieve an optimal efficiency of physiological processes on the cellular level, metabolic intermediates are directly transferred (channeled) from an enzyme that catalyses its production to another one that uses it as a substrate.

We have studied this phenomenon under different pH using rat psoas muscle myosin ATPase coupled with myofibrillar creatine kinase (CK). ATP produced by CK reaction (transfer of phosphate from creatine phosphate to ADP) is channeled to myosin ATPase, which makes difficulties during enzymatic measurement of CK activity by a classical method (Bergmeyer, Methods of enzymatic analysis, Vol. III, 1983). The analysis of PCr and ATP concentrations before and after CK reaction (Bergmeyer, Methods of enzymatic analysis, Vol. VII and VIII, 1985) gives an alternative method for CK activity measurement. The comparison of both methods gives a quantification of ATP "channeling" between MM CK and myosin ATPase.

Estimation of the size of the substrate channeling effect, using the comparison of both methods, measured in pH 7,5, revealed a discrepancy between the racing portion of PCr by more than 50 % (1). The evaluation of this result within the pH range 6.0 — 7.5 indicates a dependence of the substrate channeling degree on pH. The substrate channeling expressed in percentages of the starting substrate concentration showed a tendency of growing from pH 6 to 7, and than as off pH 7 it declines continually as off pH 7,5, there is a sharp decline. The maximum value of substrate channeling reaches 48.5 % in pH 7.0. Supported by the CR Ministry of Education Grant J13/93: 113100003.

Jansk_ L¹, Vávra V¹, Kunc P², Knížková I², Jansk_ P³, Jandová D⁴, Slováček K⁴

The aim of the project was to monitor dynamics of temperature changes in various areas of the skin after local peripheral cooling and to find out whether or not they can be influenced by repeated cooling of lower extremities. Infrared camera AGA 570 Demo and software Irwin 531were used for this purpose. Naked subjects (man, 21 years, 67 kg, 182 cm) were immersed standing into cold water (12^oC) up to the knees for 1 hour. Thermograms were taken at different times of cooling. Cooling of legs temporarly lowered skin temperature within the first 5 min of the experiment in all areas of the body. Fluctuations in skin temperatures were observed afterwards, indicating cold induced vasodilation. On thighs, vasoconstriction appeared within the first minute already and then extended to arms and to trunk subsequently. In contrast to other parts of the body, skin temperatures on the neck and on the head increased. After immersion of subjects into the cold water repeatedly (20 times during 4 weeks - total time spent in the cold water being 10 h), skin temperatures in all areas of the body were generally lower than in controls and fluctuations of skin temperatures were more visible. Data indicate that the local cooling induces a generalized activation of the sympathetic nervous system. Repeated coolings of legs induce a greater vasoconstrictor tone and, consequently, an insulative type of cold adaptation.

Jansk_ P¹, Jansk_ L², Vávra V², Slováček K³, Jandová D³,

Young male subjects (21 years, 67 kg, 182 cm) were immersed into the cold water (12^oC) up to the knees for 45 min. Changes in skin temperatures, heart rate and blood pressure were continuously monitored using a computerized system. Immersion of legs into the cold water increased heart rate (by 15 %) and brain temperature (by 0,4^oC) permanently, while systolic and diastolic blood pressure increased only temporarily within the first 10 min of the experiment. Skin temperature decreased slightly in all areas of the body. Slight cold induced vasodilation (CIVD) was observed in nonimmersed areas of the body. Data indicate that a generalized vasoconstriction due to permanent activation of the sympathetic nervous system occured during a single local cooling. Adaptation to cold by repeated coolings of legs (20 times during 4 weeks - total time spent in the cold water being 10 h) attenuated heart rate and blood pressure at rest as well as during cold immersion. Initial increases in heart rate and blood pressure did not manifest. Skin temperatures in all areas of the body were lowered and decreased more during local cooling than in controls. Cold induced vasodilation appeared to be more prominent. Results suggest that repeated local peripheral coolings lower the reactivity of the sympathetic nervous system, while increasing sensitivity of target organs to its action.

The aim of study was to ascertain the association between heart rate decrease after acute exercise and HRV parameters obtained before and after exercise.

Heart rate was recorded (using VariaCardioTF4, Sima Media, Olomouc, Czech Republic) in 17 healthy male subjects (mean age: 20 years) during pre-exercise phase (25 min of supine rest, 5 min of standing), during exercise (8 min of step test with ascending frequency corresponding to 70% of individual maximal power output) and in recovery phase (30 min in supine position). HRV analysis in time and frequency domain and evaluation of newly developed complexity measure (sample entropy (SampEn)) were performed on selected segments of heart rate time series.

HRV gradually increased but did not regain rest values during analysed recovery period. The heart rate complexity was slightly reduced after exercise and attained rest values after 30 minutes of supine recovery. The rate of cardiodeceleration did not correlated with pre-exercise HRV parameters, but was positively correlated with HRV measures and SampEn obtained from early phases of recovery.

Inst Norm Pathol Physiol, Slov Acad Sci, Bratislava, Slovakia

Movements can be intended or imagined as well as simulated without being executed. Internally generated movement representations the so-called motor images are internal images under voluntary control and kinesthetic in nature. The possible influences of the mental movement simulation as well as of active movement observation upon the slow cortical movement related potentials (MRPs) were studied in healthy undergraduates.

The experimental procedure was divided into 3 sessions. In the first one (motor task) subjects were asked to perform voluntary self paced finger movements. In the second session (imagery and motor task) they were asked to simulate mentally the same movements and to perform them simultaneously. In the third one (observation and imagery task) they were instructed to watch these movements on video and simulate them mentally, simultaneously. EEG electrodes were placed over the anterior midline, midfrontal, vertex and parietal regions (10-20 system) and also over the pre- and post-central hand representations.

The waveforms of MRPs from mental tasks were, as a whole, similar to those from the motor task alone. Interindividual differences of premovement potential changes were registered. Taking into account the subjective reports describing the fulfillment of tasks it is suggested that the relative changes of cortical activation during preparatory processes do not depend on factors connected with motor functions only. Various psychophysiological processes (individual strategy of movement initiation, intentional involvement, attention, working memory, physiological arousal level and others) can play a role also. Presented results support the view that the mental motor tasks activate and employ nearly the same human brain areas as the voluntary motor performance does.

1Ježová D, ¹Dunčko R, ²Loder I, ¹Makatsori A, ¹Monček F

Stress stimuli occurring in daily life are very variable, but the most frequent and the most threatening ones are those psychosocial in nature. Stress response involves neuroendocrine activation, which can be recorded in several models using somatic stressors. Surprisingly, there are not many mental stress models inducing apparent and reproducible rise in stress hormone release. The aim of the present study was to evaluate three stress situations with different types of mental load in healthy volunteers. Special attention has been given to the changes in cortisol levels in saliva, which represent a suitable, non-invasive approach to the monitoring of the stress response. Combination of short memory test with static exercise was found to be accompanied with a rise in salivary cortisol levels only in male subjects investigated in late afternoon. The following study was therefore performed in the evening and the mental load used consisted of the performance of short intelligence test and the Stroop test in a noisy environment for 15 min. In spite of the enhancement of blood pressure and heart rate, no changes in cortisol release were noticed. In the third series, the Trier’s social stress test, based on a public speech, was performed. The subjects were exposed to a 15 min anticipation period followed by a 15 min speech on a given topic recorded by a camera in front of a jury. This stress model was associated with a consistent rise in salivary cortisol. Similarly, increased heart rate, blood pressure and plasma cortisol levels concentrations were observed in all subjects studied. Other stress hormones were activated as well. Thus, the Trier‘s social stress test is an appropriate model to study the neuroendocrine activation during mental stress in humans. This study was supported by grants of EC ICA1-CT-2000-70008 and VEGA 2/2007.

Introduction: Application of negative expiratory pressure (NEP) at the mouth during tidal expiration provides a simple non-invasive method for detecting expiratory flow limitation (EFL) during spontaneous breathing.

Method: 70 children aged 3.7±1.9 yrs were studied. 31 were admitted for different respiratory diseases (bronchial asthma, cystic fibrosis, recurrent bronchitis), 39 were without any respiratory disease. No differences between the groups regarding age, body height and body weight were found. All pts were in a good clinical condition at the time of study. Children were examined in a semirecumbent position without any sedation. After minimally two consecutive tidal breaths a preset NEP of —5cm_H2O was applied and triggered about 20-40 miliseconds from the onset of expiration. The total duration of NEP application did not exceed duration of tidal expiration. EFL was considered when tidal expiratory flow does not change with applied NEP.

Results: EFL was found in 14/31 (45%) children of RD group and in 6/39 (15%) of the NRD group. It was found a significant difference between the groups ( p = 0,006).

Conclusions: The children with respiratory diseases exhibited EFL more frequently compared to non-respiratory disease group. NEP method represents a reasonable approach for EFL testing in non-cooperating children.

Jurčovičová J¹, Roman O¹, _ere_ J², Zeman M³

Adjuvant arthritis (AA), an experimental model of rheumatic diseases is characterized by the activation of inerleukins which consequently affect the endocrine functions. These, in turn regulate the immune reactions. Prolactin (PRL), growth hormone (GH) exert pro-inflammatory effects, while testosterone (TEST), corticosterone (CORT) and melatonin (MEL) have anti-inflammatory activity. The aim of this study was to reveal the effect of AA on 24-hour secretory profile of these hormones. Intact or arthritic male Long Evans rats on day 23 of the disease were used. AA was induced by a single injection of 50 mL of heat killed Mycobacterium Butyricum (5 mg/ml mineral oil) at the base of the tail. The rats were housed 3 or 4 per cage in animal room with 12 h light/dark cycle (light on 6 a.m. to 6 p.m.) Trunk blood was collected by decapitation in 4-h intervals starting at 6 a. m. The secretory pattern of GH was muted in arthritic rats as was the release of IGF-1. Plasma PRL did not show any difference from intact rats. The rhythm of TEST was impaired compared to normal animals. CORT levels in plasma and adrenal gland in arthritic rats was high in all studied intervals and did not follow the secretory pattern of normal rats. These results show that during AA the normal activity of pituitary regulation is impaired. The more activated immunosuppressive hormone CORT obviously plays a role to protect the organism from immune hyper-reactions.

Kádasi L¹, Plá_ilová M², Feráková E², Poláková H¹, Ferák V²

Primary congenital glaucoma (PCG) is a devastating autosomal recessive eye disease with extremly high incidence in Slovak Roms (1 : 1250). Recently, we linked the disease in this poulation to the GLC3A locus on chromosome 2p21. At this locus mutations in the cytochrome P4501B1 (CYP1B1) gene have been identified as a molecular basis for this condition. Sequencing of CYP1B1 gene in affected Rom patients revealed a homozygous G > A transition in nucleotide 1505 in the highly conserved region of exon 3. This mutation results in the E387K substitution, affecting the conserved K helix region of the cytochrome P450 molecule. Determination of the CYP1B1 polymorphic background showed a common DNA haplotype in all patients, thus indicating that the E387K mutation in Roms has originated from a single ancestral mutational event. Population structure and history of Slovak Roms are in good agreement with this observation.

An ARMS-PCR assay has been developed for fast detection of this mutation, allowing direct DNA based prenatal diagnosis as well as mutation-carrier detection in this population. Screening of 158 healthy Roms identified as high as 11 % carriers, explaining the high incidence, and at the same time idicating that the frequency of PCG in this population my be even higher than originally estimated.

Kadlecová M, Dobe_ová Z, Zicha J, Kune_ J

In conclusion, our results suggested that Igf 2 gene could be a candidate gene for metabolic abnormalities in hypertension. Supported by grants no. A7011711 and A7011805 (Grant Agency of AS CR).

L-type calcium channel Ca_v1.2 undergoes Ca²⁺-dependent inactivation with calmodulin (CaM) as its mediator. The critical molecular determinant for Ca²⁺-dependent inactivation is the carboxyl-terminal tail of Ca_v1.2 subunit containing two CaM binding domains, a CaM binding region (CBR) close to an IQ like CaM binding motif. During resting state of the cell, apoCaM is tethered to the CBR. Following channel opening and Ca²⁺ influx CaM binds also to the IQ motif. It has been suggested that the N- and C-terminal lobe of CaM bind to the CBR and IQ motif, respectively. Therefore, we tested whether the N- or C-terminal lobe have the capability to function as a dominant negative mutant.

The possible contribution of the Ca²⁺-activated K⁺ channel to the resting membrane potential has been investigated by means of the potential sensitive fluorescent probe 3, 3´-dipropylthiodicarbocyanin iodide (diS-C₃-(5)) and the incorporation of Ca²⁺-specific permeant chelator (BAPTA/AM) in several mammalian cells. In human erythrocytes, the incorporation of BAPTA caused the shift of the membrane potential from -13 to -16.8 mV. A shift to more negative values were observed in human leucocytes (from -34 to -36.5 mV) and human leucocytes in the presence of 0.1 mmol/l Ca²⁺ (from -31 to -38 mV). On the other hand, in other cells the opposite shift was observed (from -18 to -15 mV in L1210 cells and from -43 to -36 mV in L1210 vincristin resistant cells). These results suggest that the activity of Ca²⁺-activated K⁺ channel may not generally contribute to establishing the resting membrane potential. In some cells (L1210) other mechanisms may be involved.

Kassayová K, Bračoková I, _vorc P

This study deals with the investigation of the influence of the examination stress on the heart activity parameters of presumably healthy students of the Faculty of Medicine. The studied group consisted of 22 subjects (18 male, 4 female) in the age of 19—21 years. The method of study was 24-hour Holter monitoring. The acquired data were analyzed using the Premier IV Holter Monitoring (Diagnostic Monitoring, Inc., USA). The monitors were applied at 12:00 one day before the examination on physiology and removed at 12:00 after the morning examination. The results were compared with the measurements obtained on the same subjects using the same equipment during normal semestral working days under conditions of usual psychical and physical load.

The set of measured parameters included average heart rate (HR), minimal and maximal HR, average RR interval, heart rate variability, occurence and circadian distribution of ectopic activity, and other changes of the heart rhythm.

The elevation of the average HR before and during the examination (from 74.0 ± 7.6 min^-1 to 82.4 ± 8.9 min^-1; p < 0.001) has been observed. The indices of the HR variability (SDNN, SDNN-index, rMSSD, pNN50) during the examination were significantly lower than during normal activity.

Sporadic ectopic disorders in the form of supraventricular and ventricular premature beats were observed in 50 % of students during the normal activity (up to 15 per person during 24 hours) and in 77.3 % of students during the examination (up to 46 per person during 24 hours). In 18.2 % of male students pauses longer than 2.0 s (up to 2.6 s) were observed during sleep.

Other complex disorders of the heart activity were not registered.

Komínková V¹, Ravingerová T², Máleková L¹, Ondria_ K¹

The ATP-sensitive mitochondrial potassium (mito-K_ATP) channels have been shown to be an important component of the endogenous cardioprotection, known as ischemic preconditioning. Activation of the mitochondrial K_ATP channels was found to increase the influx of potassium into mitochondria, which results in both matrix expansion and mitochondrial depolarization. It was associated with increased survival of cardiac cells following ischemia and improved post-ischemic recovery of heart muscle. Calcium preconditioning inhibits mitochondrial permeability transition pore (PTP) and apoptosis. The precise mechanism responsible for such an outcome remains only partially understood.

Therefore we studied single channel properties of the potassium channels, particularly mito-K_ATP and PTP channels. In our study we isolated submitochondrial particles (SMP) from the inner mitochondrial membranes of the rat heart myocytes. These SMP were incorporated into bilayer lipid membrane (BLM). We observed several potassium channels derived from the SMP. The amplitude of the single channel current varied from 1.1 to 1.6 pA at asymmetrical 250 mM KCl in cis chamber and 50 mM KCL in trans chamber, at 0 mV. Conductance of the channels varied from 42-65 pS. Activity of the channels was inhibited by 1 mM ATP, but in 40% of the experiments the channels were not affected. We also observed channels with high conductance (300-600 pS), supposing to be PTP channels.

Koprovičová J¹, Petrá_ová D¹, Kuchta M^1,2

Background: The major goal of this pilot trial was to evaluate the actuall status in the serum concetration of apo C-II, apo C-III, vitamin C and to determine the degree of lipid disorders in adult patients with combined familiar hyperlipidemia (CFHL).

Methods: To the study were admited 42 patients of medium age (45 years old) and 20 probands as control group of the some age. Besides apo C-II, apo C-III and vitamin C, the serum concentration of apo B and some lipids was estimated. Apo C-II and apo C-III were quantified by the RID and apo B by the EIA. Vitamin C were determined by the colorimetric method.

Results: The mean value of apo C-II in patients with CFHL was significantly arising on 150% against the value in control group (p<0.01). The serum concentration of apo C-III was exponentially increased on 190% in 95 percentile, concomitantly with arising of the serum concentration of TG (p<0.001). Naturally, significantly increased serum concentration of TCH and LDL-CH were also. The deficiency of vitamin C was found significant, because it drop by 11% in comparison with control group.

Conclusions: From our results we may indirectly suggest that in patients with CFHL TG-rich lipoproteins are accumulated in circulation for delayed it clearense. This phenomenon has certainly mising causes, which see explained in the text of the lecture. We may suggest that significantly high serum concentration of apo C-III may be the important clinical biomarker for CFHL.

Since the discovery of pancrein by Paulesco and insulin by Banting with Best in 1921,insulin is administered almost exclusively subcutaneously (SC) or intravenously (IV) in diabetic patients and all trials of administering insulin via gastrointestinal tract were without effect. With aim to alleviate the difficulties connected with SC administration of insulin in diabetic patients, author administered soluble, rapid acting Bovine insulin SIGMA in Thiopental anesthetized normal and 46 STZ-diabetic rats of both sexes aa in dose 10U/kg with enhancers of absorption POESET or NOET sublingually (SL) and in all rats, glycemia ranging from 4.2 mmol/l up to 30.1 mmol fell at 30 min. by 10 up to 31% and at 60 min. up 30%, in the same rats. Stimulated by these results, author on himself, non-diabetic, observed a decrease of fasting glycemia 4.5-5.5 mmol to 3.5-4.5 mmol at 30 and 60 minutes after 25 u SL., and a decrease in postprandial glycemia from 7.2 to 5.8 or from 8.8 to 7 mmol after 30 and 60 min. and 50 u of rapid acting human insulin with POESET or NOET SL. In 10 diabetic patients with hyperglycemia ranging from 8.1 up to 22.9 mmol/l the decrease in glycemia after 20 u SL. after one hour was less, with mean 1.4 mmol.

Conclusion: In normal and diabetic rats absorption of rapid acting insulin administered SL with innocuous enhancers of absorption is rapid and nearly equals to the same dose of SC. administered insulin, in normal and diabetic men the effect is less, probably caused by histologically documented greater thickness of human sublingual mucosa.

Ko_ka J¹, Ježová D¹, Pacák K², Kvetňansk_ R¹, Viga_ M¹

Changes in ambient temperature induce thermoregulatory response to maintain body temperature within physiological interval. Both heat and cold exposure can act as stressors. Stay in a cold environment is associated with increase sympathetic activity while heat exposure results in the release of a variety of stress hormones. Moreover, changes in ambient temperature influence body responses to other stressors. In healthy subjects, we have shown that external hyperthermia augments neuroendocrine responses to swimming and insulin-induced hypoglycemia. In contrary, hypothermia reduced adrenaline and totally prevented prolactin response to the latter stimulus.

The role of peripheral vs. central thermoreceptors in triggering hormonal release was studied using head-out and forearm warm-water immersion. Even local heat application induced elevations of catecholamine and growth hormone concentrations. Systemic hyperthermia induced an augmentation of hormone responses to local heat and an additional release of prolactin. Metabolic consequences of neuroendocrine activation during cold exposure in abdominal subcutaneous fat were studied using microdialysis technique. No changes of glucose levels were observed in plasma and subcutaneous fat. Twice as high response of plasma noradrenaline in women compared to men together with similar lipolysis rate indicate increased sensitivity of lipolysis to sympathetic activation. Thus, neuroendocrine activation induced by changes in ambient temperature seems to have metabolic consequences of apparent clinical relevance.

Kostyuk E, Kruglikov I, Shishkin V, Voitenko N

Ca²⁺ transients evoked by membrane depolarisation were measured in primary and secondary nociceptive neurones of rats with streptozotocin-induced diabetes (serum glucose level 16-25 mM), showing neuropathic syndroms. Definite prolongation of the decay phase of the transients was observed in diabetic conditions in primary (DRG) and secondary (spinal dorsal horn) neurones. To analyse the mechanisms of these changes, alterations in the Ca²⁺-accumulating function of endoplasmic reticulum and mitochondria were measured. In secondary neurones Ca²⁺ uptake by the endoplasmic reticulum was found to be substantially decreased: application of caffeine elevated cytosolic Ca²⁺ by only 41±9 nM compared with 269±nM in control. In primary nociceptive neurones endoplasmic reticulum did not play substantial role in the formation of Ca²⁺ transients; however switching-off mitochondrial Ca²⁺ accumulation by protonophore CCCP abolished the described prolongation. Thus alterations of Ca²⁺ uptake and release by both endoplasmic reticulum and mitochondria may be the reason for changes in neuronal calcium signalling during diabetic complications, leading to neuropathic potentiation of synaptic transmission of afferent volleys from primary to secondary nociceptive neurones.

Kostyuk PG, Shishkin VA, Potapenko ES, Kostyuk EP, Girnyk OV, Voitenko NV

Calcium ions are the most universal intracellular messenger. The steep transmembrane gradient of these ions inject them at high speed into the cell during physiological activity and create "calcium signals" — transient elevations of their cytosolic level. By binding to different protein structures, the injected ions trigger or modulate practically all function s of the excitable cells; therefore their amplitude and kinetic characteristics are of critical importance. The latter are determined by complex interaction of several molecular mechanisms following the opening of plasmalemmal channels: uptake and subsequent release by intracellular compartments and extrusion from the cell by active transport and ion-exchange systems. The relative contribution of these mechanisms is extremely variable in different types of excitable cells, leading to corresponding changes in cell functioning. In the present work we compared their contribution to the formation of calcium signals in primary and secondary of rats, which form the main synaptic switch for the processing of nociceptive information to higher brain structures, using electron microscopy and fluorescent Ca²⁺-indicators. Substantial differences have been detected in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurons. The functional implications of such differences for the transmission of afferent volleys will be discussed.

The hypothalamic paraventricular nucleus (PVN) contains visceromotor cell types that are in a position to coordinate neurosecretory and autonomic responses to stress. Magnocellular neurosecretory neurons produce arginine vasopressin (AVP) that contributes to the defense of arterial pressure and volume. Parvocellular neurosecretory neurons synthesize corticotropin-releasing hormone (CRH) and AVP to govern pituitary adrenocorticotropin secretion. We have described differential activation and feedback sensitivity of these genes to physical stressors. Ether stress results in a rapid increase of CRH transcription, followed by delayed activation of AVP expression. Evidences are emerging on the role of immune challenges such as allergic insults to drive the stress-related central circuits. Using intron specific probes we followed the expression of CRH and AVP genes in the PVN in response to systemic allergic reaction, anaphylaxia. In contrast to the situation seen after ether stress, allergic insults resulted in a rapid and parallel induction of CRH and AVP genes in the parvocellular compartment and a sustained increase of magnocellular AVP expression, suggesting stressor-specific regulation of neuropeptide gene expression. Activation of these genes to allergic reactions might have long term impact on the neuroendocrine regulation when allergen exposure became repeated or chronic.

Kretschmannová K, Zemková H

Suprachiasmatic nucleus (SCN) has been identified as a mammalian biological clock, which regulates circadian and seasonal rhythms in many behavioural and physiological functions. Based on structural and functional differences, two parts of the SCN can be distinguished — dorsomedial (DM) and ventrolateral (VL) part. DM part appears to function as intristic clock per se. VL part receives direct input from the retina through glutamatergic terminals of retinohypothalamic tract (RHT) and it synchronizes intristic clock to 24 h light—dark cycle. Fast synaptic transmission between neurons of the SCN is mediated by GABA, nevertheless, the type and functional properties of GABA_A receptor are not yet well understood.

The present study was undertaken to investigate possible day-night difference in GABA_A receptor function in two SCN subdivisions, and to characterize its sensitivity to zinc. It is known that Zn²⁺ is co-released together with glutamate in VL part of the SCN and that it affects GABA_A receptors in dependence on their subunit composition. Whole-cell patch clamp technique and rapid application perfusion system was used to test the effect of Zn²⁺ on currents induced by GABA in VL and DM parts of the SCN in slices.

Application of Zn²⁺ in concentrations 1—1000 mM had competitive inhibitory effect on GABA-evoked (30 mM) currents in all SCN neurons measured. Sensitivity to Zn²⁺ was similar in both VL and DM parts. During the day, the inhibitory effect of Zn²⁺ was higher (IC₅₀ = 43.9 ± 4.7 mM) in comparison with the inhibitory effect of Zn²⁺ during the night (IC₅₀ = 58.6 ± 3.8 mM). It seems that Zn²⁺ can modulate synaptic activity in neurons of the SCN in dependence on circadian time.

Kristek F¹, Fáberová V², Varga I³

Experiments were designed whether long-term administration of exogenous donors of nitric oxide (NO) have beneficial effect on both general haemodynamic parameters and geometry of conduit arteries of spontaneously hypertensive rats (SHRs). We used: (1) Control Wistar rats, (2) SHRs, (3) SHRs treated by molsidomine (100 mg/kg in tap water, by gavage), (4) SHRs treated by pentaerythrityl tetranitrate (PETN) (200 mg/kg in tap water, by gavage). Blood pressure (BP) was measured by the tail plethysmographic method. After six weeks we evaluated content of cyclic guanosine monophosphate (cGMP) in platelets in all three experimental groups. Then the animals were sacrificed, perfused by glutaraldehyde fixative (120 mmHg), and arteries were processed for electron microscopy.

Increase of BP in experimental groups was found (p<0.01). No differences in BP were observed among the experimental groups. Increase of cGMP content was observed in molsidomine and PETN groups (p<0.01 to SHRs). The values of wall thickness and cross sectional area of thoracic aorta, carotid artery and coronary artery in experimental groups were increased (p<0.01) in comparison to control group. NO differences were observed among the experimental groups.

Long-term administration of exogenous NO donors did not evoke beneficial effect on BP and geometry of conduit arteries of SHRs. We suggest that pathogenetic background of SHRs is probably not intimately associated with NO deficiency.

Kroupová V, Trávníček J

Optimal nutrition of farm animals exposed to the high production stress and the extreme life conditions is based not only on calculation of the nutrients balance in comparison with the nutritional norms but also on evaluation of real nutrients status and their metabolites in organism.

The goal of this study was to elaborate virtual models of deciding tables for disclosure of surplus or primary and secondary deficiency of energy, digestible protein, and minerals. By the experimental verification of quantitative relationship between intake of nutrients in diet and status of selected parameters in blood, urine, and feces in cattle we came to conclusion that among valuable indicators belongs in blood plasma: urea, selenium, zinc, cooper, manganese, and beta-carotenes, in urine: consistency, calcium, phosphorus, magnesium, natrium, potassium, and iodine, in feces: all above mentioned minerals. The built up model represent an initial database for making of software that enable evaluation of nutritional failures.

Kubalová Z, Pavelková J, Zahradník I, Zahradníková A

Calcium tail current after a short prepulse to the reversal potential of the calcium current (I_Ca) is the preferable stimulus to study release-dependent inactivation (RDI) of I_Ca, as it induces RDI with minimal contribution of voltage- and current-dependent inactivation. However, the short prepulse duration requires considering the finite membrane charging time.

Quantitative characterization of I_Ca inactivation in cardiac myocytes is complicated by the presence of three inactivation mechanisms that are difficult to separate. Part of the problem is in the complex character of the inactivation stimuli, usually consisting of the voltage prepulse itself, the calcium current during the prepulse, and the tail calcium current following the prepulse. We show in isolated rat ventricular myocytes that the tail calcium currents evoked by very brief prepulses induce negligible voltage- and Ca current-dependent inactivation of I_Ca, leaving calcium release-dependent inactivation (RDI) as the dominant inactivation mechanism. In these experiments the degree of peak I_Ca inactivation increased in a dose-dependent manner (n_H ~ 2) with the Ca charge injected by the tail currents and reached maximal values of ~75%. Slower tail currents at less hyperpolarized interpulse potentials were more effective in inducing RDI of I_Ca than faster tail currents at more hyperpolarized interpulse potentials, despite having a smaller single-channel amplitude. Kinetics of RDI during the test pulse I_Ca could be reproduced by simulation of excitation-contraction coupling units (ECCUs) containing multiple DHPRs. In the model, the changes in RDI kinetics were due to a decreased rate of release activation arising from an increased proportion of inactivated DHPR channels. At the level of the E-C coupling unit, these findings suggest that efficiency of cardiac E-C coupling is more sensitive to changes in the duration of Ca influx than to changes in the amplitude of the single Ca channel current activating release, and that local Ca²⁺ signaling within ECCUs is impaired by interventions reducing the density of active DHPRs.

Kubovcakova L, Micutkova L, Krizanova O, Kvetnansky R

Catecholamines are crucial mediators of the stress response. In our facility we are breeding three types of genetically altered mice: c-fos knockout mice, unable to synthesize fos-protein, a part of A1 complex, DBH knockout mice lacking dopamine-ß-hydroxylase, the enzyme that converts dopamine to norepinephrine and CRH knockout mice unable to synthesize corticotropin-releasing hormone. Such mice can serve as an interesting model for studying mechanisms involved in the response of a hypotalamic-pituitary-adrenal axis to stress.

Our data revealed significant differences not only between species, but also between +/+ and -/- homozygots. These results suggest that the amount of TH/PNMT immunoprotein in adrenal medulla of Sprague-Dawley rats, CRH knockout mice and their controls are really different.

Kukačka J¹, Bíbová J², Ruskoaho H³, Pelouch V¹

The quantity and quality of extracelullar matrix (ECM) proteins in myocardium is determined by the balance between their synthesis and degradation - mediated by matrix metalloproteinases (MMPs) and regulated by endogenous tissue inhibitors (TIMPs). Exposure of rats to chronic hypoxia (H) results in ventricular hypertrophy characterized by remodelation of ECM.

The objective of this study was to examine the effects H on gene expression of collagen types: I, III and XV, MMP-2, TIMP-1 and natriuretic peptides (ANP, BNP) in both right (RV) and left (LV) ventricles of myocardium. Wistar male rats were exposed 2 weeks to hypoxic condition (10 % O₂) in isobaric chamber.

From Northern blot analysis followed that H increased in hypertrophied RV the expression of mRNA for collagens a₁ (I), a₁ (III) and a₁ (XV). Furthermore, higher levels of gene transcripts indicated co-expression of MMP-2 and TIMP-1 again only in RV. The markers of cardiac injury, mRNA levels of ANP and BNP, were significantly elevated by H in RV as well. On the other hand, no such changes were in hypertrophied LV of H rats, however, mRNA for collagens III and XV were significantly lower (as compared with controls). The present study showed that gene expression of ECM in both RV and LV of H is affected by different mechanisms; that probably explained diverse functional and structural differences of both parts of ventricle myocardium.

Kuncová J, Slavíková J

Dept of Physiology, Faculty of Medicine, Charles University, Plzeň, Czech Republic

The present study was aimed to investigate the effect of two neuropeptides, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), on the spontaneous and K⁺-stimulated release of noradrenaline (NA) from isolated rat heart atria. VIP is known to be localised in the intrinsic cardiac neurones particularly in the atria and coronary vasculature (1), CGRP serves as a neurotransmitter of cardiac sensory neurones (2).

Heart atria of female adult Wistar rats were sliced and superfused with oxygenated Krebs-Henseleit solution containing neuronal uptake blocker desipramine in the presence or absence of VIP in concentrations 10^-8 and 10^-7 mol/l, CGRP (10^-8 and 10^-7 mol/l), and specific VIP antagonist [D-p-Cl-Phe⁶,Leu¹⁷]-VIP (10^-6  mol/l ).

Neuropeptides did not influence spontaneous release of NA. However, in K⁺-stimulated preparations NA output was significantly increased by VIP. In addition, VIP antagonist by itself slightly inhibited K⁺-stimulated NA release, which probably reflects the blockage of the action of endogenous VIP, released during the stimulation.

In conclusion, both direct and indirect effects of VIP might be involved in the regulation of cardiac chronotropy and inotropy. These findings provide the first evidence that the indirect effect of VIP in the rat heart atria during stimulation is mediated most probably via presynaptic specific receptors.

(1) Weihe E et al: Cell Tissue Res 236: 527-540, 1984.

(2) Mulderry PK et al: Neuroscience 14: 947-954, 1985.

Kuri_čák E, Trojan S, Wünsch Z

Time precision and reliability with which neurons generate action potentials (AP) are limited mainly by the probabilistic nature of synaptic transmission, electrical properties of neuronal membrane and by neuronal noise.

Taking advantage of computer modelling, we built in the software environment GENESIS 2.1 a multicompartmental model of a rat hippocampal neuron. The model consisted of soma, axonal initial segment (IS) and of a large dendritic tree (DT) on which synapses of various cluster size (determined by number of multiple synapses belonging to one axon) and with variable probability of vesicle release (P) made evenly spread contacts. In the model was implemented membrane noise (channel and thermal noise) which corrupted propagation of PSPs along dendritic processes and AP initiation at the IS. During simulations, random patterns of spatio-temporal synaptic activity were repeatedly presented to the neuron, producing spike pattern being further statistically processed using MATLAB 5.3, assessing thus the influence of the P, cluster size and membrane noise level on time precision of AP generation.

According to our results, the time precision was significantly improved if the size of synaptic cluster and the value of P increased. Membrane noise had on the precision smaller effect, when considering its influence on soma and dendritic tree, but at the IS the reliability of AP generation was influenced mainly by the membrane noise.

Stress can be extremely harmful when excessive and it can lead to many ailments. In fact, it has been proposed that nearly two-thirds of the ailments seen by physicians are either stress-induced or stress-related. Stress activates neuroendocrine, nervous, immune, and other systems. The specific activation of two components of the sympathoadrenal system (adrenomedullary and sympathoneural) by various stressors is nowadays intensively studied. The aim of the present work was to investigate changes in catecholamine (CA) synthesizing enzymes - tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) - gene expression, immunoprotein levels and activities in the adrenal medulla (AM) of rats after a single or repeated exposure to various stressors. Immobilization for 2h (IMO), cold 4°C (COLD), administration of insulin 5IU (INS) or 2-deoxyglucose 500 mg/kg (2DG) were used. A single exposure to IMO, COLD, INS or 2DG was found to induce increases in both TH and PNMT mRNA levels in AM. Increased transcription rate is responsible for TH and PNMT gene expression. Repeated exposure to these stressors elevated besides mRNA also activity and immunoprotein levels of the enzymes. Various stressors regulate TH gene expression by different mechanisms.

One day cold exposure elevated TH mRNA levels in AM, however, 28 day cold exposure did not show any changes. Cold-adapted rats, however, responded to heterotypic novel stressors (IMO, INS, or 2DG) by exaggerated responses.

Thus, our data suggest an adaptation of TH gene expression during long-term exposure to stressors. An exposure of adapted rats to novel stressors induces exaggerated responses which is an important adaptive phenomenon. Supported by EU Centre of Excellence Grant ICA1-CT-2000-70008 and VEGA 2-6109.

Lacinová Ľ¹, Klugbauer N², Hofmann F²

Voltage dependence of on- and off-charge movement could be fitted by a single Boltzmann function. For on-charge, a half-maximum was reached at +12.9±1.4 (n=25) mV with a slope of 22.4±0.4 mV. Off-charge reached half-maximum at +12.3±0.7 mV with a slope of 18.1±0.4 mV. The difference in steepness was significant (p<0.001, paired t-test).

Lajdova I¹, Chorvat D, Jr.², Spustova V¹, Chorvatova A^1,2

Purinergic receptors are cationic channels modulated by external ATP, implicated in the activation of T lymphocytes. Their regulation by 4-aminopyridin (4-AP), known to induce calcium influx and apoptosis, was examined.

4-AP induced a dose-dependent rise in [Ca²⁺]_i, determined by Fluo 3 fluorescent probe, and stimulated Ca²⁺ influx in cells where internal stores were emptied using thapsigargin. These results suggest that 4-AP is responsible for enhancement of Ca²⁺ entry from extracellular space rather than release of calcium from its internal stores.

Brilliant blue (50 μM), a specific inhibitor of pore-forming purinergic receptors (P2X7), blocked 4-AP-induced intracellular calcium rise. Application of ATP evoked comparable responses as 4-AP, as ATP also stimulated Ca²⁺ entry independently on the calcium release from its internal stores. The effect of 4-AP and ATP was non-additive when studied on the same population of cells, suggesting implication of the same type of channel. Furthermore, 4-AP allowed entry of ethidium bromide (314 Da), but not propidium iodide (414 Da) into the cell, pointing to the involvement P2X7 pore.

Presented results demonstrate for the first time in human lymphocytes from healthy volunteers that P2X7 channel pore is implicated in the calcium regulation and that 4-AP is able to activate the receptors and induce sustained calcium entry.

Langmeier M¹, Bene_ová P^1,2, Betka J², Trojan S¹

Using NADPH-diaphorase staining we studied effects of i.p. administration of kainic acid (KA) on individual areas of the hippocampus and on the auditory cortex. One day prior to the KA application, rats were exposed to chronic hypoxia. Rats were exposed to long-lasting repeated hypoxia in a hypobaric chamber at a simulated altitude of 7000m for 8 hours a day from their 2^nd postnatal day till the age of 17 days. At the age of 18 days, animals received a single i.p. injection of KA (2,5 mg/kg). Four days later, animals were sacrificed by the transaortal perfusion of 4% buffered paraformaldehyde. Cryostat sections were stained to prove NADPH-d positive neurons, which were then quantified in individual parts of the hippocampus (CA1, CA3, hilus, dorsal and ventral blades of the dentate gyrus) and in the auditory cortex.

Our results brought the evidence that hypoxia as well as KA given to the normoxic animals increased the density of NADPH-d positive neurons in the hilus, CA1, CA3 areas and in the auditory cortex, compared to the control group. Contrary to it, KA given to the hypoxic animals resulted in the smaller density of these neurons in the hilus, dorsal and ventral blades of the dentate gyrus.

This study suggests that hypoxia stimulates the nitric oxide (NO) production because of its influence on nitric oxide synthase (NOS) gene expression. KA then possibly causes NO synthesis enhancement due to its binding on KA channels (subpopulation of non-NMDA receptors). Our results show that hypoxia also lowers the density of KA receptors that may indirectly contribute to the low NO production after the KA administration.

Lantos J, Temes G, Rőth E

Granulocyte activation and adhesion molecule expression is a causal factor in microcirculatory disorders during myocardial reperfusion. While the dynamics of these processes have been well defined during acute reperfusion, there is very little data regarding long lasting reperfusion. The aim of this study was to investigate granulocyte CD11a expression during myocardial ischemia followed by 4 weeks of reperfusion. The left descending coronary artery (LAD) was occluded in dogs for 1 hour (Group I. n=8) and 3 hours (Group II. n=9) that was followed by 4 weeks of reperfusion. Sham operated animals served as control (Group III. n=5). Leukocyte expression of CD11a was measured by flow cytometry in peripheral blood samples. CD11a expression started to increase at the second day of reperfusion with the maximal elevation by the third postoperative day 141,2±11,6% in Group I. and 170,6±30,3% in Group II. (p<0.05). The similar elevation seen in sham operated animals normalised within the first postoperative week, however, CD11a expression was elevated in both groups of ischemic animals even after 4 weeks reperfusion. The increased CD11a expression during first postoperative week may reflect the effect of surgical trauma, but the increased granulocyte function during later reperfusion may indicate the activated state of cells and the prolonged healing of injured myocardium.

Lewiński A, Karbownik M

A mutual relationship between the pineal gland and the thyroid has - for a long time — been the subject of intensive experimental investigations. The abundant to-date’s evidence relates mostly to the inhibitory action of melatonin (N-acetyl-5-methoxytryptamine) - the main secretory product of the pineal gland - on the thyroid growth and function and — to a lesser extent — to the stimulatory effects of thyroid hormones on the pineal gland. There are clear experimental data that both the pineal and the thyroid are involved in oxidative processes under physiological or pathological conditions. Therefore, the role of free radicals in the mutual relationship between these two glands, in particular between their secretory products - although not yet clarified - should be taken into account. The basic premise for this consideration is the fact that melatonin is a well known antioxidant and free radical scavenger, widely distributed within the organism. Melatonin protects against enhanced oxidative stress in tissues and organs, and this indoleamine is supposed to participate in the regulation of oxidative stress at physiological level. As regards the thyroid, it is known that reactive oxygen species and free radicals participate in physiological processes in the gland, e.g., in thyroid hormone synthesis. Moreover, several thyroid disorders are accompanied by oxidative stress. Highly probable is an assumption that, under pathological conditions, an enhanced melatonin access to the thyroid tissue occurs and - in consequence - melatonin protective role in the gland may be observed. In conclusion, the possible mechanisms of the regulatory and protective actions of melatonin in the thyroid gland appear to be related to oxidative stress processes.

Loew HG¹, Schmid D², Plass CA² , Spieckermann PG² 

1Dept. of experimental physics, Dept. of radiotherapy Univ.Vienna
²Inst. of Physiology, Univ. Vienna

Lukyanetz EA^1,2, Lukyanets IA¹, Koval LM¹, Tokar SL², Yavorskaya EN¹

Steroid hormones are well known agents participating in numerous metabolic regulations in living organisms. Dysfunction in adrenocortical system leads to numerous human pathologies. One of the factors that can influence on secretory properties of adrenocortical cells is changes of cell volume observing in many pathological processes. In this study, we tested the hypothesis that osmotic stress (cell volume changes) initiates changes in intracellular Ca²⁺ signaling and subsequent changes in secretion of steroids in adrenocorticocytes. Using electron microscopic and morphometric methods as well as microfluorometric measurements with Fura-2, cell ultrastructure, Ca_i and cell volume were monitored in adrenocortical cells exposed to hyper and hipo-osmotic solutions. Further experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as increase of Ca_i transients. Calcium-free media eliminated Ca_i increases. Pretreatment with thapsigargin, or CCCP (blockers of the internal calcium stores) significantly decreased the percentage of cells responding to osmotic stress. The peak cell volume change as well as the percent recovery of cell volume, was dependent on Ca_i. Our electron microscopic experiments have shown that increase in intracellular Cai induced the changes in ultrastructure of adrenocorticocytes which were characteristical for stimulation of steroidogenesis. Especially were remarkable activation of lipid droplets and mitochondria. These findings indicate that significant volume changes causes increase in Ca_i in adrenocortical cells and may such way stimulate steroidogenesis.

Macejová D¹, Lí_ka J², Dvorčáková M¹, Brtko J¹

Retinoids, as potentional anti-cancer drugs, can through their RARs start redifferentiation or are capable to prevent further dedifferentiation of neoplastic tissues. RARs have been shown to play a role in both the regulation of cancer development and the anticancer activities of retinoids. Experimentally MNU-induced carcinogenesis of mammary gland in rat is widely used model of breast cancer in human. In rat, MNU-induced tumours were found to have some common features with that of in human. In the model used in our laboratory, we have determined the status of RARs in several histologically defined types of mammary gland tumours in female Sprague-Dawley rats. Scatchard’s analyses have shown marked differences in both Bmax and Ka of retinoic acid binding to their cognate receptors in different types of tumours. Concentration of RARs was the highest in papillary ductal tumours with cribriform patterns (PDC-Cr) (Bmax 1,5 pmol.mg^—1), lower in invasive ductal tumours with cribriform and comedo patterns (IDC-CrCo) (Bmax 0,34 pmol.mg^—1) and the lowest in invasive ductal tumours with comedo patterns (IDC-Co) (Bmax 0,11 pmol.mg^—1). On the other hand, the afinity of RA-RAR complex was the lowest in PDC-Cr (Ka 0,0834.10⁹ dm³.mol^-1), higher in IDC-Co (Ka 0,513.10⁹ dm³.mol^-1) and the highest in IDC-CrCo (Ka 0,6175.10⁹ dm³.mol^-1). Electrophoretic mobility shift assays with the retinoic acid responsive element (DR2) have confirmed increased expression of RARs which occurs in different types of tumours when compared to control mammary gland. In conclusion we can summarize that status of RARs differs in different types of MNU-induced mammary gland tumours in rats.

Macho L¹, Červenáková Ž¹, Zórad _¹, Patterson-Buckendahl P², Kvetňansk_ R¹ 

In rats, alcohol intake affects consumption of food, weight gain, and response of insulin to glucose administration. In this experiment the effect of chronic ethanol consumption on plasma levels of insulin (I) and glucose (G), and on insulin receptors in tissues was studied. Adult male Sprague-Dawley rats were fed:

a/ control diet ad libitum(CLD), b/ ethanol liquid diet (5% of ethanol by weight, fed ad libitum, ELD) c/ pair-fed rats (PF) offered an amount of CLD equal to the amount consumed by ethanol group. The animals were on these diets for 9-12 days. At the end of experiment animals were sacrificed by decapitation, blood and adipose, liver and muscle (m. quadriceps) tissues were immediately frozen. Plasma I (Linco-RIA kit, USA) and G (Super GL kit, Germany) were determined. Plasma membranes were isolated from adipose, liver and muscle tissues and binding of I¹²⁵ labeled insulin was determined. Lower weight gain and increase of plasma G levels were observed in animals fed ELD. Plasma I concentrations were not affected by ethanol intake. The binding of I to plasma membrane of adipocytes was elevated after ELD. The binding capacity of insulin receptors in liver membranes was slightly elevated in PF rats, however, was not affected by ethanol intake. Specific binding of I in muscle was not changed in rats fed ELD. In conclusion, the ethanol intake increased plasma G levels and insulin binding capacity in adipocytes. The binding of insulin in muscle and liver was not affected by ELD consumption.

Máleková Ľ, Ondria_ K

The aim of our work was to characterize potassium and chloride single channels from sea urchin eggs microsomes. We incorporated the microsomes into bilayer lipid membrane using solutions (in mmol/l): 250 Hepes, 53 Ca(OH)₂, 1 KCl, pH 7.4 (trans side), and 550 KCl, 250 Hepes, 113 Tris, 1 EGTA, 0.7 CaCl₂, pH 7.4 (cis side).

We observed several potassium and chloride channels. Small potassium channel had conductance 22 pS, at the range from —60 mV to +60 mV. Single channel current at 0 mV was 1.7 pA. Open probability (P-open) was voltage dependent. It was high at —60 mV (P-open was 0.7-0.8), and with increasing voltage, P-open gradually decreased, and it was <0.05 at +50 mV. Open dwell time of the channels at 0 mV was 27 ms.

The potassium channel with conductance 113 pS, at the range from —40 mV to +30 mV was also observed. Single channel current at 0 mV was 4.7 pA. P-open was not voltage dependent, and it was high (P-open=0.95—0.99) at the studied voltages.

Chloride single channel had conductance 90 pS, at the range from —30 mV to +30 mV. Single channel current at 0 mV was 4 pA. P-open was voltage dependent. It was low at —30 mV (P-open was <0.02), and with increasing voltage, P-open gradually increased, and it was 0.6 at +20 mV.

Mare_ J¹, Pometlová M¹, Brožek, G²

1Institute of Normal, Pathological and Clinical Physiology, 3^rdFaculty of Medicine and ²Institute of Physiology, 2^nd Faculty of Medicine, Charles University, Prague, Czech Republic

Central nervous system could be affected by hypoxia as well as epileptic seizures. Some implications of the both stressors are also expressed as impaired behavioural performance. Rapid repetition of intense oxygen insufficiency could worsen tissue impairment in comparison with repetition in longer intervals (hours, days). This preconditioning was described on the heart. We tested if it play some role also in nervous tissue. Morrison water maze is a good tool for testing changes in learning after different experimental impairments of the brain. The combination of the epileptic seizures and hypoxia was also tested.

Adult male Wistar rats (No 85) were divided in 3 experimental and 3 control group. Experimental animals were exposed to 2times-repeated hypobaric hypoxia (9 000 m). The schedule of experiment was hypoxia — 1 h (or 72 h) in normal conditions — hypoxia —24 h — learning in the water maze. In next group the second hypoxia was interchanged by epileptic seizure elicited by fluorothyle. Control groups: only 1 seizure; 1 hypoxia; without hypoxia or seizure were elicited. Performances were statistically compared in individual days and as the whole learning curves.

Hypobaric hypoxia (if repeated in short interval) impaired the spatial learning more then with longer interval. Unexpected was small difference between learning after hypoxia followed by seizures and learning after seizures. It also seems that epileptic seizure influences spatial learning more than hypoxia itself. Preconditioning may be elicited also in brain tissue.

Maťa_eje A, Béder I, Kittová M, Okkelová J, Važan R

The aim of the study was the erythrocyte deformability monitoring as one of important factors securing the appropriate tissues perfusion.

Measurements were accomplished in 7 healthy subjects and 16 diabetic patients with insulin dependent diabetes mellitus. Erythrocyte deformability was determined by filtration method with centrifugation and erythrocyte filtrability was calculated as percentage of filtered erythrocytes from the erythrocyte count before centrifugation. Diluted blood suspensions were filtered by centrifugation through membrane filters with the pore diameter 5 μm. The speed and duration of-centrifugation 1400 rpm (188 g) and 5 min respectively were selected as the best ones for filtration because of the lowest value of the coefficient of variance.

The arithmetic mean and standard deviation of erythrocyte filtrability in normal subjects were 72,2±7,9%. In the group of diabetic patients with the long-lasting insulin therapy the values amounted to 69,1±4,4%. In diabetic patients the mean value of glucose in the blood was 11,7 mmol.L^-1 and the glycosylated hemoglobin 9,04%. From the viewpoint of the reference values this fact indicates a good compensation of diabetes mellitus. In normal subjects the erythrocyte count was 5,1±0,4.10¹².L^-1, hemoglobin concentration 168,9±9,1 g.L^-1 and the mean cell volume 87,6±1,6 fL. In diabetic patients the erythrocyte count was 5,3±0,5.10¹².L^-1, hemoglobin concentration 167,2±11 g.L^-1 and the mean cell volume 87,8±3,5 fL. The experimental data were statistically tested by non-parametric Kolmogorov - Smirnov Test using STATGRAPHICS 4.0 programme.

Mátéffyová A¹, Najvirtová M¹, Oliver C², _trbák V¹

Objective. TRH is present and synthesized (1) in the islets of Langerhans within the b cells producing insulin. There are indications that TRH could play important role in insulin secretion.

Design: 1. Inhibition of TRH biosynthesis by TRH mRNA antisense oligonucleotide (1mM) to target a specific mRNA and block the expression of the proTRH by sequence specific hybridization in vitro 2. inhibition of a-amidation of TRH by Disulfiram (inhibitor of rate limiting enzyme peptidylglycine-a-hydroxylating monooxygenase (PHM) in vivo (5-days, dose:200mg/kg). At the end of experiments isolated pancreatic islets were exposed to stimulation by 16.7mM glucose and 30% hypo-osmolar medium in presence or absence of TRH to test the insulin response.

Results: We observed no insulin response to 16.7 mM glucose after incubation pancreatic islets in medium containing TRH anti-sense and TRH sense, and in islets from rats treated with Disulfiram. Response to glucose challenge was recovered by TRH (1nM) supplementation.

Conclusion. Our results demonstrated that pancreatic TRH is essential for adequate insulin response to glucose stimulation.

Supported by the grant from Slovak Academy of Sciences (VEGA) 2/7178/20 and the European Commission ICA1-CT-2000-70008..

The aldosteronE receptor blocker spironolactone prevented hypertension and remodeling of the left ventricle in L-NAME induced hypertension

Matuskova J, Luptak I, ¹Pechanova O, ²Babal P, Simko F

&#The aldosterone receptor blocker spironolactone attracts the interest of both experimental and clinical cardiology in the recent years. However, despite the great success of the RALES study, the mechanism of protection by spironolactone is still not quite clear.

&#The present study investigated the effect of spironolactone on the heart remodeling in the model of L-NAME induced hypertension. Four groups of Wistar rats were investigated: control, spironolactone (200mg/kg), L-NAME (40 mg/kg) and L-NAME + spironolactone. Animals were killed after 5 weeks of experimental manipulation.

&#Systolic blood pressure, the weight of the left ventricle (LV), DNA concentration in the LV were increased, while NOS activity in the LV, kidney and aorta were decreased in L-NAME treated rats. The addition of spironolactone to L-NAME did not prevent diminution of NOS activity induced by L-NAME in the left ventricle. Despite that spironolactone partly prevented the development of hypertension, hypertrophy of the LV and DNA concentration enhacement in the LV.

We conclude that aldosterone receptor blocker spironolactone prevented in part hypertension development and remodeling of the left ventricle in NO-deficient hypertension.

Mazancová K, Mik_ík I, Kune_ J, Pácha J

The data suggest that the placental glucocorticoid barrier is not decreased in SHR and DS rats in comparison with normotensive WKY and DR counterparts and that inactivation of corticosterone predominates during gestation but is different among various rat strains.

Intact tissue (intestinal slices) converted B to 20-dihydrocorticosterone (20-diB) but not to 11-dehydrocorticosterone via 20-hydroxysteroid dehydrogenase (20HSD) located predominantly in the cytosol of enterocytes. The activity of 20HSD was blocked by carbenoxolone (CBX), the known inhibitor of 11HSD, and was not significantly different along the intestine with the exception of coprodeum where the activity was lower. The stimulatory effect of B on induction of aSCC was lower in the presence of CBX than in its absence and the only two steroids found in the medium were B and 20-diB.

In conclusion, 20HSD seems to play an important role in the avian intestine: (1) it protects the intestine to glucocorticoid access and (2) regulates the magnitude of mineralocorticoid-induced Na⁺ retention.

Micutkova L, Rychkova N, Mravec B, Krizanova O, Kvetnansky R

Institute of Experimental Endocrinology SAV Bratislava, Slovakia

Stellate ganglia are directly involved in innervation of cardiac tissue.

The aims of this work were to quantify the precise amounts of TH and DBH mRNA levels in stellate ganglia in control rats. We also examined different time intervals after the end of the single two-hour immobilization, as well as the effect of short-term repeated immobilization on gene expression of these enzymes.

TH and DBH mRNA levels were quantified by competitive RT-PCR.

In stellate ganglia about 30 times less amount of TH mRNA compared to adrenal medulla was found (0.15 amol/ng RNA), whereas the amount of DBH mRNA was comparable to adrenal medulla (19.5 amol/ng RNA). After single two-hour immobilization, the highest elevation of TH and DBH mRNA levels occurred 24 hours after the termination of the stress stimulus. Repeated immobilization (7 days 2 hours daily) did not produced further increase in TH and DBH mRNA levels compared to adapted control.

In our study we compared for the first time the amounts of TH and DBH mRNA in stellate ganglia in control conditions and after immobilization stress.

Moeslinger T, Friedl, R, Spieckermann, PG

Introduction: Atherosclerosis is a major cause of morbidity and mortality in chronic renal failure and is associated with the proliferation of macrophages within atherosclerotic lesions.

Methods: Because the progression of atherosclerosis as a consequence of decreased nitric oxide synthesis has been described, we investigated the correlation between the inhibition of inducible nitric oxide synthase (iNOS) by urea, macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein, and macrophage apoptosis.

Results: Urea induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide stimulated mouse macrophages (RAW 264.7) with concomitant macrophage proliferation. Macrophage proliferation as determined by cell counting became statistically significant at 60 mM urea corresponding to a blood urea nitrogen level of 180 mg/100 ml, concentrations seen in uremic patients. iNOS protein expression showed a dose dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of urea. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with urea shows that the proliferative actions of urea are associated with a decrease of NO-induced apoptosis.

Conclusions: These data demonstrate that inhibition of iNOS dependent nitric oxide production caused by urea enhances macrophage proliferation as a consequence of diminished NO-mediated apoptosis. This fact may be important for the development of atherosclerotic lesions during chronic renal failure.

Mojži_ová A, Križanová O, Ondria_ K

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP), metabolite of NADP, activates Ca²⁺ release from intracellular stores in number of tissues very effectively. Effect of NAADP was first described by Lee and coworkers in sea urchin homogenates. Later studies showed that NAADP mobilizes intracellular Ca²⁺ in mammalian cells including cardiomyocytes.

Despite of intensive research of this compound, it is still unclear what is a target for this agent, what is the "channel" responsible for NAADP-induced calcium release.

In our study we have tried to answer that question. We have used the method of incorporation of native microsomal vesicles into artificial lipid bilayer and measured calcium currents flowing through the calcium channels derived from these internal membranes.

We have used microsomes derived from dog heart, rat brain and sea urchin eggs. In heart and brain preparations we observed mostly RyR channels. 45% of these channels were slightly activated by NAADP. However, the activation was mostly delayed, so we suggested, that it could be indirect or needs some another mediator, sometimes present in preparations, sometimes not.

In sea urchin eggs preparation, we observed RyR channels very rarely and they were not NAADP activated. Till now, we have not observed "NAADP channel".

Molnár Z, Petheő GL, Fülöp Cs, Spät A

The carotid body is responsible for sensing arterial pO₂, pCO₂ and pH and plays a substantial role in the appropriate control of ventilation. It has already been demonstrated that osmotic changes also affect the function of the organ. We tested the effects of osmotic changes on the chemosensory cells, known as chemoreceptor or type I cells of the carotid body.

Experiments were performed on primary cultures of chemoreceptor cells isolated from 10-20 day old Wistar rats. By using single cell microfluorimetry cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) and cytoplasmic pH (pH_i) were monitored in bicarbonate-buffered extracellular solutions.

Decreasing the osmolality of the extracellular solution from 300 mOsm to 250 mOsm caused marked elevation of [Ca²⁺]_c in all tested cells (n=18), and at the end of the 10 min stimulus 11 cells showed elevated [Ca²⁺]_c or repetitive Ca²⁺ spikes were observed. Short-term (100 s) decrease in osmolality by 15 to 50 mOsm resulted in graded Ca²⁺ responses. Ca²⁺-free medium abolished the Ca²⁺ response to the 50 mosM hypotonic challenge, and Nifedipine (10 µM), a blocker of the L-type Ca²⁺ channel, inhibited the response by 74% in average. Niflumic acid (300 µM), an inhibitor of the swelling activated Cl^- channel, abolished the Ca²⁺ response. Decreasing the osmolality by 50 mOsm resulted in slight, sustained intracellular acidification, while increasing by 50 mOsm had the opposite effect.

Our data provide evidence that hypotonic challenge evokes elevation of [Ca²⁺]_c and intracellular acidification in the chemoreceptor cell, and the initial step of the hyposmosis induced Ca²⁺ response is the activation of the swelling activated Cl^- channel.

Monček F, Dunčko R, Ježová D

Different stress stimuli result in neuroendocrine activation of different type and intensity. Some stressors can have long lasting effects on the function of neuroendocrine systems. We hypothesized that the stressor-specific changes could be in part due to changes in serotonergic neurotransmission. We tested this hypothesis by exposing rats to three different stress stimuli and using the selective serotonin reuptake inhibitor citalopram to test serotonergic function as seen by its influence on hypothalamic-pituitary-adrenocortical axis activation. The stressors used were a single immobilization 6 days prior to the experiment, repeated restraint 2 h daily and unpredictable stress stimuli changed twice a day for six days. Blood samples were taken from cannulated freely moving animals. A single dose of citalopram (10 mg/kg, i.p.) resulted in a significant increase in plasma corticosterone and adrenocorticotropic hormone (ACTH) levels in at 15 and 30 min, as well as increase in corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus of the hypothalamus. After a weeklong administration of citalopram, the CRH mRNA and ACTH responses were attenuated, though corticosterone levels remained similar to those after a single dose. No differences in plasma hormone levels and CRH and proopiomelanocortin gene expression between citalopram induced responses in stressed rats compared to nonstressed animals were found. In conclusion, the stress stimuli used did not influence serotonergic system in the extent that would change neuroendocrine response to citalopram challenge.

Mravec B, Mičutková L, Rychkova N, Kiss A, Križanová O, Kvetňansk_ R

The area A5 and other brainstem noradrenergic cell groups are activated by stressful stimuli, resulting in an increased release of norepinephrine in the hypothalamus and leading to the activation of hypothalamic-pituitary-adrenocortical axis. The A5 group of noradrenergic neurons plays a key role in autonomic mechanisms like cardiovascular regulation, nociception and respiration. The aim of this work was to detect the gene expression of catecholaminergic enzymes in A5 brain nuclei. We also investigated the effect of various time intervals after the end of the single two-hour immobilization, as well as the effect of short-term repeated immobilization (7-times) on tyrosine hydroxylase gene expression in this structure.

In this study we detected for the first time the presence of DBH mRNA in micro dissected A5 cell group. We also showed for the first time how the gene expression of tyrosine hydroxylase changed with the function of time after the single immobilization exposure.

Mysliveček J^1,2, Říčn_ J¹, Tuček S¹ 

(1) Mysliveček J., Trojan S., Tuček S.: Physiol. Res., 48, Suppl. 1, S98, 1999.

(2) Mysliveček J., Trojan S., Tuček S.: Physiol. Res. 49, P11, 2000.

Nagyová K¹, Tanczos J², Rievaj J¹, Waczulíková I¹, Ziegelhöffer A³

Norepinephrin (NE) and diabetes (DIA), both induce changes in energy transport through the heart mitochondrial membranes (MM). However, the accompanying changes in MM properties are still little understood. The present paper is a study of DIA- and NE-induced changes in membrane fluidity and ATPase activities of the heart MM. Methods: Adult male rats (234+19 g b. wt.) were treated either with NE (single dose, 1 mg.kg^-1 s.c.) or made diabetic with streptozotocin (45 mg.kg^-1 i.v.). Heart mitochondria were isolated according to Lehninger (1973) procedure. MM fluidity was assessed using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Mg²⁺-ATPase activity in absence and presence of 2,4-dinitrophenol (DNP, 0.01 mmol.l^-1) were measured by the amount of P_i liberated during ATP splitting at 700 nm. Results: NE and DIA decreased the DPH anizotropy (increase in fluidity) vs. healthy controls by 10 and 7 %, p<0.01. Mg²⁺- and DNP stimulated ATPase activities in healthy controls, amounted 16.8±0.9 and 1.1±0.1mmol P_i.g prot^-1.h^-1. NE and DIA induced changes were within the range of 3-9 % (p>0.05). Conclusion: NE and DIA induce significant decrease in MM fluidity without any effect on ATP-ase activities.

Najvirtová M, Baqi L, Strbák V

The aim of present study was to test if ethanol induced TRH release from rat posterior pituitary (PP), hypothalamic paraventricular nucleus (PVN) and isolated pancreatic islets (as non-neural tissue) involves cell-swelling and to characterize the pathway of induced secretion.

Stimulating effect of 80mM ethanol on TRH release in vitro in a short term static incubation system from PP, PVN and pancreatic islets was found only in isosmolar but not in hyperosmolar (prevents cell swelling) medium thus indicating involvement of cell-swelling inducing mechanism. L-Canavanine (incorporated into clathrin on immature secretory granules makes them unable to shed clathrin coats) in 30 mM concentration increased basal and 30% hyposmosis induced TRH secretion from PP and PVN and basal and ethanol induced TRH secretion from isolated pancreatic islets, thus indicating presence of both, constitutive-like and regulatory secretory pathway.

Our data demonstrated that ethanol in clinically relevant concetration induced TRH release from PP, PVN and isolated pancreatic islets by a mechanism involving cell volume changes. Increase of secretion by L-canavanine suggests exocytosis from clathrin coated vesicles after cell-swelling stimulation.

Nánási PP, Bányász T, Magyar J, Fülöp L, Szentandrássy N

Endocardial action potentials (AP) exhibit a prominent plateau, while spike-and-dome appearance is characteristic to the AP of epicardial cells in several mammalian species. The aim of this study was to compare the amplitude and kinetic properties of the L-type calcium current (I_Ca) in epicardial versus endocardial canine and human ventricular myocytes using AP-clamp and square pulse voltage clamp techniques at 37 ^oC.

The time course of I_Ca recorded in canine and human myocytes using AP-clamp differed from that reported in guinea pig cells. Rapid activation of the current in canine and human cells were followed by complete inactivation during the early phase of plateau, in sharp contrast to the prominent late component of I_Ca observed in guinea pig myocytes. Following the rapid inactivation of I_Ca a second activation of the calcium current occurred in epicardial but not in endocardial canine and human cells. The maximum of this second current peak strictly coincided with the raising phase of the dome, and its amplitude was proportional to the depth of the notch. When epicardial AP was applied as voltage command to endocardial cells, I_Ca displayed biphasic configuration, similar to those observed in epicardial cells. No differences were observed in the inactivation and recovery kinetics of I_Ca between epicardial and endocardial cells when analyzed under square pulse voltage clamp conditions.

Němec P¹, Burda H², Oelschläger HHA³

Whereas there is abundant evidence from behavioral studies for magnetic compass orientation among vertebrates, its sensory and neural basis remains enigmatic. Magnetic orientation in diverse representatives of birds, reptiles, amphibians, fishes, and insects has been intensively studied over past three decades, but in mammals it is poorly understood and most of the evidence is still controversial. The best demonstrated case of magnetic compass orientation in mammals so far has come from studies on Zambian mole-rats (Cryptomys anselli, Bathyergidae). Spatial orientation of these subterranean, behaviorally blind rodents is based on a magnetic, light-independent, polarity compass.

Here, in an unprecedented study, we combined two established methodological approaches: a) our behavioral test designed to assess magnetic compass orientation in mole-rats, and b) immunocytochemical visualization of the transcriptional regulatory protein c-Fos as a marker of neuronal activation. We focused on the superior colliculus, a prominent subcortical sensorimotor integrator, which plays an important role in orientation towards diverse sensory stimuli. We show that the superior colliculus of the mole-rat is involved also in magnetoreception: It contains a population of neurons that are responsive to magnetic stimuli, directionally selective, and orderly organized within a discrete sub-layer. This is the first evidence for involvement of a specific mammalian brain structure in magnetoreception.

The developed model biom n 2001, simulates the body mass growth of any virtual homeoterm defined by its initial body mass (g₀), its genetically limited body mass (gli), the dry matter fraction of proteins (cpr) and the ash (cas) in the lipid free empty body mass (lfebm). Metabolizable energy intake (mei) and energy uptake in the form of the obligatory total heat production (thp) are declared as a multiple of the basal metabolism (bm=kbm.g ^_ ). The amounts of proteins and lipids deposited in the body are derived from the value of net energy for production (nep=mei-thp) and the biological variable actual growth potential (agp=1-g/gli), the value of which decreases with the progress of the body mass maturing (g/gli).

The universal use of the biom n 2001 model is documented on results obtained in the growth of broiler chickens, hybrids ross 208. The body mass growth from the day of hatching up to the body mass of 4,0 kg, raised in the regulated temperature optimum, do precisely cope with the amount of the feed consumed. The validity of the model in simulation of proteins and lipids deposition is documented in the growth of pigs, hybrids large white x landrace, they were fed with different amount of metabolizable energy. A good fit of experimental and simulated data of the body mass growth, the metabolizable energy intake, the heat loss and the mass of proteins deposited has been obtained.

Simulation of the body mass growth as a result of intake and uptake of metabolizable energy and the value of the actual growth potential yield also new features in simulation of growth and the rational regulation of the body mass index in man. Preliminary results obtained in modeling the body mass reduction, increase or maintenance on the desired level is presented.

Novák P, Zahradník I

Passive electrical characteristics of isolated cells represented by their membrane capacitance, membrane resistance, and series resistance determine the quality of the patch-clamp measurement. On the other hand, they may also serve as a quantitative measure of the overall physical state of the cell, which may be related to its metabolic activity. We have tested these assumptions on isolated rat ventricular myocytes.

The passive electrical parameters were estimated on-line using a method based on the measurement of partial electrical charges accumulated in the myocyte under square wave voltage stimulation. Simultaneous records of passive parameters revealed time course of adaptation of the ventricular myocytes to equilibration with the pipette solution during a patch-clamp experiment. Large and abrupt changes were typical for the membrane resistance, while the series resistance and the membrane capacitance tended to change gradually. Independent behavior of the membrane resistance and the resting current measured at the holding potential of —50 mV was observed. In experiments with hypo-osmotic stress a specific increase of the membrane capacitance was detected.

We have found that monitoring of the passive electrical parameters provided new insight on stability of the myocyte characteristics during the experiment. On-line recording was useful for setting and correction of the amplifier parameters and for development of experimental protocols. Records of the passive parameters reflect correlation of experimental interventions with changes in the state of myocytes.

Our previous experiments with sigma receptor ligands in rat myocytes and rat isolated hearts suggested that these receptors may play a role in modulation of contractility. The aim of the present study was to examine the effect of sigma ligand haloperidol in the multicellular guinea pig heart preparations.

Ten adult male guinea pigs (270-330 grams) were sacrificed under ether anaesthesia. The hearts were rapidly removed, left atrium was cut and placed in thermostatically controlled horizontal bath (30°C) containing 95% O₂, 5% CO₂ aerated Krebs-Henseleit solution (1.25 mM Ca²⁺) The preparation was attached to the transducer in the bath and stimulated at the rate of 1Hz. Contractions were recorded isometrically. After 30-45 minutes of stabilisation, sigma ligand haloperidol in concentration 10^-8 M was administered to the bath for 30 minutes.

The effects of haloperidol on the amplitude of contraction and on the restitution of contractility were investigated. In all experiments we observed marked positive inotropic effect previously described in rat cardiomyocytes (increase in the twitch amplitude ranging from 50 to 90 %, with maximal effect in the 5^th minute of perfusion). The mechanical restitution pattern under the effect of sigma receptor ligand is not altered.

In conclusion: the positive inotropic effect of sigma receptor ligand haloperidol was confirmed also in guinea pig atrial preparations. According to its effect on the mechanical restitution we can assume that it is mediated via increased cytoplasmatic Ca²⁺concentration.

Nováková Z¹, Závodná E¹ , Novotn_ J², Honzíková N¹

Interrelationships among the variability in systolic blood pressure (SBP), pulse intervals (PI), and baroreflex sensitivity (BRS) in untrained children (Co) and trained swimmers (S) were studied.

Blood pressure was recorded in 84 children (S: 28; Co - age-matched: 56; 13-16 years of age) for 5 min (Finapres, frequency of breathing 0.33 Hz). The power spectra of SBP (mmHg²/Hz) and of PI (ms²/Hz) were calculated. BRS was determined by spectral method. Children were divided into subgroups according to the spectral power at the frequency of 0.1 Hz. The following limits were used: high - hSBPÒ100, hPIÒ12500; middle - 100ÒmSBPÒ55, 12500ÒmPIÒ5400; low - lSBP<55, lPI<5400. Relationships among groups A(hSBP, hPI), B(mSBP, hPI), C(lSBP, hPI), D(hSBP, mPI), E (mSBP, mPI), F(lSBP, mPI), G(hSBP, lPI), H(mSBP, lPI), I(lSBP, lPI) were analysed. Comparing Co and S, longer PI (p<0.01), higher PI-variability at 0.1 Hz (p<0.01) and lower SBP (p<0.05) were in S. Variability of PI correlated with BRS (p<0.01); this relationship was shifted to the higher PI-variability at the same BRS in S. The number of children in particular groups was higher in hPI-groups (p<0.01) and lower in lPI-groups (p<0.01) in S, no child was in groups G and H in S. The variability of PI increased with increasing BRS in each triplet of subgroups with the identical variability in SBP in both, S and Co. This relationship was shifted to the higher values of PI variability at the same BRS: 1) with the increasing variability in SBP in Co and S; 2) for each triplet of subgroups with the identical variability in SBP in S in comparison to Co.

development of tubular agGregates in fast skeletal muscle of CK^-/- mice - A Morphological study

Novotová M, Pavlovičová M, Zahradník I.

A characteristic feature of many diseased skeletal muscles is the presence of tubular aggregates (TAs), a well-defined membranous structures consisting of packed tubules or sacs. Recent immuno- cytochemical evidence shows, that TAs contain marker enzymes of the sarcoplasmic reticulum and the mitochondria. In this study we provide morphological view of steps leading to formation of TAs. As a model muscle was used m. gastrocnemius of CK^-/- mice, ultrathin longitudinal sections of which were viewed in transmission electron microscope.

In addition to TAs, several other features distinguishing CK^-/- from normal muscles were observed. First, damaged mitochondria that could be seen near TAs and other locations showed signs of opened outer membrane and spiralized internal membranes especially when in direct contact with triads and T-tubules. In parallel, sarcoplasmic reticulum appearing normaly as a chain of small vesicles along sarcomeres turned at some loci to wide sacs of single or multiple layers. Deteriorating mitochondria frequently formed clusters of vesicles of dense or light appearance. At these locations normal SR was difficult to recognize. More often SR formed vesicles similar to vesiculated mitochondrial membranes. At many locations, abundant membranous material formed large aggregates in which vesicles were transformed to prolonged sac-like structures, often forming parallel layers.

We hypothesise, that TAs develop in the process of deterioration of mitochondria and SR, presumably at reduced catabolic metabolism of the muscle.

Ngozi Nwanosike¹, Táňa Ravingerová², Oľga Križanová¹

Myocardial ischaemia causes metabolic waste accumulation and changes in energy production, and affects a variety of systems, e.g. calcium transport systems. Reperfusion is capable of reverting ischaemia depending on ischaemic severity and duration. Hence, using ischaemia and reperfusion (IR) models we aimed to study changes in mRNA levels in selected calcium transport systems. Isolated hearts of male Wistar rats were perfused in a Langendorf mode at a constant pressure of 70 mmHg and 37^o C. After stabilisation, the hearts were subjected to global ischaemia (I) and reperfusion (R) by clamping and declamping of aortic inflow. 1, 2 and 4 cycles of IR consisting of 5 min I followed by 5 min R were induced. Tissue samples obtained from both atria and ventricles were stored at —70^o C until used in the assays. Gene expression of IP3 receptors and AT1 receptors were measured using RT-PCR and quantified relative to housekeeper GAPDH. Proteins were detected using Western Blot analysis and ECL technique. Type 1 IP₃ receptor mRNA (IP₃R1 mRNA) levels were highest in left atrium. After 1, 2 and 4 IR cycles, mRNA levels in both atria decreased. Similarly, we noted reductions in IP₃ protein levels. AT₁R mRNA also manifested reductions after IR. The most significant decrease was observed after 1 IR cycle, 4 IR cycles led to a near return to control values of AT₁R mRNA in both atria. Our results are in conformity with conclusions that the ischaemic heart’s incapability to fully revive its function is associated with changes in calcium transport systems. The effect of ischaemia and reperfusion is time dependent.

Obuchowicz R, Pawlik M, Sendur R, Szczepański W, Pawlik WW

Aim of our study was to clarify possible role of capsaicin sensitive fibers in ischemic preconditioning (IPC) evoked protection in the intestine of the rat. Anaesthetized rats were used. Capsaicin was administered acutely on perivascular neurons 15 min before anterior mesenteric artery occlusion. Rats underwent IPC procedure — four periods of 5 min total mesenteric artery occlusion with intermittent reperfusion (5min). IPC was followed by 30 min total ischemia with subsequent 60 min reperfusion (30/60 I/R). After reperfusion 10 cm ileal segments were excised and exposed. Area of mucosal lesions was determined with the use of computerised planimetry. Arterial pressure (MAP), intestinal blood flow measured (LDBF) and total mesenteric blood flow (MBF) were measured.

Experimental groups: First - control with 30/60 I/R alone; second -with 30/60 I/R proceeded by IPC; third group with 30/60 I/R pretreated with capsaicin; Fourth 30/60 I/R proceeded by IPC pretreated with capsaicin.

In the first group marked intestinal mucosal impairement was observed. In the second group mucosal lesions area was diminished by 43±7 % (p<0,05) in comparison to control I/R group. In the third and fourth group mucosal lesions area was diminished in comparison to control I/R by 19±4% and 31±7% (p<0,05) respectively. Capsaicin administration was followed by marked increase of MBF and LDBF by 27±7% and 65±4% respectively. Above results indicate that c fibers play a role in the maintenance of the mucosal integrity under I/R stress and at least in part are modulators of IPC induced protective action against mucosal injury.

THE CONTENT OF PROTEIN CARBONYL GROUPS (PCG) FOLLOWING REVERSIBLE BILATERAL CAROTID OCCLUSION IN THE GERBIL BRAIN
Ondrejičková O¹, Ziegelhoeffer A², Breier A³, Horáková Ľ¹, _tolc S^1
1Inst of Exp Pharmacology SAV,²Inst for Heart Research SAV, and ³Inst of Mol Physiol and Genetics SAV, Bratislava, Slovakia

It was assumed that besides enhanced production and local accumulation of lactate, reversible ischemia (I) is also accompanied by increased PCG formation due to oxidation of amino acids by reactive oxygen species. The aims of our study were: a) to elucidate whether the reperfusion (R)—induced decrease in brain cortex lactate level is accompanied by a diminution of the PCG content and b) to find out whether there exists any correlation between the I/R induced changes in lactate and PCG contents. Methods: Brain I was induced in adult male gerbils (60-70 g) by 20 min bilateral carotid occlusion. Determinations: Lactate-Randox kit UK, PCG-spectrophotometry and immunoassay (Oxyblot kit, Quantumgene, France). Results: I produced a significant increase (142.8 %; p<0.01) in brain cortex lactate. The elevation of lactate was accompanied by a significant increase (73.4 %; p<0.05) in PCG. R induced a significant (65.3 %; p<0.05) decrease in lactate and also in PCG content p<0.01) even down to control values.

Conclusion: Brain ischemia induces a simultaneous and significant increase in tissue lactate and PCG contents. R decreases the ischemia-induced lactate accumulation significantly and reverses the oxidative damage to proteins in the brain.

Ondria_ K, Máleková Ľ

The aim of our work was to characterize IP3R calcium release channels from rat brain microsomes. We incorporated the microsomes into bilayer lipid membrane. Using 53 mmol/l Ca²⁺ as ion carrier, we observed three different behaviors of the IP3R channels after activation by IP₃. A.) Classical regular channel behavior: The channels were regularly gated after application of 5 μmol/l IP₃, and their activity was prolonged for more than two minutes. B.) "Digital" channel behavior: The channel after activation by 5 μmol/l of IP₃ opened only once for a few ms and after that remained closed for more than one minute. Increased concentration of IP₃ up to 15 μmol/l did not activate again the channel. Conductance 1.2-1.5 pA and open time (a few ms) of the "digital" channels were similar to the regular channels. C.) Coupled channels: After application of 5 μmol/l IP₃ calcium channels were observed which had double conductance of the regular channels. The coupled channels were inhibited by 20 μmol/l Heparin. We supposed that the double channel conductance results from the cooperative openings of two single IP3R channels.

Orlick_ J¹, Gmucová K², Thurzo I², Pavlásek J³

The aqeous solution is the simplest model for understanding charge transfer (ions and electron ) in biological systems including biomembranes.L-ascorbic acid(AA) can serve as an electron donor for a trans-plasma membrane oxidoreductase activity. Ampero-metric determination of AA is based on its electrochemical oxida- tion.The common problem in the AA oxidation is that it requires large overpotential(+0.5 V) and the repeated use of glassy carbon electrode suffers from the fouling effect resulting in poor selecti- vity and sensitivity.We have solved this probleme by profiting from the kinetics-sensitive double-step voltcoulometry(DSVCM) [1]. DSVCM voltamograms of AA exhibited the reversible peak at negative potential -0.25 V and the irreversible positive one at +0.25 V.A different linear relationship between the both peaks were found in a dose-response of AA and the peaks height on the voltamograms in the tested range of 0.02-2 mM.The measurment of AA vere also made in a mixcture (1-nitro-manitol,dopamine), in commercial orange drinks and/or in the fresh fruits.Our results showed that the AA peak at negative potential is very sensitive with respect to the composition of the analysed solutions (e.g.the ionic strenght,the presence O₂ and CO₂). This peak is detected only in the fresh deareted solutions and/or the fresh fruits.

Ostatníková D¹, Celec P¹, Putz Z², Kúdela M³, Bursk_ P⁴, Stárka L⁵, Hampl R⁵ 

Testosterone was proved to influence not only physical characteristics, but also behaviour and mental functioning as well. It is true for both sexes. Testosterone is known to exhibit circadian rhythm and annual rhythmic variations in man. The aim of our study was to investigate possible infradian variations of salivary testosterone. Salivary testosterone reflects free testosterone fraction in plasma. Non-invasive saliva sampling enables frequent collection.

Four ml of saliva from 31 healthy men (18-25 years of age) were collected every second day during 1 month and every third day during the following 1,5 months. Testosterone levels were determined by radioimmunoassay. Data were statistically analysed on infradian rhythmic changes using two different methods: moving averages-zones of extremes and analysis of rhythmic variance.

The results showed rhythmic variations with 2 different infradian periods. The differences between extreme values of the circatrigintan and circavigintan cycles were found to be highly relevant.

According to our knowledge this study is the first to find the existence of infradian rhythms of salivary testosterone in human male in fertile age.

The spinothalamic tract (STT) is regarded as a major ascending pathway involved in transmission of nociceptive information from the spinal cord level to higher brain areas, while the dorsal column (DC) pathway is considered to be mainly involved in conveying information about innocuous stimuli. Recent studies showed that a DC lesion alleviates pain of visceral origin in cancer patients and that postsynaptic dorsal column pathway (PSDC) neurons in the vicinity of the central canal are activated by noxious and innocuous stimuli applied in viscera and on the skin.

To evaluate the role of these two ascending pathways in visceral and cutaneous nociception we used electrophysiological, immmuno-histochemical and behavioral techniques combined with mechanical and chemical stimulation of skin and different visceral organs.

Although neurons projecting in both the STT and the PSDC path can be activated by noxious stimuli of cutaneous or visceral origin, our results suggest that the STT plays a crucial role in the perception of acute cutaneous pain and that the DC pathway is important for transmission of visceral pain.

Paulmichl M^1,3, Ritter M¹, Fürst J¹, Jakab M¹, Gschwentner M¹, Konrat R², Botta G³, Meyer G³

Pavelková J, Kubalová Z, Zahradníková A, Zahradník I

We have characterized the fast-inactivating component (release-dependent inactivation, RDI) of the L-type calcium current (I_Ca) by three different kinetic models, assuming the concept of independent excitation-contraction coupling units (ECCUs), and assuming that inactivation of I_Ca by the released calcium is a pseudo-first order process. The models differed in their quantitative description of coupling between I_Ca activation and triggering of Ca²⁺ release. We have tested these models to fit calcium currents measured in isolated whole-cell patch-clamped rat ventricular myocytes at different stimulus potentials.

Model 1 assumed that calcium release is activated in all ECCUs synchronously and it is delayed with respect to the stimulus by a specific time t_d. Model 2 assumed tight coupling between activation of I_Ca and activation of Ca²⁺ release and the presence of a coupling time, t_c, necessary for diffusion of Ca²⁺ from the Ca channel to the ryanodine receptors (RyRs) and for RyR activation. Model 3 assumed that activation of Ca²⁺ release in individual ECCUs is stochastic, with an average time delay with respect to the stimulus, t_d, equal to the mean of a Gaussian distribution, and a synchrony of release, S_R, inversely proportional to the standard deviation of this distribution.

Pavlovičová M, Novotová M, Valachovičová K, Ventura-Clapier R¹, Zahradník I.

The aim of this work was to study the reaction of ultrastructure of cardiac muscle to elimination of creatine kinase (CK), a key enzyme in transport of energy in muscle cells. As a model, we used left ventricles of CK^-/- mice with blocked expression of the mitochondrial and cytosolic isoforms of the CK genes. Cardiac function of CK^-/- mice was nearly normal.

The electron-microscopic images of CK^-/- cardiac myocytes revealed frequent disorganisation of the myofibrils with irregular network of sarcoplasmic reticulum (SR) around the A and I bands. Mitochondria formed abundant clusters instead of the typical longitudinal columns along the myofibrils.

As the contact area between mitochondria and myofibrils was not modified, we conclude that deficiency of CK did not cause such changes in the microarchitecture of myocytes, which would break up transfer of energy between mitochondria and myofibrils.

Pawlik WW, Sendur R, Biernat J, Obuchowicz R

Dept. Of Physiol. Jagiel. Univ. Med. School, Krakow, Poland

The splanchnic vasculature is innervated by the intrinsic fibers from enteric nervous system and extrinsic nerves. Within the extrinsic visceral vascular nerves are visceral sensory afferent nerves. Such peripheral peptide containing fibers appear to participate in the transmission of sensory impulses in visceral reflexes. This innervation plays also an important role in the modulation of splanchnic local vascular responses. These afferent neural fibers when stimulated can elicit visceral vasodilation via a local axon reflexes releasing vasodilator peptide-neurotransmiters (mainly CGRP ). It has been demonstrated that afferent sensory fibers can modulate resting intestinal blood flow and oxygen uptake and intestinal vascular autoregulatory responses including reactive hyperemia, functional hyperemia and post stimulatory escape. We found that capsaicin when applied periarterially elicted a significant vasomotor response from the splanchnic arteries that appears to have two components: an initial transient dialatation that fades and a sustained constriction of lesser amplitude than observed vasodilation. During early hyperemic response blood flow was redistributed into the mucosal-submucosal compartment of intestinal wall. Whereas during the late vasoconstrictive response blood flow was distributed into the muscular compartment. The observed changes of the blood flow distribution in the vascular compartements in the intestinal wall may be due to grater density of afferent C fibers in the mucosal-submucosal layer.

Afferent C fibers play a significant role in the control of gastric, pancreatic and hepatic vasculature at basal conditions and during ischemia/reperfusion induced vascular damage of visceral organs.

Pecháňová O, Bernátová I

The aim of the present study was to examine whether treatment with the NO synthase substrate (L-arginine) or ACE inhibitors can prevent hypertension and cardiac hypertrophy in spontaneous hypertensive rats (SHR). The involvement of NO/cGMP pathway in this prevention was investigated. Six-week-old SHR were divided into 4 groups: control, group receiving L-arginine (150 mg/kg/day) and groups receiving captopril or enalapril (100 mg/kg/day) during 6 weeks. NO synthase activity, cGMP level and the concentration of free radical marker - conjugated dienes (CD) were determined in the heart and aorta. At the end of experiment, systolic blood pressure (SBP) and HW/BW ratio increased by 41 % and 23 %, respectively, in the control group. Similarly, in the L-arginine group the above parameters increased by 39 % and 25 %, respectively. On the other hand, captopril and enalapril prevented SBP increase and hypertrophy development. However, SBP in the captopril group (112±5 mmHg) was significantly lower than that in the enalapril group (131±6 mmHg). All three substances increased NO synthase activity in the heart and aorta. Noteworthy only captopril and enalapril increased cGMP concentration by 22% and 34% in the heart and by 31% and 50% in the aorta, respectively. Significantly lower CD concentrations in the captopril group compared to both L-arginine and enalapril groups in the above tissues were detected.

We conclude that ACE inhibitor captopril with —SH group, except its direct effect on ACE, prevented hypertension and hypertrophy also by increasing NO synthase activity with simultaneous decrease of oxygen free radicals which together led to higher increase in cGMP concentration.

Pelouch V¹, Pechová M², Jirmář R³ 

Contractility of myocardium is determined by complex interactions of non-collagenous (metabolic and contractile fractions) and collagenous proteins (different collagens, glycoproteins, proteoglycans and elastin) in the presence of Ca- ions under sufficient energetic potential (ATP and CP). Many pathological situations in cardiac muscle (e.g. ischemia, hyper-trophy or congenital heart disease) are accompanied by remodelation (qualitative and quantitative changes) of collagenous proteins localized in extracellular matrix. (ECM) of myocardium. There are many possibilities how to determine this one: isolation of pepsin-soluble (newly synthetized collagen) and -insoluble fractions of ECM proteins (maturated forms of collagens and other ECM proteins decorated by different glycosaminoglycans), determination of concentration of both protein and hydroxyproline in these fractions, acrylamide electrophoresis (for types of collagens) and zymography (determination of metalloproteinase’s) are more frequent. Recently, carboxyterminal propeptide of collagen (PICP), aminoterminal propeptide of procollagen I (PINP), carboxyterminal telopeptide of type I collagen (ICTP) and type III procollagen (PIIINC) have been developed by ORION Diagnostica, Finland; these RIA kits have been used for determination of bone remodelation. However, we have used these possibilities for analysis of speed both synthetic and degradation procedures on the level of myocardial collagen in groups of patients after cardiac infarct (blood was taken the 1, 2, 7 and 30 days from infarct); biochemical data were correlated with function parameters. Furthermore, cardiac tissues were analyzed from children with congenital heart disease (samples from both right ventricular and right atrial cardiac tissue were taken during surgery of different normoxemic and hypoxemic patients).

Using the pancreatic acinar cell as an example, I shall illustrate how different types of cytosolic Ca²⁺ signals, ranging from repetitive short-lasting local spikes in the apical secretory pole to global sustained elevations, can be used to control three entirely different processes in the same cell.

[1] Local Ca²⁺ spikes can be induced by physiological concentrations of acetylcholine or cholecystokinin and are due to short-lasting openings of Ca²⁺ release channels in apical extensions, into the granular region, of the endoplasmic reticulum (ER) (Petersen, Tepikin & Park TINS 24, 271-276, 2001). These cytosolic Ca²⁺ rises are confined to the apical pole by a mitochondrial firewall around the granular region and elicit both exocytosis and fluid secretion through opening of Ca²⁺-activated Cl^- channels localized specifically in the apical plasma membrane (Petersen & Fedirko Curr. Biol. 11, R520-R523, 2001; Park, Lomax, Tepikin & Petersen PNAS 98, 10948-10953, 2001).

A global rise in the cytosolic Ca²⁺ concentration, induced by high cholecystokinin or bile acid concentrations and sustained by opening of store-operated Ca²⁺ channels in the plasma membrane, can lead to intracellular protease activation. This causes autodigestion and is the basis of the human, and often fatal, disease Acute Pancreatitis (Raraty et al. PNAS 97, 13126-13131, 2000).

The oxidant menadione, which releases Ca²⁺ from the ER and also depolarizes the inner mitochondrial membrane by opening the permeability transition pore, causes global repetitive Ca²⁺ waves. These events elicit programmed cell death (apoptosis) (Gerasimenko et al. J Cell Sci. 2002, in press).

Petrá_ová D¹, Hijová E¹, Kuchta M^1,2, Koprovičová J, Majlingová S² 

Coeliac disease is an immuno-pathological disease characterised by an inflammatory affection of the small intestine, which is caused by the hypersensibility to the grain aleurone component-gluten. It has pronounced manifestations of MAS (from the picture of coeliac crisis to substantially easily running clinical pictures) being often diagnosed also in late adulthood. Regarding variability of the clinical picture and intensity of its symptoms with reflection to the immunological profile, the serum concentrations of prealbumin and transferrin as non-enzymatic antioxidant were examined. Both proteins are used for evaluation of the nutrition status of the hospitalised Romany children.

Methods: Two hundred and forty seven Romany children aged from 1 month to 14 years were examined and divided according to various stages of the disesase and age categories.

Results: The positive of antigliadin antibodies (AGA) was found in both classes IgG and IgA. The BMI (kg/m²) value was found to be lower by 13% in the individual age categories in comparison with the control group represented by non-Romany children from East Slovakia. The concentration of prealbumin ranged from 0.10-0.27 g/L in all the age categories. The lowest mean value was found in the children to one year of life (0.18±0.05 g/L). The mean value of serum transferrin was 3.26±0.84 g/L (min.=1.8; max.=5.0 g/L).

Conclusions: It turns out that the values of transferrin and prealbumin may be very useful prognostic markers of success of the aleurone free diet in ill children at various staged of coeliac disease.

LDL Oxidation by thiols and transition metals
Pfanzagl B, Koller E, Möslinger T, Tribl F

Reduced forms of thiols like cysteine and homocysteine can have pro- and antioxidant properties, the prooxidant activity being caused by rapid autooxidation in the presence of oxygen whereby superoxide radicals and hydrogen peroxide are generated. It is still a matter of discussion wether copper-dependent oxidation of low density lipoprotein (LDL) is enhanced or inhibited by thiols.

We show that thiols inhibit LDL oxidation by copper(II) only at copper concentrations which cause a rapid increase in lipid peroxidation in the absence of thiols. This was shown by measurement of formation of lipid conjugated dienes and thiobarbituric acid reactive substances (TBARS). In contrast, cysteine and homocysteine stimulated copper-dependent LDL oxidation at copper concentrations causing only a slight oxidation of LDL lipids. LDL oxidation by homocysteine and copper(II) is further enhanced by the addition of cystine at plasma concentrations, although cystine alone has an inhibitory effect on copper-catalyzed LDL oxidation. The enhancement of homocysteine-stimulated copper-dependent LDL oxidation by a thiol mixture similar to the one found in plasma is especially pronounced at homocysteine concentrations of 30 µM or higher and can be reduced but not completely abolished by plasma concentrations of the copper-chelating amino acid histidine. Stimulation of LDL oxidation by cystine in the presence of homocysteine was also observed with iron(III) as the oxidizing agent at acidic pH.

RAT PAPILLARY MUSCLE FORCE AND ACTION POTENTIAL ACQUISITION AND ANALYSIS USING LABVIEW
Plass CA¹, Schmid D¹, Loew HG², Spieckermann PG¹

To automate analysis of force and action potential data we used LabVIEW^TM to create applications that digitize and display data recorded from a force transducer and a microelectrode in a modified setup according to Hoffmann and Kelly¹ to investigate contractions of rat papillary muscles.

Direct indication of the measured signals during data acquisition in graphical form and online extraction of the most important parameters are performed. Full recall and full replay of the entire protocoll for detailed automated or manual evaluation using cursors has been implemented. Applications calculate all relevant isotonic and isometric contraction parameters (PS, tPS, PV, tVC, VR, tVR, AT, tAT, PdF, tPdF, NdF, tNdF, tHR, tOC) and also all relevant action potential parameters (Umem, OV, dU/dtmax, APD10, APD50, APD90) and draws onscreen diagrams. Various data export features following automatic parameter processing are established.

Precise measurement of force development and electrical activity after field stimulation was easily accomplished using a data acquisition sample rate of 5000 S/s. Programmable triggering was also managed via our LabVIEW^TM virtual instrument.

These analyses have simplified and automated the process of evaluating papillary muscle preparations in a very cost effective way. Representative results will be shown and discussed.

Pokorn_ J, Hrachovina V, Langmeier M, Trojan S

Genetic program of the neuroplastic mechanisms of develop-ment, adaptation, and recovery is controlled by the state of nerve tissue microenvironment. Its stability is assured by reactions of the vascular system, based on perfusion and blood-brain barrier (BBB) reactions. An increase of permeability may result from the impairment of blood vessels or that of surrounding tissue. Exact data about the dynamics of permeability changes are still missing.

Brain lesion was induced by a fluid injection into the infragra-nular layer of the dentate gyrus. Permeability of BBB in various time intervals was assayed using Evans blue as a tracer. The dye binds to plasma albumins, forming a complex (MW=68 500 Da), which cannot penetrate through an intact barrier. Distribution of the tracer was detected in a fluorescent microscope.

Results have shown a marked increase of the BBB permeability. Evans blue was detected not only in the extracellular space of the nerve tissue, but in significant concentrations also in the nerve cells in the vicinity of the lesion. The region of increased permeability had an irregular shape, its extend decreased with time, and it persisted at least 3 to 4 weeks.

The lesion induced elevation of BBB permeability and its long-lasting persistency need not reflect only the local impairment of the vascular network, but it can also represent the pathway, which enables spreading of neurotrophic factors and other signalling molecules activating neuroplastic mechanisms of recovery and reorganisation of neuronal circuits.

Poláček H¹, Vrána J¹, Rachmanová R¹, Tintěra J², Stančák A¹

The aim of our study was to show the spatial correspondence between the hemodynamic and electrocortical responses during repeated painful electrocutaneous stimulation.

Ten healthy right handed men (age 22±4 years) took part in one EEG (BrainScope^®, 88 electrodes) and one fMRI (1.5-Tesla Siemens Magnetom Vision, 29 slices, 4_1.8_1.8 mm resolution) experiment. The subjects were stimulated on the 2nd right finger using intradermal needle electrode in 4 blocks each lasting 384 seconds. The stimulus intensity for painful stimuli was set to 20% above pain threshold. Preprocessed data were analyzed using BESA™ and BrainVoyager™. The functional maps were corregistrated with 3-D anatomical MRI. In each subject, topographical differences between electrical sources and clusters of hemodynamic response were evaluated.

In 9 subjects, corresponding EEG and fMRI activations were found in contralateral parietal operculum, mesial frontal cortex and supplementary motor areas. The mean distance between fMRI cluster maxima and EEG sources was 14.7 mm in the contralateral parietal operculum, 4.3 mm in the cingulate cortex and 8.8 mm in the supplementary motor area.

The results suggest a good spatial correspondence between electrical and hemodynamic responses in the parietal operculum and mesial frontal cortex.

Poliaček I¹, Jaku_ J¹, Stránsky A¹, Tomori Z², Baráni H¹

The neural mechanisms regulating the laryngeal muscles during mechanically induced cough are still obscure.

Electrical activities of diaphragm (DIA), abdominal muscles (ABD), laryngeal abductor (m. cricoarytaenoideus posterior - PCA) and laryngeal adductor (m. thyroarytaenoideus - TA) as well as pleural and blood pressures were recorded in 43 anaesthetized (sodium pentobarbitone 35-40 mg/kg i.p.) spontaneously breathing cats. Microinjections of kainic acid (50-200 nl) were applied to the medullary raphe nuclei (RN), lateral tegmental field (LTF) and pontine respiratory group (PRG) under stereotaxic control.

Our data confirm that the cough reflex elicited both from the laryngopharyngeal and tracheobronchial regions consists of four phases : 1) deep initial inspiration characterized by glottal dilation (PCA activity) and DIA activity, 2) compressive phase (the glottal narrowing) caused by co-contraction of ABD and TA generating high subglottal pressure, 3) expulsive phase with glottal dilation (PCA activity) and rapid expulsion of air by high pleural pressure (ABD activity) and 4) subsequent glottal constriction (TA activity). After kainic acid lesion of RN, LTF or PRG cough could not be elicited, however, there were residual reflex changes in PCA and TA activities during mechanical stimulation.

Pometlová M, Romsauerová A, Mare_ J

The epileptic seizures cause some alterations of cognitive functions. The aim of our experiments was to find out which age in early ontogenesis is more sensitive to elicit these alterations.

The epileptic seizures were induced by repetitive injections of subconvulsive doses of pentylenetetrazol (PTZ) until onset status epilepticus (SE). There were 3 experimental groups (Wistar, males). First group, SE was induced only at 14^th postnatal day (14PND) second group only at 18PND and third group, SE was induced repeated at 14PND, 16PND and 18PND. Control groups (siblings) received saline at the same age. All rats were subjected to behavioural testing, between the 17 - 23PND and then as adult.

In comparison with the control group, animals after repeated SE showed impairment in the simple learning in childhood (homing- p<0,05) as well as in adulthood (Olton´s radial arm maze - p<0,05; Morris´s water maze - comparison of non-linear regression curves, p<0,001). Animals after one SE at 14PND exhibited more moderate alterations in learning in childhood (homing) and in adulthood (Morris water maze). Learning in animals after one SE at 18PND was without changes compared with their controls.

Impairment of learning in our experiments was not a pure function of number of epileptic seizures. The age also seems to play very important role.

Popelar J, Mazelova J, Syka J

Previous studies have demonstrated that descending fibers from the inferior colliculus (IC) project to the superior olivary nuclei. Fibers originating in the superior olive form the olivocochlear bundle and innervate sensory cells in the cochlea. Activation of the olivocochlear system by contralateral acoustical stimulation has been shown to suppresses the amplitude of otoacoustic emissions (OAEs). A simple method of activating the olivocochlear pathway by contralateral electrical stimulation of the round window (RW) was used in this study, with the aim of comparing the efficacy of acoustically and/or electrically evoked suppression of OAEs. Another aim was to test the effect of electrical stimulation of the IC on the OAEs in guinea pigs. OAEs were recorded in guinea pigs with an ILO 96 analyzer. The contralateral ear was stimulated with continuous broad-band noise, the RW was electrically stimulated through an implanted electrode using a continuous train of rectangular pulses. The same type of electrical stimuli was used for bipolar IC stimulation. Acoustical stimulation of the contralateral ear resulted in otoacoustic amplitude suppression by 1 dB on average. The same amount of suppression was obtained by electrical stimulation of the RW or the IC. The maximal effect was obtained when the stimulating electrodes were located in the ventral layers or rostral parts of the IC. The results demonstrate that: i/ electrical stimulation of the RW can be used as an alternative for activating of the olivocochlear efferent system, and ii/ the descending fibers from the IC are functionally connected to the olivocochlear neurons, and electrical stimulation of the IC can suppress OAEs similarly as does acoustical stimulation of the contralateral ear. Supported by grants of the Czech Ministry of Health 6454-3 and GACR 309/01/1063.

The ontogeny of intracellular heart structures is not terminated by the time of birth. Especially the sarcoplasmic reticulum (SR), the main structure of Ca²⁺ storage and release in the adults, is not fully developed during the perinatal period and neonatal myocardium therefore exhibits different Ca²⁺ handling in comparison with adults. While in the mature cardiomyocytes is a main source of contraction activator calcium SR, in the newborns play these role a transsarcolemmal influx of Ca²⁺. During first postnatal month take place a rapid maturation of SR and origin of T-tubules. The aim of our study was to analyse the postnatal age dependent changes in the calcium economy and sensitivity to the block of calcium current (ICaL). The experiments were performed on the right ventricle papillary muscles of newborn (age less than 3 days after birth) and adult (age over 8 month) cats. The action potential (AP) and appropriate contraction (MG) were measured. The elevation of extracellular Ca²⁺ (from 2 to 6 mM) causes four-fold increase in MG of newborn cat myocardium as compared with the adult ones. The underdeveloped myocardium was much more sensitive to the presence of verapamil, use dependent blocker of ICaL. The results indicate that the sensitivity of ventricular myocardium to the interventions changing calcium economy is significantly higher in newborn cats. In the correspondence with increasing role of SR to accumulate and release Ca²⁺, the dependence of contraction/relaxation cycle on extracellular calcium decreases. During postnatal ontogenesis the immature ECC (so called sarcolemmal type) converts to mature ECC (reticular type), which is mainly depend on SR function and transsarcolemmal calcium fluxes play in the "beat-to-beat" regulation only supporting roles.

Ravingerova T¹, Neckář J², Strnisková M¹, Barančík M¹, Kolář F² 

2Inst Physiol, Acad Sci of the CR, Prague, Czech Republic

We have previously demonstrated an enhanced resistance to ischemic arrhythmias in rats with diabetes mellitus (DM) suggesting activation of some cardioprotective mechanisms in the diabetic myocardium. Mitogen-activated protein kinases (MAPK) signaling activated by various forms of stress have been implicated in the mechanisms of cardioprotection, however little is known about their role in DM. The present study was designed to extend our knowledge to the protection against myocardial infarction and to investigate the changes in levels and activation of MAPKs. Adult male Wistar rats were rendered diabetic (D) with streptozotocin (45 mg/kg, i.v.). After 1 week, anaesthetised open-chest D rats and controls (C) were subjected to 30-min occlusion of LAD followed by 4-h reperfusion for the evaluation of the infarct size (IS) by tetrazolium staining. The contents and phosphorylation state of extracellular signal-regulated protein kinases (ERKs) and p38-MAPK were investigated by Western blot analysis. The IS normalised to the size of area at risk was significantly smaller in D than in C (47.2 ± 2.8% vs 70.2 ± 2.+%, resp.; P<0.05). No differences in the total levels of ERKs and p38-MAPK, as well as in the phosphorylation of p38-MAPK between D and non-D myocardium were observed. On the other hand, phosphorylation of ERKs was significantly increased in D as compared to C. We demonstrate for the first time that rat hearts appear to be more resistant to irreversible cell damage in the acute phase of experimental insulin-dependent DM, and this situation is associated with the activation of ERKs pathway.

Dopaminergic modulation of blood flow in the ciliary body, aqueous humor production and intraocular pressure in rabbits.
Reitsamer HA^1,2, Kiel JW^2 
1Inst Physiol, Univ Vienna Med School, Vienna, Austria,, and ²Dept Ophthalmology, Univ Texas Health Science Center, San Antonio, Texas, USA

Ritter M¹, Ravasio A, Danzl JG, Rudzki J, Scandella E, Fürst J, Chwatal S, Jakab M, Paulmichl M^1,2

The ICln protein has been shown to act as ion channel and to be essential for cell volume regulation. Using fluorescence resonance energy transfer (FRET) between a fusion protein of cyano fluorescent protein and ICln (CFP-ICln) as donor and a yellow fluorescent protein and ICln (YFP-ICln) as acceptor FRET can be detected, indicating self association of the ICln proteins. In native cells ICln is located in the cytosol and a minor fraction resides in the cell membrane. Using FRET between CFP-ICln as donor and a membrane tagged YFP (YFP-Mem) as acceptor clear FRET can be detected under isotonic conditions. Upon cell swelling the FRET signals significantly increase, indicating cell swelling induced cytosol to membrane shift of the ICln protein. This shift is sensitive to changes in extracellular pH.

In C.elegans the ICln gene is organized in an operon and transcribed with two other proteins termed Nx and Ny. The transcript of the ICln gene is alternatively spliced to yield two protein variants, termed IClnN1 and IClnN2 (encoded by exon 2a). IClnN1 is highly homologues to all ICln proteins identified so far, whereas IClnN2 bears additional 20 AAs close to the inner mouth of the channel pore. In contrast to IClnN1, IClnN2 exhibits strong voltage dependent channel inactivation if reconstituted in lipid bilayers. Reconstitution of IClnN2 and protein Nx reveals suppression of this voltage dependent inactivation indicating a close interaction between these proteins.

Roman O¹, _ere_ J² Pometlová M², Stančíková M³, Jurčovičová J¹

The aim of present experiments was to compare the effects of stress of repeated psychological challenge (PS) and of chronic food restriction (FR) on the development of adjuvant arthritis in the Long Evans male rats. Four groups of animals were used: Vehicle treated control (VC) and arthritic (AA) rats, and two analogous groups with random daily exposures to isolation, overcrowd cage, food or water deprivation, foot shock, tilting, fear, for 14 days before the cFA or vehicle injection and 12 days thereafter (PS-C and PS-AA). In FR series the stress was induced by a chronic 40 per cent food restriction. Control, AA, both with free access to food and water, FR and FR-AA groups of rats were used. All rats were killed 22 days following cFA injection. Psychological stress induced depressed exploratory activity as measured by the method of elevated +maze at the end of the stress period. At the end of the studied period serum corticosterone (CORT) did not differ between VC and PS groups, it was elevated in AA and to the same extent in PS-AA group. AA-induced hind paw swelling was significantly potentiated in PS-AA group. The reduced albumin levels were similar in AA and PS-AA groups. On the contrary FR associated with chronic CORT elevation prevented hind paw swelling and restored reduced albumin and enhanced NO serum levels found in AA. These results show that FR mitigated and PS worsened the development of adjuvant arthritis and underline the specific effects of different stress exposures.

Rőth E¹, Jaberansari MT¹, Jancsó G¹, Borsiczky B¹, Kiss K², Szeberényi J²

We have previously demonstrated that subthreshold (2x2 min) preconditioning (PC) can confer delayed protection if pigs were pretreated with the ACE inhibitor perindoprilat (P). We now aim to confirm that P acts via inhibition of bradykinin breakdown, thus augmenting subthreshold PC stimuli. Using PTCA, pigs were subjected on day 1 to sham or ischemic PC protocols. On day 2 all animals underwent 40-min LAD ligation and 3 hours of reperfusion, followed by infarct size analysis using TTC staining. Furthermore we investigated possible modulation in binding activity of nuclear factor (NF)-κB and activator protein (AP)-1 by electrophoretic mobility shift assay (EMSA). Group I was sham operated (control). Group II was preconditioned with 4x5 min LAD occlusion, leading to almost 50% limitation in infarct size vs. controls (p<0,05). 2x2 PC stimuli (Group III) did not confer significant protection. Treatment with P (0,06 mg/kg, IV) in conjunction with 2x2 PC (Group IV) however significantly limited infarction (p<0,05). Pretreatment with Hoe140 (bradykinin receptor antagonist) prior to treatment with P and 2x2 PC (Group V) completely abolished the protection. Drug control studies did not show significant protection. NF-κB and AP-1 investigations correlated with protection against infarction. The results demonstrate increased bradykinin activity via ACE inhibition can augment subthreshold PC, perhaps through downstream NF-κB and AP-1 activation, conferring delayed protection.

Prolactin (PRL) together with steroid hormones is an important factor in neuroendocrine regulation of immunity and plays a role in the pathogenesis of autoimmune disorders. PRL release increases during gravidity, plasma concentration reaches maximal level at the third trimester and continues in post-partum period in relation with breast-feeding. Decrease of pituitary and placental production of immunosuppressive corticoids after delivery and prolonged augmented production of immunostimulatory PRL increases the risk of onset of rheumatic disease.

Increased plasma PRL levels were found in one third of the patients with SLE, Rheumatoid Arthritis (RA), Sjogren’s Syndrome and in other rheumatical diseases and co-relation between increased plasma PRL and disease activity was reported occasionally. Our study showed worsed response to basic therapy and increased need of corticosteroids in patients with higher basal plasma PRL levels.

Mechanism of elevated PRL release in patients with rheumatic diseases is not very clear yet. Central regulation of PRL can be geneticaly determined or inflammatory cytokines induced.

We have not found significant difference in PRL secretion in SLE patients neither on pituitary level nor on hypthalamic level comparing to gender and age matched control group. On the other hand, patients with RA showed normal PRL release on pituitary level but decreased PRL response on stimulus mediated on hypothalamic level.

Rybalko N, Mazelova J, Syka J

Gap detection threshold (GDT) was tested in eight adult (8-13 months) female pigmented rats (strain Long-Evans) by an operant conditioning technique with food reinforcement. Control experiments found that the GDT depended on the spectral characteristics and intensity levels of the continuous noise in which the gaps were embedded. The mean values of GDT measured in white noise of 70 dB SPL were significantly smaller than GDT in low-frequency noise of the same intensity and amounted to 1.57±0.07 ms and 2.9±0.34 ms, respectively (p< 0.05). Decreasing the noise intensity from 80 dB SPL to 20 dB SPL produced an increase in GDT. Bilateral ablation of the primary auditory cortex (complete destruction of Te1 and partial destruction of Te2 and Te3 areas) resulted in an increase in GDT values. GDT retesting conducted five days after ablation showed that all rats were able to discriminate gaps in noise; however, the number of their false alarm reactions increased. The values of GDT amounted to 250-300% of preoperative levels and reached on average 4.2±1.1 for white noise and 7.4±3.1ms for low-frequency noise. During the first month after cortical ablation, recovery of the GDTs was observed. However, GDTs remained slightly higher than control data (1.8±0.18 for white noise, 3.22±0.05 for low-frequency noise, p<0.05). A decrease in GDT values during the subsequent month was not observed.

Rychkova N, Micutkova L, Krizanova O, Kvetnansky R

Adrenal medulla is the main source of catecholamines: dopamine, noradrenaline and adrenaline. It synthesizes all enzymes of catecholamine biosynthesis: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). It is known that gene expression of those enzymes increases under the influence of stress.

The aim of this work was to establish quantitatively the effect of single and repeated immobilization stress on mRNA levels of TH, DBH and PNMT in adrenals of rats by RT-competitive PCR method. In RT-competitive PCR, a constant amount of transcribed RNA is mixed and co-amplified with fixed amounts of a synthetic competitor that differs from the target cDNA.

After the single immobilization the amount of mRNA of individual enzymes has been increased. The maximal increase of mRNA levels of TH, DBH, PNMT was observed 3 hours after the termination of stress stimulus. TH gene expression increase has been 10-fold, DBH - 2.6-fold and PNMT - 8-fold. 24 hours after the termination of the first exposure to immobilization returned to levels in unstressed controls. The last immobilization did not increase the mRNA levels of all these enzymes.

In our work we quantified the amount of mRNA of catecholamine synthesizing enzymes in adrenal medulla. Our results indicate: 1) the ratio of TH mRNA was much lower than PNMT mRNA; 2) during the single immobilization stress the TH gene expression achieved the highest level compared to PNMT and DBH gene exspressions.

ELECTROPHYSIOOGICAL ALTERATIONS IN VENTRICULAR MYOCYTES INDUCED BY OXIDIZED LDL
Schaffer P¹, Pelzmann B¹, Bernhart E¹, Lang P¹, Zorn-Pauly K¹, Ledinski G², Greilberger J³, Jürgens G², Koidl B¹

It was our aim to investigate effects of human LDL, copper-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs. LDL oxidation was performed for 2-24 h and the content of total lipid hydroperoxides (LPO) was estimated. Guinea pig ventricular myocytes were kept under cell culture conditions (at 37°C) and incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h. Afterwards myocyte damage, action potentials and transmembrane ionic currents were studied and compared with control myocytes. All recordings were made using the patch-clamp technique in the whole-cell recording mode at a temperature of 37°C. Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect on myocyte viability. Myocyte damage did not increase with duration of LDL oxidation, but depended on the content of total LPO, which reached a maximum value in moderately (4h) oxidized LDL. Ox-LDL induced intense electrophysiological effects including: prolongation of action potential duration; depolarization of resting membrane potential; spontaneous activity; generation of afterdepolarizations and modification of transmembrane ionic currents. In conclusion, this study showed that ox-LDL was able to induce both, severe myocyte damage and irregular electrical activity in isolated ventricular myocytes. The mechanisms of the myocyte damage are not clear at present.

Schmid D¹, Plass C¹, Loew HG², Spieckermann G¹

Introduction: At higher stimulation frequencies contraction force declines after an initial increase in rat left ventricular papillary muscle (negative staircase). However, the reasons for this phenomenon are largely unknown. Rat left ventricular papillary muscle represents a model with long diffusion distance for oxygen and substrates. Our hypothesis is that frequency associated changes in energy balance may lead to activation of ATP-sensitive potassium (K_ATP ) channels and reduction of action potential duration (APD₅₀) mediating reduction in contraction parameters.

Methods: Rat left ventricular papillary muscles were mounted in a vertical organ bath superfused with Krebs-Ringer-Solution at 29°C. Cardiac action potentials were recorded using conventional intracellular glass microelectrodes simultaneously with isometric force.

Results: Glibenclamide (Glib, a specific K_ATP blocker) induced a transient increase of developed force from 4.2 mN to 5.5 mN at 1.2 Hz with a maximum after 20 min, thereby abolishing negative staircase. There is no effect at 0.2 Hz. APD₅₀ was correspondingly prolonged from 44 to 56 ms after addition of glibenclamide, At 0.2 Hz APD₅₀ was not changed significantly after addition of Glib (35 vs. 36 ms)

Conclusion: Glibenclamide-sensitive force and APD₅₀ reduction at higher frequencies suggests endogenous K_ATP activation probably constituting a protective mechanism at diffusion limited conditions.

Schwendt M, Pirník Z, Dunčko R, Ježová D

Receptors for glutamate are thought to be involved in a variety of higher brain functions including learning, cognition, stress response, reward and addiction. Nevertheless, little is known about the functional modulation of glutamate receptors at the molecular level. The aim of the present study was to investigate molecular changes of glutamate receptors in situations involving reward mechanisms namely during spontaneous anhedonia and after acute opiate exposure. Gene expression of NMDA and AMPA receptor subunits was measured in specific brain regions implicated in reward, addiction, stress and memory. Furthermore, glutamate receptor in the adrenals known to be involved in the stress response, were evaluated as well. The experiments were performed on adult male or female rats. Hedonic behavior was assessed by measuring intake rate of sweet solution (sucrose preference test). Morphine was administered as a single injection (10 mg/kg s.c.) and its biological activity was shown in Straub-tail test in mice. Using RT-PCR technique, it was found that spontaneous anhedonia (low intake of sucrose) is accompanied by a decrease of NR1 receptor subunit mRNA levels in the ventral tegmental area and increased GluR1 mRNA in the hippocampus. In another experimental series, single morphine injection up-regulated NR1 mRNA in the adrenals 24h later. Surprisingly, no changes were detected in the hippocampus. In conclusion, modulation of glutamate receptor subunit gene expression might represent one of neurobiological mechanisms underlying spontaneous anhedonia rats and development of addiction.

Sedláček M, Vyklick_ Jr., L

Primary afferent fibers transduce nociceptive stimuli to sensory information that is conveyed to neurons located in the dorsal laminae of the spinal cord. The aim of the present study was to characterize synaptic responses induced in lamina I neurons by stimulation of dorsal roots. Lamina I neurons were visualized in the hemisectioned spinal cord with attached dorsal roots prepared from 12-15-day-old rats using IR/DIC microscopy. The neurons formed a thin layer of cells adjacent to the tract of Lissauer. Whole-cell patch-clamp technique was used to record current responses induced by electric stimulation of dorsal roots. Four types of postsynaptic responses were observed: (i). spontaneously occurring excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs); (ii.) EPSCs induced by electric stimulation of dorsal roots that were characterized by constant latency after stimulation, fast kinetics and sensitivity to AMPA receptor antagonists; (iii.) mixed EPSCs/IPSCs induced by electric stimulation of dorsal roots and characterized by variable latency (occurring mainly within 50 ms after the stimulus) fast kinetics 2 — 25 ms and sensitivity to both AMPA and GABA_A receptor antagonists; (iv.) EPSCs induced by stimulation of dorsal roots and characterized by a very slow kinetics (half decay � 1 s).

The results of our experiments suggest complex synaptic input to lamina I neurons induced by primary afferent stimulation —monosynaptic (presumably glutamatergic), polysynaptic (glutamatergic, GABAergic and glycinergic) and slow transmission (with yet unidentified neurotransmitter).

Sendur R, Pawlik MW, Biernat J, Obuchowicz R, Pawlik WW

The aim of this study was to evaluate the possible protective effects of the inhibition of Renin Angiotensin System against ischemia/reperfusion (I/R) induced gastric mucosa injury in rats.

Experiments were performed on 60 fasted anesthetized Wistar rats. Systemic arterial blood pressure (AP) and gastric mucosal blood flow (GMBF) were measured. The area of gastric lesions was determined planimetrically. In all experimental groups the gastric mucosal I/R lesions were induced by clamping the celiac artery for 0,5 h followed by 3h of reperfusion.

Ischemia alone failed to induce gastric lesions but when it was followed by 3h of reperfusion, gastric erosive lesions occurred. The GMBF after release of total ischemia was reduced by 59±8% (p<0,05)i n comparison to preocclusive value. After 3h of reperfusion the GMBF was reduced by 27±6% (p<0,05) and acute gastric mucosal lesions area was 17±3 mm². After pretreatment of animals with ACE-inhibitor Lisinopril (MSD)(1,0 mg/kg i.p.) GMBF at the beginning of reperfusion period was reduced by 36±7% and after 3 h of reperfusion was reduced by 18±3%. Acute gastric mucosal lesions area was reduced by 39±5% (p<0,05) in comparison to the vehicle treated group. AII receptor blocker Losartan (MSD) (1,0 mg/kg i.p.) significantly reduced mucosal lesions area by 37±6% and GMBF was reduced by 32±5% after gastric ischemia was completed, and by 17±4% at the end of reperfusion.

_evecová D¹, Čalkovská A¹, Javorka K¹, Javorka M¹, Drgová A², Petrá_ková M¹

During HFJV can be moveable substances directed into the lungs (inpulsion regime) or outside of the airways (expulsion regime) according to the duration of inspiration and expiration phases of respiratory cycle. We compared the effects of lung lavage by means of HFJV regimen and conventional ventilation (CV) on gas exchange, right-to-left pulmonary shunts (RLS) and recovery of instilled meconium in rabbits after meconium aspiration.

Methods: Anesthetized, paralyzed, tracheotomized and cannulated adult rabbits were used in experiments. Suspension of human meconium was instilled into the tracheal tube. When respiratory failure developed, saline lavage was performed during HFJV or CV. Animals were further ventilated for 2 hours with either HFJV or CV.

Results: Administration of meconium caused a significant drop in gas exchange and lung compliance in both groups. HFJV improved gas exchange and decreased RLS in comparison with CV. In the amount of removed meconium there was no significant difference between groups.

Conclusion: Although HFJV led to improvement of gas exchange and reduction of right-to-left shunts, combination of inpulsion and expulsion regimen of HFJV was not more effective in removal of meconium than CV.

Autoimmune diseases are conditions in which the immune system damages normal components of the individual. Initially it was thought that autoimmune disease was the inevitable outcome of the presence of clones of lymphocytes with receptors that recognize self-antigens. Thus tolerance to self, the state of non-autoimmunity, was due to the absence of self-recognizing lymphocytes, the 'forbidden' culprits of autoimmune disease. Autoimmune diseases were found to be multifactorial in their etiology. For practical reasons these factors are classified into four categories: Genetic, which entail the MHC class I, II, and III. A case in point will be the haplotypes of HLA-DR3, B8 which are prevalent in many classical diseases. In addition Gm allotype, idiotypes and some complement deficiencies may have a genetic background. Immune deficiencies: C₁q C2, C4 and IgA deficiencies are among the most common defects associated with diverse autoimmune conditions. Hormonal state, most autoimmune diseases are detected in females at the child bearing ages. The role of estrogens will be delineated. In addition other hormones play a role i.e. prolactin. Environmental causes: Those are the most important as a trigger factors determining the time and type of disease. They entail infectious agents, chemicals, drugs and even vaccines.

The type of disease in an individual, in an autoimmune prone family, will be determined by the specific combination of the different factors mentioned above.

A special case report of a 45 years old lady (HLA DR3, B8) who developed 8 autoimmune diseases and 10 different autoantibodies, upon accidental exposure to a chemical, compound (a polyclonal activator), will be detailed.

Simko F, Matuskova J, Luptak I, ¹Pechanova O, ²Stvrtina S, ²Babal P

&#Introduction: 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce cardiovascular mortality associated with hypercholesterolemia. Yet, the mechanism of this beneficial effect is far from being clear.

&#Material and Methods: We investigated the effect of simvastatin on the heart remodeling in the model of L-NAME induced hypertension. Four groups of Wistar rats were investigated: control, simvastatin (10mg/kg), L-NAME (40 mg/kg) and L-NAME + simvastatin. Animals were killed after 6 weeks of experimental manipulation.

&#Results: Systolic blood pressure, LV weight, DNA concentration in the LV and thickness of aortic wall were increased, while NOS activity in the LV, kidney and brain was decreased in L-NAME treated rats. Addition of simvastatin to L-NAME prevented partly the hypertension development and diminution of NOS activity in the kidney and brain. Nevertheless, simvastatin did not prevent either the LVH development, aortic wall thickening or DNA concentration enhancement and fibrosis development of the LV.

&#Conclusion: HMG-CoA reductase inhibitor simvastatin prevented in part hypertension development, yet without any effect on the remodeling of the left ventricle in NO-deficient hypertension.

_imonová Z^1,2, Lai L-J³, Bjelke B³, Syková E^1,2 

Fluoxetine is a widely used antidepressant which inhibits serotonin re-uptake and acts when administered chronically. It was also reported to enhance the number of granule cell precursors in the dentate gyrus of adult rats (1). We studied the effect of fluoxetine pretreatment on cell proliferation after a photochemical lesion. Rats used in the study were divided into four groups: one group was treated with fluoxetine for 14 days at a total dose of 50 mg per animal, the second group was subjected to beam walking for 14 days, the third group received both treatments and the fourth group was untreated. On the 15th day, a focal lesion in the right sensori-motor cortex was induced photochemically using Bengal Rose as a photosensitive dye. On the 19^th day, bromdeoxyuridine (BRDU) was injected a total of four times, at a dose of 75 mg/kg every two hours. Rats were killed on the 20th day and processed immunohistochemically. Neurons were detected by NeuN antibody, astrocytes by anti-GFAP, and dividing cells by anti-BRDU. Beam walking enhanced the number of BRDU positive cells, mostly neurons, in the subgranular zone in the dentate gyrus. Fluoxetine induced a massive migration of dividing cells, both neurons and astrocytes, to the whole ipsilateral hemisphere, particularly in layers II and III, and to the vicinity of the lesion. This study supports a role for serotonin in neurogenesis.

.(1) Manev H. et al.: Eur. J. Pharmacol. 411(2001)67-70.

The content of releasable calcium in cardiac cells appears to be maintained constant at a frequency dependent level by a negative feedback modulation of sarcolemmal calcium transport via Ca channels and Na-Ca exchange. A mathematical model of Ca-turnover was designed that explicitly incorporates this (Ca²⁺ transient mediated) feedback mechanisms. The model operates with the amounts of Ca transferred among compartments during partial phases of cardiac cycle which are expressed as time integrals of Ca-fluxes or measurable ionic currents. Most processes were approximated by simple (mostly exponential) functions and the integrals could be replaced by functions of variable parameters. The model thus contains both discrete quantities and continuous quantities calculated during each cycle as a numeric solution of differential equations. Although the model is rather uncomplicated it still retains the physiological meaning of all parameters involved.

The primary objective of the model was to get a better insight into the frequency-dependence of electrical and mechanical behaviour of the heart. Its input is an arbitrary sequence of intervals between excitations. The model makes it possible to simulate the known effects of cardiotropic drugs and agents or to predict their unknown mechanisms by visualizing the changes in individual Ca compartments. By altering the individual parameters the model simulates also the well known prominent species differences in the frequency effect and tissue differences (atria vs. ventricles).

Slavíček J¹, Trojan S¹, Trefn_ Z², Kittnar O¹, Dohnalová A¹ 

Most of works study the influence of drugs (statins) on the decrease in risk factors of cardiovascular diseases, while the lifestyle is less investigated (1,2). The aim of the present work was to ascertain if the 6 days-lasting stay NEW START (low fat-low energy vegetarian diet, physical activity, without smoking, without alcohol and coffee) is able to decrease some risk factors of cardiovascular diseases. In 308 volunteers 50 years — old (73 men, 235 women) the body weight, BMI, blood pressure, heart rate, serum cholesterol and blood glucose were measured before and after stay. Body weight decreased (n=239) from 71.0 to 70.6 kg (p<0.0001), BMI (n=224) from 24.7 to 24.6 (p<0.001), blood pressure syst. (n=243) from 126.4 to 122.6 mm Hg (p<0.001), blood pressure diast. (n=242) from 79.9 to 77.2 mm Hg (p<0.001), serum cholesterol (n=184) from 5.01 to 4.32 mmol/l (p<0.0001), blood glucose (n=103) from 4.60 to 3.78 mmol/l (p<0.0001), heart rate (n=218) from 72.9 to 71.5 (p=NS). Our results showed, that the short lasting stay NEW START containing low energy diet and physical activity significantly decreased some risk factors of cardiovascular diseases in 50 years — old volunteers.

Hooper L. et al.: Brit. Med. J. 322, 757-763, 2001

Fraser, GE, Shavlik D. J.: Arch. Intern. Med. 161, 1645-1652, 2001

Slavíková J¹, Kuncová J¹, Reischig J²

The present study was aimed to reveal putative developmental interrelationships among three types of cardiac neurons: noradrenergic sympathetic neurons, dopaminergic small intensely fluorescent (SIF) cells and sensory neurons. After birth, animals were subjected to sympathectomy by guanethidine, sensory denervation by capsaicin and combined sensory + sympathetic denervation. Intact rats served as controls. At the age of 40 days, concentrations of noradrenaline (NA), adrenaline (A) and dopamine (DA) were determined in tissue extracts of right and left atria (RA, LA) using commercial radioimmunoassay method and expressed in ng/g wet weight.

In control rats, NA and A concentrations were higher in RA than LA, whereas DA levels were higher in LA than RA, suggesting multiple neuronal origin of DA.

Sympathectomy led to at least 95% reduction of NA and A concentrations in the atria while DA levels decreased only slightly. Thus the SIF cells seem to be unaffected by guanethidine. In contrast, sensory denervation led to a significant increase in all catecholamine concentrations in both atria. While DA levels increased 6 — 8 fold, NA and A concentrations augmented by factors 1,2 — 1, 9 only.

In conclusion, sensory innervation may have an inhibitory effect on development of the sympathetic postganglionic neurons. Removal of the sensory input to SIF cells seem to increase DA concentrations independently on NA and A levels in both atria.

_majda B, Paulíková E, Slivková E, Lacková M

Male Sprague-Dowley rats were irradiated with a whole-body dose of 6 Gy of gamma-rays. The irradiation caused a significant increase in paw-licking response latency in the hot plate test performed 30 min after irradiation. The count of blood lypmhocytes and the hematocrit value were decreased in irradiated and hot plate tested animals. The administration of 4 mg/kg b.w. (i.p.) naloxone 15 min before testing had no effect on paw-licking latency in irradiated animals, but enhanced the eosinophils and neutrophils in irradiated and the number of blast cells in sham-irradiated animals.

Administration of 8 mg/kg of naloxone reversed the analgetic effect of radiation in hot-plate test and increased the phagocytic activity and phagocytic index without changes in the number of phagocytes in irradiated and also in sham-irradiated rats given naloxon.

These findings support the hypothesis, that opioids play an important role in radiation-induced analgesia in rats and could modify the reaction of the immune system to stress.

Spät A, Tóth A, Makara JK

The most important stimuli of aldosterone secretion by adrenal glomerulosa cells are extracellular K⁺, angiotensin II and ACTH. Early in vivo animal experiments suggested that changes in plasma osmolarity may influence aldosterone secretion as well, however, the cellular mechanism of this effect remained unknown.

Alteration of osmolarity by sucrose addition in the 250-330 mOsm range did not influence basal aldosterone production by cultured rat glomerulosa cells. However, hyposmosis increased the hormone response evoked by raising [K⁺]_o from the control 3.6 to 5 mM, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic [Ca²⁺] of single glomerulosa cells was not affected by hypo- or hyperosmotic exposure but the Ca²⁺ signal evoked by elevation of [K⁺]_o to 5 mM was augmented by hyposmotic solution. The osmosensitivity of the T-type and L-type voltage-gated Ca²⁺ currents was studied using the nystatin-perforated voltage-clamp technique. Hyposmosis (250 mOsm) increased the amplitude of the T-type current within 20 seconds and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosM) reduced L-type current amplitude. Fluorimetric measurement of cell area after calcein loading showed changes in response to changing osmolarity to 250 or 350 mOsm within a few seconds. Raising [K⁺]_o to 8.6 mM also increased cell area, moreover, some cells already responded with swelling to [K⁺]_o elevation by 1 mM only.

Spi_ák B, ¹Spi_ák B jr., ²Petrá_ová D

According to literary date, pets (cat, dogs and other animals) keeping indoor in the last years increase. We suppose, that therefore will increase allergy to pets.

The goal our work was to determined total and specific IgE antibodies again cat and dog epithelium and skin prick tests.

Material and methods. Total IgE and specific IgE againts cats and dogs allegens were examined in 50 patients with repiratory allergosis. Average age was 7,9 years. Total IgE in the serum was examined by the method Elisa fi Immunotech, specific IgE antibodies by the set Allergocoat Sanofy, Pasteur Inc. Positive skin prick test we take measure induration average more than 3 mm.

Results. Total IgE in the groups with keeping pets indoor we were found in average 227,18 g/l and patient without keeping ones in average 192,6 g/l. Specific IgE in 25 children with keeping dogs or cats indoor were found in 21 children (84%). In 4 (16%) children were negative. Skin prick test in this group was positive in 15 patients (60%), negative in 10 (40%). In 25 children without keeping pets indoor we found specific positive IgE in 17 (68%)children and in 8 (22%) patients negative. Positive skin prick tests we were found no patient, negative in 25 patients.

Conclusion. Explanation interesting findings between specific IgE and skin-prick test in patient with keeping pets indoor will need next studium.

Stančák A¹, Vrána J¹, Poláček H¹, Rachmanová R¹, Králík J¹, Tintěra J²

We analyzed in two studies (1) the cortical changes related to five different intensities of electrocutaneous stimulation of the right-finger (13 subjects), and (2) differences in hemodynamic (fMRI) and EEG responses following painful and innocouous intracutaneous right-finger stimulation (10 subjects).

The innocuous stimuli were followed by desynchronization of the EEG alpha (8-12 Hz) and beta (16-32 Hz) rhythms. The cortical desynchronization was larger over the contralateral as compared to ipsilateral sensorimotor hand area, and differentiated the stimuli of weak and strong intensities. The sources of the somatic evoked potentials were found in S1, contra- and ipsilateral S2 and in SMA. The peak amplitudes of the EEG sources changed in parallel with stimulus intensity.

Pain-related fMRI activation was found in the right and left insula and S2, contralateral thalamic ventrobasal nuclei, ipsilateral cerebellar cortex and vermis, and in the cingulate gyrus. EEG sources were localized in the right and left parietal operculum, cingulate sulcus, and precentral gyrus. Painful stimuli as compared to innocuous stimuli were followed by a strong contralateral synchronization of beta rhythms over the lateral frontal cortex.

CALCIUM RELEASE IN PORCINE PURKINJE CELLS
Stankovicova T^1,2, Sipido RK¹

1Lab Exp Cardiol, Katholieke Univ Leuven, Leuven, Belgium and ²Dept Pharmacol, Comenius Univ, Bratislava, Slovak Republic

The primary function of cardiac Purkinje cells is fast conduction of the impulse, rather than generating contractile force. They are known to have a different cellular architecture with a less dense myofilament network. EC coupling in these cells is expected to be different from that in myocytes of the working myocardium.

In the present study, we have examined the spatial characteristics of [Ca²⁺]_i in single enzymatically isolated Purkinje cells of pig hearts using whole-cell patch clamp technique and laser scanning confocal microscopy. Staining with di-8-ANEPPS showed clearly surface membrane of the Purkinje cells lacking transversal tubules, which are typical for striated working cardiomyocytes.

Under voltage clamp conditions, depolarization of Purkinje cells to 0 mV induced an inward current followed by an increase of the Ca²⁺-dependent fluorescence. Transverse confocal line-scan images revealed simultaneous increase of fluorescence in regions just below the membrane, spreading across the cell with delay and attenuation in the central part of cytoplasm giving the picture of U-shaped profile.

Application of caffeine induced a spontaneous transient rise of Ca²⁺ transients accompanied by a transient current. The line-scans showed that Ca²⁺ increase started in one region of the cells and propageted along the entire cell as a Ca²⁺ wave.

_timmelová J, _vorc P, Bračoková I

Hypoventilation (HV) decreases the electrical stability of the heart and probably produces the substrate for arrhythmogenesis. Therefore the aim of this study was to investigate the effect of HV on time intervals and amplitudes of ECG in the dependence on the alternation of light and dark in female Wistar rats.

The experiments were performed in the anesthetised (ketamine-100mg/kg i.m. + xylazinum-15 mg/kg i.m, Léčiva, Praha) female rats and adapted to the light-dark regime 12:12 hours. The rats (light n=8, dark n=8) were subjected gradually to the following experimental interventions and results were divided into: 1. group of results without artificial ventilation, 2. group of one (after tracheothomy and thoracothomy with 5 min. of period of stabilization with artificial ventilation (parameters of ventilation: 40 breaths/min., 1 ml/100 g). 3. group of one from 20 min. HV (parameters of ventilation: 20 breaths/min., 0,5 ml/100 g). The ECG parameters were analysed from the II. limb lead of ECG. The significance of differences were evaluated by Student´s t-test.

At the comparison of time intervals of ECG from light and dark phases, only the duration of QT and QTc intervals were significantly (p < 0.05) prolonged in the dark phase against the light one. The PQ and RR interval durations were decreased, but not on the significant level. There were no differences in the wave amplitudes in this comparison.

Our results show that HV in the dark (active) phase of the rat regime day increases the vulnerability of the heart to the ventricular arrhythmias from the electrophysiological point of view.

Many studies have been carried out to delineate the impact of the sympathetic and sensory nervous system in arthritis. In animal studies, alpha2-adrenergic and A1 adenosine receptor mechanisms are involved in sensitization of primary afferent sensory nerve fibers leading to proinflammatory substance P release into the lumen of the joint. On the other hand, other studies indicated that experimental arthritis is attenuated by administration of beta-adrenergic agonists. In this respect, the sympathetic nervous system plays a dual role which presumably depends on the ligation of sympathetic neurotransmitters at either alpha2 or beta-adrenoceptors and A1 or A2 adenosine receptors, respectively. The differential effects of sympathetic neurotransmitters (norepinephrine, epinephrine, adenosine; see above) fit well to the concept that stimulation of beta-adrenoceptors or A2 adenosine receptors increase intracellular cAMP which is antiinflammatory.

In a recent extensive study in long-term RA patients (mean duration 10 years), we found a highly significant reduction of sympathetic nerve fibers in RA as compared to OA patients. It may be that reduction of sympathetic nerve fibers in the chronic disease leads to uncoupling of the local inflammation from the antiinflammatory input by the CNS. The direct comparison of substance P positive fibers and sympathetic fibers revealed a preponderance of approximately ten to one for primary sensory afferents as compared to sympathetic efferents which depends on local inflammation. In conclusion, such a preponderance may lead to an unfavorable proinflammatory situation supporting the disease process of RA.

_trbák, V¹, Najvirtová, M¹, Benick_, J¹, Mátéffyová A¹, Baqi, L¹, Greer MA²,

Cell swelling induced by various techniques (hyposmotic medium, isosmotic concentration of permeants e.g. ethanol, urea) evokes an immediate high amplitude secretory burst of hormones stored in secretory vesicles with dynamics indistinguishable from those induced by specific secretagogues. We have evaluated sensing and signal transduction mechanisms involved in this phenomenon. In contrast to most types of regulated secretion, that induced by cell swelling in normal cells does not require a rise in intracellular Ca²⁺ through opening L-type Ca²⁺ channels. Using various tissues (pituitary, pancreatic islets, hypothalamic structures, brain septum) we found that hormone secretion induced by cell swelling is not depressed by inhibition of stretch mechanoreceptors (10-20 mM GdCl₃), mercury-sensitive aquaporins (100 mM HgCl₂), Na⁺ or K⁺ channels, and it is not associated with a rise in cAMP, IP₃, or prostaglandins. Participation of such a general biophysical phenomenon in physiological reactions rises question of specificity. In most cells volume expansion induces non-specific exocytosis from secretory vesicles. However, the reaction of neurons controlling osmoregulation is more restricted (e.g. isosmotic ethanol stimulated TRH but not oxytocin secretion by the PVN and posterior pituitary).

In conclusion: Cell swelling induced by extracellular hyposmolar challenge or permeants in isosmotic concentration consistently stimulate peptide secretion. This regulated exocytosis follows a different transduction pathway than that delineated for other secretagogues as well as for cell volume regulation.

Sulkova I¹, Hubka P³, Benedekova M², Hulinova S², Camborova P¹

Gastrointestinal motility is coordinated by oscillation of transmembrane potential in smooth muscle cells. Such oscillations are paced by interstitial cells of Cajal (ICC) and can be registered noninvasively using electrogastrography (EGG). Our study was aimed on developmental changes in EGG pattern in neonates after birth.

EGG was recorded in 6 healthy full-term (9-30 days after birth) and in 16 prematurely (27th-36th week of gestation) born neonates (recorded 3-67 days after birth) from 5 cutaneous Ag-AgCl electrodes (Hewlett-Packard) placed on the epigastrium.

In all full-term neonates we have observed stable activity in frequency range 2-4 cycles per minute (cpm) already 9 days after birth(3,24 ± 0,34 cpm). Contrary, no dominant stable activity was found in the group of prematurely born neonates except of one.

Electrogastrographical image of postnatal spontaneous electrical oscillations, was qualitatively different in prematurely born neonates in comparison to full-term ones. No stable dominant frequency was found in pre-term neonates few weeks after birth. Our results suggest that appearance of adult-like activity in EGG of prematurely born neonates is postponed following birth in comparison to the full-term ones.

Sumová A, Jáč M, Sládek M, Illnerová H

Animals were entrained to photoperiod with either 12 h (LD12:12), 16 h (LD16:8) or 8 h (LD8:16) of light and released into constant darkness. In another experiment, either a 9-hour light pulse starting before midnight or melatonin/vehicle at the time which corresponded to circadian time (CT) 10 was administered to one group of animals entrained to LD12:12, while the other groups of animals remained untreated as controls. Using free floating immunohistochemistry, a rhythmic production of PER1-immunoreactivity (ir) was followed during the next cirkadian cycle.

Su_ánková K, Lyfenko A, Vlachová V, Teisinger J, Vyklick_ L

The vanilloid receptor (VR1) is expressed in a sub-population of small DRG neurones in mammals and serves as the common transducer of the pain producing stimuli. VR1 exhibits sensitivity to noxious heat (43-52°C), acids (pH₅₀~5.5) and capsaicin (EC₅₀ ~0.3 µM). Little is known about the mechanisms by which this channel translates noxious stimuli into structural rearrangements leading to altered gating. The existence of three cysteine residues located at positions 616, 621 and 634 of VR1 led us to explore whether the conformational stability of VR1 can be regulated by redox active substances.

Here we report that dithiothreitol (DTT) (2— 60 mM), an agent that maintains —SH groups in a reduced state, greatly facilitates whole cell membrane currents induced by noxious heat and capsaicin (1 µM) in cultured rat DRG neurones of small diameter (< 20 µm) and in VR1 transfected HEK293 cells. The effects of DTT are concentration dependent and fully reversible. In DRG cells, the presence of 30 mM DTT increased the average inward membrane current induced by 1 µM capsaicin from 187 ± 53 pA to 380 ± 87 pA at 25°C (n = 8). At 45°C, the average increase was from 1260 ± 345 pA to 2130 ± 562 pA (n = 8). In VR1-HEK293 cells, the average ratio of the capsaicin induced current in the presence of 2 mM DTT as compared to the control capsaicin response significantly increased by 9.1 ± 5.8 and 1.5 ± 0.2 at 25°C and 45°C, respectively.

It is suggested that the ratio between free —SH groups and disulfide bonds of the cysteine residues of VR1 presets sensitivity of primary nociceptors to algogens and may represent a potential therapeutic target for treating specific pain states in humans.

_ustrová I, Vaněček J

The endogenous pacemaker of mammalian circadian system is located in the suprachiasmatic nucleus (SCN) of hypothalamus. Arginine-vasopressin (AVP) is the most abundant peptide secreted by the SCN neurons located in the dorsomedial subdivision of the nucleus.

We have studied effects of the pineal hormone melatonin on AVP release from organotypic SCN slice cultures. The spontaneous AVP release is low during the subjective night and peaks in the middle of subjective day. The rhythm persists for about 4 weeks in vitro. Endogenous period of the AVP rhythm in our cultures ranges between 23 — 24 h. The cultures were treated with Melatonin 10nM for 4-h period starting at ZT 2,6,10,14,18 and 22.

Melatonin 10 nM phase-shifted the AVP rhythm. When applied at zietgeber time (ZT) 2 and 22 melatonin delayed AVP rhythm, at ZT 6 and 14 it advanced AVP rhythm an at ZT 10 and 18 it had no effect. The phase-shifts persisted for 3 days after withdrawal of melatonin.

_uta D, Kva_ňák E, Popelář J

, Syka J

The medial geniculate body (MGB), the thalamic auditory nucleus, is usually divided into three subdivisions: ventral, medial and dorsal. The activity of 240 MGB neurons was recorded extracellularly in 28 guinea pigs anaesthetized by a ketamine-xylazine mixture.

The responses to pure tone stimulation at the neuronal characteristic frequency (CF) were recorded and afterwards divided into six PSTH patterns: onset, sustained, onset-sustained, ‘chopper’, ‘pauser’ and inhibitory responses. In the whole MGB, more onset and ‘chopper’ responses than sustained were found. Onset responses prevailed in the ventral MGB, whereas in the dorsal MGB ‘chopper’ responses were more often present.

The hearing threshold, given by the envelope of an area containing the thresholds of neurons at the CF, was found not be significantly different among the MGB subdivisions. The lowest average thresholds and the shortest average latencies were found in the ventral MGB, considered as a primary auditory MGB subnucleus. In the ventral and dorsal MGB the highest Q₁₀ values, related to the sharpest neuronal tuning curve, were found. These findings are consistent with the primary auditory role of the ventral subdivision of the MGB.

In summary, the MGB, similarly as the inferior colliculus (IC), is organized into three subdivisions, with the ventral MGB characterized by the quickest onset reaction with the lowest thresholds to auditory stimulation.

Svoboda J¹, Králík J², Sovka P², Stančák A¹

We analyzed the in§uence of force of right index Þnger isometric extension on EEG coherence.

Twelve healthy right-handed subjects (age 23.4±2.5 years) performed isometric extension of the right index Þnger (~2.5 min) at different loads (0, 100, 200 and 300g). Monopolar EEG (82 channels) was recorded and the Laplacian operator method was applied to Þlter out the low spatial frequencies. The coherence functions were computed in the 8—13 Hz band for all combinations of EEG channels. The monotonic changes in pair-wise coherence were tested using regression analysis.

An increase of coherence paralleling load was observed between the contra- and ipsilateral S1/M1, contralateral S1/M1 and SMA, and between contralateral S1/M1 and the posterior parietal cortex. The above changes of EEG coherence were conÞrmed using analysis of variance for repetead measures.

The results suggest enhanced coupling between the primary and higher-order motor cortical areas during increased motor activation.

_vorc P, _timmelová J, Bračoková I, Kassayová K

Extrasynaptic transmission plays an important role in communication between neurones, axons and glia. It is mediated by the diffusion of neuroactive substances in the extracellular space (ECS). In this way, transmitters can reach high-affinity receptors located outside synapses and on glia. Cell volume changes, the structure of cellular aggregates and the extracellular matrix (ECM) affect the migration of molecules in the ECS, so that diffusion in the CNS is inhomogeneous, facilitated or slowed down, or in certain regions facilitated in one direction rather than in another. This diffusion anisotropy, found in myelinated white matter, cerebellum and hippocampus, may be of importance for neurone-glia communication and ‘cross-talk’ between synapses.

Changes in neuronal, glial and, consequently, ECS volume and geometry accompany physiological neuronal activity, development and aging, as well as pathological states, e.g., anoxia/ischemia, seizures, injury, demyelination and tumours. Ionic changes and amino acid release result in cellular swelling, compensated for by ECS shrinkage and a decrease in the apparent diffusion coefficients of neuroactive substances or water as determined by diffusion analysis using ion-selective microelectrodes or diffusion-weighted NMR. The structural changes, particularly astrogliosis and ECM changes, increase diffusion barriers. Demyelination and gray and white matter pathologies often result in the loss of anisotropy.

It is evident that the movement of substances is hindered not only by the size of the pores between the cells, but also by the cellular structure. The swelling and movement of glial processes towards active synapses result in changes in synaptic efficacy.

Potassium channel openers (KCOs) are agents, discovered in the early 1980s, that act by stimulating ion flux through K⁺ channels. KCOs act on two types of ion channels: plasma membrane ATP-regulated K⁺ channels and Ca²⁺-activated BK-type K⁺ channels. KCOs were first identified by their antianginal or antihypertensive mode of action. Now, they are at various stages of development as antiasthmatic and cardioprotective agents.

Until recently, the effects of KCOs were believed to be attributable entirely to the modulation of K⁺ channels in cell surface membranes. It is now apparent, however, that new targets for KCOs exist in intracellular membranes including those of sarcoplasmic reticulum, zymogen granules, and mitochondria. Mitochondria seem to be particularly important targets for KCOs because the interaction of KCOs with these organelles appears to mediate the cardioprotective action of these compounds.

A small-conductance potassium channel, with properties similar to the ATP-regulated potassium channel from the plasma membrane, has been described in the inner mitochondrial membrane (mitoK_ATP channel). The mitoK_ATP channel is blocked, similarly to the plasma membrane K_ATP channel, by antidiabetic sulfonylureas and activated by KCOs such as diazoxide. An increased influx of K⁺ into mitochondria in the presence of KCOs was observed. Despite the effect on K⁺ transport, diazoxide also exhibits a direct effect on mitochondrial energy metabolism by inhibition of respiratory chain. The presence of a mitochondrial target for KCOs in brain and skeletal muscle mitochondria has been recently observed.

Telezhkin VS¹, Shuba MF^1,2

The present study was designed to investigate electrical and contractile responses of rabbit main pulmonary artery smooth muscle cells (PASMC) to K⁺-channel blockers tetraethylammonium (TEA) and 4-aminipyridine (4-AP), and nitric oxide (NO) donor nitroglicerin (NG). The research was realized under inhibition of adrenergetic and cholinergetic neuro-muscle transmission with phentolamine, propranolol and atropine. TEA and 4-AP acted in a dose-dependent way evoking depolarization and smooth muscle contraction. The simultaneous action of TEA and 4-AP caused significant depolarization, action potentials (AP) generation and increased contraction. NG applied together with TEA and 4-AP almost completely inhibited APs and the contraction, whereas the degree of the depolarization did not change.

These data indicate that Ca²⁺-dependent K⁺-channels of large conductivity (K_Ca-cannels) play more significant role in the resting membrane potential forming of rabbit PASMC than voltage-gated 4-AP-sensitive potassium channels (K_V-channels). They also suggest that in the presence of TEA and 4-AP the depressing action of NO (released from NG) on the AP generation and on the contraction is due to the voltage-gated L-type Ca²⁺-channels inhibition and to the lowering the Ca²⁺-sensitivity of the contractile apparatus.

Tomá_ová, H, Herget J¹ , Novotná J,

Hypercapnia inhibits the development of hypoxic pulmonary hypertension (HPH) [1] and the collagen breakdown in the peripheral pulmonary arteries. We hypothesize that CO2 inactivates production of radicals induced by hypoxia [2]. Radicals are activators matrix degrading proteinases. In the present study we measured the proteolytic activity activity of serine proteinases [3], collagenase [4] and activity of tissue extracts elastase [3] in lungs of rats exposed for 4 days to hypoxia (group H, FiO2 = 0.1, n = 6), hypoxia and hypercapnia (group H+CO2, FiO2 = 0.1, FiCO2 = 0.045, n = 8) and normoxia (group N, FiO2 = 0.21, n = 5).

The serine proteinases activity did not difer between the groups. The collagenase activity was significantly increased in the group H (10,5 ± 1,6umol/l, SEM, P<0,05) compared to the groups N ( 6,63 ± 1,7umol/l) and H+CO2.(7,9 ± 2,1umol/l). The elastase activity was significantly higher in both hypoxic groups than in normoxic controls.

Inhibition of hypoxia induced collagenolytic activity by hypercapnia is of clinical interest because hypoxia combined with hypercapnia (lung disease) produces less severe HPH than hypoxia with hypocapnia (sojourn in high altitude).

Tomori Z¹, Beňačka R¹, Szabóova E², Donič V¹

Introduction: Cardio-respiratory failure, induced by repeated inhalation of hypoxic mixtures (93 % N₂ with 5 % O₂, and 2 or 7 % CO₂) in cats and episodes of sleep apnoea arising in patients, are characterised by cardiac dysrhythmias.

Methods: Acute experiments were performed in 9 anaesthetised adult cats (Pentobarbital 40mg/kg i. p.) and whole-night polysomnographic recordings were completed in 12 patients suffering from obstructive or central sleep apnoea. The aim was to analyse: 1) the incidence and character of cardiac dysrhythmias occurring in both groups, 2) their gradual development depending on the severity of hypoxia and asphyxia, 3) their probable reversibility and management.

Results: Various types of sino-atrial and atrio-ventricular conduction blocks, as well as ventricular and supra-ventricular extrasystoles developed gradually in both conditions. The development and character of these cardiac arrhythmias were very similar in sleep apnoea patients and hypoxic animals and were frequent but potentially reversible. The severity of dysrhythmias correlated with various indices of the degree of hypoxia (Sat O2, paO2). Hypercapnia in both experimental and clinical conditions increased the incidence and severity of these potentially life- threatening dysrhythmias.

Conclusion: The similar character, development and reversibility of dysrhythmias indicate common pathogenic mechanisms in cats and sleep apnoeic patients. Hypercapnic mixture proved to be more suitable for model studies in animals.

It has been suggested that nitric oxide (NO) may be released in the cerebral vessel wall from endothelium and perivascular nerves, and that it may be involved in the regulation of vascular tone and modulating vascular reactivity. The present study was performed to examine whether or not NO participate in the nonadrenergic noncholinergic relaxation induced by electrical field stimulation (EFS) of rat pulmonary and mesenteric arteries. Isolated rings of arteries were suspended in organ baths and connected to a force transducer for the recording of isometric tension. Under resting conditions EFS of perivascular nerves elicited a tetrodotoxin-sensitive contractile response in both tested arteries. In the arteries treated with guanethidine (10mM) and indomethacin (1mM), and precontracted with phenylephrine (1mM), EFS produced a frequency-related relaxation. EFS-induced relaxation was not influenced by atropine and propranolol, but it was partially inhibited by capsaicin, a toxin for sensory neurone. NG-nitro-L-arginine (L-NNA, 0.1mM), an inhibitor of NO production, inhibited the EFS-induced relaxation in the pulmonary artery while that in the mesenteric artery was not affected. Preincubation with L-arginine (1mM) antagonized the inhibitory effect of L-NNA in the pulmonary artery. These results suggest that the EFS-induced relaxation in the rat pulmonary artery, unlike the mesenteric artery, is predominantly mediated by NO or NO releasing substance.

Travnickova-Bendova Z^1†, Cermakian N¹, Reppert SM², Sassone-Corsi P¹

The phase resetting of the mammalian circadian clock in response to light stimuli involves changes in gene expression and apparently includes the activation of mPeriod(mPer)1 and 2 genes. Here we present the promoter structure of three mPer genes and compare their regulation in a luciferase reporter system. Although all three promoters are activated by the CLOCK/BMAL1 heterodimer, only mPer1 promoter is responsiveness to stimulation of intracellular signaling pathways. This promoter is synergistically activated by the cAMP and MAPK pathways. The cAMP-responsive element (CRE) present in mPer1 and mPer2 promoters can bind CREB in protein extracts from the suprachiasmatic nucleus, the site of the circadian clock. The integrity of this DNA element is necessary for the response of mPer1 promoter to signaling pathway activation. Furthermore, we show that CLOCK/BMAL1-dependent transcription can occur in the absence of an intact CRE within mPer promoters. Altogether, these results are strong evidence for CREB as a pivotal endpoint of signaling pathways for the regulation of mPer genes in response to environmental light stimuli. Importantly, this signaling activation is distinct from the CLOCK/BMAL1-driven transcription of mPer genes involved in the clock regulatory feedback loop.

Trefn_ ZM¹, Bayevsky RM², Svačinka J¹, Trojan S³, Slavíček J³, Herczegh _¹

INTERCELLULAR JUNCTIONS AND VULNERABILITY OF SPONTANEOUSLY HYPERTENSIVE RATS (SHR) TO LOW K⁺-INDUCED LETHAL ARRHYTHMIAS
Tribulova N¹, Novakova S¹, Hirosawa N², Imanaga I², Manoach M³

The present study was designed to examine vulnerability of SHR to low K⁺ elicited ventricular fibrillation (VF) as well as to investigate the role of intercellular junctions (IJ) in arrhythmo-genesis.

The experiments were performed on isolated heart preparation of 10-13 weeks old male SHR and age-matched normotensive Wistar Kyoto rats (WKY). Equilibration of the heart by perfusion with oxygenated, 37⁰C worm, standard Krebs solution at constant pressure was followed by perfusion with K⁺ deficient one. ECG was continuously monitored and ventricular tissue was analysed for ultrastructure as well as immunofluorescence and expression of major gap junction protein connexin-43.

The results showed that incidence of ventricular tachycardia, transient and sustained VF was markedly higher in SHR compared to WKY. There were no apparent differences in immunodetection and expression of Cx-43, however, WB analysis revealed signifi-cantly lower ratio of phosphorylated vs nonphosphorylated Cx-43 in myocardium of SHR. Moreover, low K⁺ perfusion itself lead to focal abolishment of immunoreaction of Cx-43 as well as to decrease of phosphorylated form of Cx-43 in WKY and further decrease in SHR hearts. Low K⁺-induced subcellular alterations of the cardiomyocytes and their junctions indicated Ca²⁺ overload and impairment of intercellular communication. These changes were heterogeneously distributed within myocardium and more pronounced in SHR.

In conclusion, IJ related alterations can contribute to increased susceptibility of SHR to lethal arrhythmias.

Turcani M¹, Skalova K², Turcani P³, Simko F² 

Potassium channel blockade as well as reductions in extracellular potassium concentration produces a hypertrophic response in myocytes in culture. Inhibition of potassium channels may contribute to hypertrophy in an in vivo model also.

To test this hypothesis, sham-operated rats and rats with ascending aorta constriction were treated with tedisamil, a potassium channel blocker (36 mg/kg/day) for 7 weeks. Left ventricular mass and geometry, myosin isozymes, hydroxyproline concentration and isovolumic ventricular function were assessed.

Rats with aortic constriction exhibited a marked increase in left ventricular weight and the diastolic pressure-volume relationship was shifted to smaller volumes. The hydroxyproline concentration remained unaltered, however, the proportion of b-myosin heavy chains (MHC) was increased (p<0.05). Hypertrophied left ventricles manifested an enhanced overall performance but depressed myocardial contractility.

Administration of tedisamil was associated with decreased heart rate (p<0.05) but the concentric left ventricular hypertrophy was not significantly altered. Hypertrophied hearts from rats treated with tedisamil expressed more a-MHC (65±4% versus 57±4%; P<0.05). Maximal rate of wall stress rise and decline was higher in tedisamil treated pressure overloaded rats (P<0.05) also.

In an experimental model of pressure overload hypertrophy tedisamil partially corrects myocardial dysfunction. Postulated mechanism of this effect is the phenotype modification of myosin filaments in hypertrophied cardiomyocytes.

Turecek R¹, Trussell LO²

Each principal cell of the rat medial nucleus of the trapezoid body (MNTB, a brainstem auditory nucleus) receives a single, excitatory, glutamatergic input (the calyx of Held), as well as several inhibitory inputs. In our experiments, electric stimulation of excitatory fibers elicited the excitatory postsynaptic currents (EPSCs) recorded from principal cells by the patch-clamp technique. Activation of inhibitory fibers led to the inhibitory PSCs that were GABA- or glycinergic, depending on the age of the animal.

Repetitive stimulation of the inhibitory fibers in 2-week-old rats potentiated subsequent EPSCs, as a result of glycine (Gly) spillover from inhibitory boutons. Presynaptic recordings from the calyceal terminals revealed Gly-activated chloride-permeable receptors (GlyRs). Presynaptic GlyRs depolarized the terminal, caused an activation of voltage-gated calcium channels, and an accumulation of intraterminal calcium, leading to an enhancement of glutamate release.

In one-week-old animals, Gly had no potentiating effect on EPSCs recorded from the MNTB. At this age, calyces instead contained functional GABA_A receptors that, when activated by exogenous GABA_A agonists, strongly potentiated glutamate release. This effect was not observed in two-week- old rats. Thus, the presynaptic GABA_A and Gly receptors are reciprocally regulated during the early postnatal development and these changes might be involved in developmental processes critical to shaping the rat auditory system.

Tybitanclova K, Macho L, Mravec B, Kvetnansky R, Szabova L, Zorad S

Prolonged cold exposure (CE) of rats leads to cold-induced hypertension. On the other hand, plasma renin activity (PRA) measurements in CE experiments provide conflicting data. Therefore, wide range of time intervals of CE (2 hr, 6hr, 1, 7 and 28 days) was used in our study. Expected angiotensin II AT₁ receptor alternations were studied in epididymal fat tissue. For the CE experiments the adult male Sprague-Dawley rats were housed at 4°C. Food intake and body weight were recorded throughout the whole time course of the experiment. PRA was evaluated as the amount of generated angiotensin I. Angiotensin I and serum insulin were determined by radioimmunoassay. Serum glucose was estimated by the orthotoluidine method. AT₁ receptor expression was measured using RT PCR method.

In the time course study the food intake in CE rats was significantly increased by day 3. Despite that these animals displayed lower body weight. Serum glucose was not significantly influenced by cold though the serum insulin showed significant decrease in 1-day-cold-exposed rats. At the same time PRA reached the highest level. AT₁ receptor mRNA displayed a significant, approximately 50%, decrease in 7 and 28-day-cold-exposed rats.

In conclusion, we assume that PRA during CE is regulated by food intake. Apparently insufficient food intake in 1-day-cold-exposed rats resulted in a decrease of serum insulin and a peak of PRA. After increasing the food intake in 7 and 28-day-exposed rats the PRA and serum insulin tended to normalize. The regulation of AT₁ expression during chronic CE seems to be unrelated to food intake and/or PRA changes. Supported by grants VEGA 2/7213, 2/2090 and CE ICA 1-CT-2000-70008.

Urminská M, Kristek F

We studied long-term effect of N^G-nitro-L-argininmetylester (L-NAME) administration on blood pressure and geometry of the rat basilar artery, and whether four week interruption of L-NAME treatment has effect on changes evoked by NO-synthase blockade.

Ten-week-old male Wistar rats were used: (1) control rats, (2) rats treated by L-NAME for six weeks (50 mg/kg b.w./day in tap water), (3) like group two plus four week interruption of L-NAME administration. Blood pressure (BP) was measured by the tail plethysmographic method. The rats were perfused with glutaraldehyde fixative (120 mmHg) and basilar artery was processed for electron microscopy. Wall thickness /WT/, cross sectional area /CSA/ and inner diameter /ID/ were measured. BP was in control rats 150±10.9 mmHg. In L-NAME group it was 211,7±11,7 mmHg (p<0.01). In third group it was 182.5±14.1 mmHg (p<0.01 to both control and L-NAME group). ID in control rats was 266±7.7 µm, in L-NAME group it was 319±9.3 µm, in third group it was 258±17.6 µm (p<0.01 between experimental groups). WT in control rats was 14.26±0.27 µm, in L-NAME group it was 17.62 ± 0.67 µm (p<0.05). WT in third group was 21.64±1.26 µm (p<0.01 to both control and L-NAME group). CSA was 12.6±0.35x10_ µm_ in controls, in L-NAME group it was 18.2±0.77x10_ µm_, in third group it was 18.9±1.6x10_µm_ (p<0.01 was found between control and experimental groups). Long-term NO-synthase inhibition resulted in pronounce changes in BP and geometry of the rat basilar artery. Four week interruption of NO-synthase inhibition partially reversed increase of BP but it had no effect on geometry of the basilar artery.

Vaculín _¹, Franěk M¹, Andrey L², Rokyta R¹

Extensive dorsal rhizotomy at cervicothoracic level (C5 — Th1) in rats is used as a model of central pain Recognition of a pain state in the deafferented animals is based on a presence of self-mutilation behavior. It has been demonstrated that after the deafferentation aprox. 60 % of neurons of the medial thalamic nuclei (pF, CL) fire in bursts. The aim of the present study was to explore a participation of the rest of the neurons in a coding of nociceptive information. Since chaotic behavior of a single neuron had been identified both experimentally and analytically, we decided to use chaodynamic methods.

Interspike intervals (ISI) were calculated after extracellular recording of activities of the neurons. From the scalar observation — ISI — 3-dimensional phase space structure (an attractor) was reconstructed using time lags (T = 2). Then the attractors were compared between the deafferented rats without any signs of pain syndrome (Gr. 1) and those exhibiting the self-mutilation (Gr. 2).

The principal difference in a shape of maximal density of the attractors between the two groups results from obtained findings. Firing patterns of neurons of group 1 were represented in a form of tricuspid maximal density (80 %) of the attractor, in the group 2 in a form of triangular-to-almost-spherical (72 %) one.

It has been demonstrated lesser susceptibility to postischemic brain injury in females likely due to effects of circulating estrogens. We tested estrogen on both genders of rats of different age by comparison of excitability of neurons.

Rat pups were kept for 8 hours a day in a hypobaric chamber at the altitude of 7 000 m since the day of birth till the 11^th or 17^th day. Excitability of the brain was tested by repeated electrical stimulation of the sensorimotor cortex in 12, 25, 35 and 90-day old male and female animals. Stimulation parameters: frequency 8Hz, pulse duration 0.5 ms, intensity 3-5 mA, duration 15 s. The stimulation was repeated five times, 1minute after the end of previously evoked cortical afterdischarges (ADs). Another groups of animals were exposed to hypoxia in the same condition, but treated immediately before each exposition to hypoxia by agofollin (4 mg/kg s.c.). Measured data were tested by ANOVA and t-test from programs Prisma.

Hypoxia prolonged the duration of ADs in all age’s groups. Estrogen pre-treatment decreased the duration of cortical ADs in 12-day-old animals, both in female and male animals (p = 0,01-0,05) and in 35-day-old females were ADs shorter than in males. Estrogen did not influence the reaction of the 25-day-old and adult rats.

Vargová L, Jendelová P, Chvátal A, Syková E

Changes in IOS are clearly related to increased neuronal activity. Neuronal activity leads to fine structural changes, e.g. of fine astrocytic processes, that may cause IOS changes.

Vavřínková H, Tutterová M, Cahová M

Elevated content of serum free fatty acids (FFA) which accompany insulin resistance to among risk factors for cardiovascular diseases. Further factors which can increase the risk of cardiovascular complications are increased plasma levels of catecholamines, decreased supply of oxygen and decreased availability of glucose. We have studied both separated and combined effects of these factors on the degree of damage of isolated perfused rat heart (Langendorf model) of adult male Wistar rats tested by release of lactate dehydrogenase (LD) into the perfusate expressed in IU per heart per 60 minutes.

In the absence of glucose the palmitate (1 mmol/l) moderately increased the LD release from the myocardium (0,5 IU ± 0,13 vs 1,21 ± 0,17). The addition of adrenaline (5,5 umol/l) under the same situation increased the LD release 10x (1,21 ± 0,13 vs 12,5 ± 1,08). In the presence of glucose (5 mmol/l) in the perfusion medium the palmitate alone or in combination with adrenaline did not increase the LD release from the heart. Palmitate with glucose and adrenalin increased LD release only under partial ischaemic conditions (0,86 ± 0,1 vs 6,14 ± 0,64).

In conclusion, palmitate lead to a moderate myocardial injury only in the absence of glucose. This damage is much greater in the concomitant presence of adrenaline in combination with partial ischeamia. These findings suggest that the decreased availability of glucose with contemporary increase of FFA and adrenaline concentration can contribute to cardiovascular complication of insulin resistance.

Važan R¹, Béder I¹, Pancza D², Styk J²

The effect of pineal hormone melatonin (MLT) on Ca-injury of the rat heart was studied using the model of calcium overload (Ca-paradox). In order to inhibit endogenic production of MLT, rats were exposed to constant light 24h before experiment. After excision, hearts were perfused at 37^oC and a constant perfusion pressure 75torr (Langendorff preparation). Ca-paradox was induced (after 30min of stabilization) by 1min perfusion with Ca-free Krebs-Henseleit (KH) solution and subsequent 20min perfusion with a normal Ca-containing KH solution. In MLT group (n=11), melatonin (10micromol/l) was in the perfusion solution throughout the experiment. In controls (n=8), there was no MLT. Variables: heart rate (HR), coronary flow (CF), systolic (S) and diastolic (D) pressure, dP/dtmax (index of contractility) and dP/dtmin (index of relaxation) were measured at the end of stabilization and then in the 5th, 10th, 15th, 20th min after perfusion with Ca-free KH solution.

RESULTS: There was no difference between MLT group and controls at the end of stabilization. After perfusion with Ca-free KH solution, there was significantly stronger depression of: SD difference (in the 10th, 15th, 20th min), dP/dtmax (in the 5th,10th, 15th, 20th min) and dP/dtmin (in the 15th min) in MLT group than in controls. There was also stronger, but insignificant, increase in CF in MLT group.

CONCLUSION: Melatonin decreases resistance of isolated rat heart to Ca injury caused by Ca-paradox.

Functional and morphological evidence has shown that some neurotransmitters or substances may be released from both synaptic and nonsynaptic sites for diffusion to target cells more distant than those observed in regular synaptic transmission. Functional evidence was obtained that there are interactions between neurons without synaptic contacts, and matches between release sites and localization of receptors sensitive to the chemical signal are exceptions rather than the rule in the central nervous system. This also indicates that besides cabled information signalling (through synapses), there is a "wireless" nonsynaptic interaction between axon terminals. This would be a form of communication transitional between discrete classical neurotransmission (in Sherrington’s synapse) and the relatively nonspecific neuroendocrine secretion. Recent findings indicate that in addition to monoamines (norepinephrine, dopamine, serotonin), other transmitters, such as acetylcholine and nitric oxide (NO), may also be involved in these nonsynaptic interactions. It has been shown that NO, an ideal mediator of nonsynaptic communication, can influence the function of uptake carrier systems, which may be an important factor in the regulation of extracellular concentration of different transmitters. The nonsynaptic interaction between neurons mediated via receptors and transporters of high affinity not localized in synapses has the potential to be an important contributor to the properties and function of neuronal networks. In addition, it was suggested that the receptors and transporters expressed nonsynaptically and being of high affinity are the target of drugs taken by the patient.

Voří_ek I^1,2,3, Hájek M^1,3, Nicolay K⁴, Syková E^1,2,3

Severe astrogliosis was found close to the injury site, mild astrogliosis in the ipsilateral but not in the contralateral cortex. Immunostaining for CSPG revealed a uniform increase throughout the ipsilateral cortex. The diffusion parameters in the hemisphere contralateral to the wound were not significantly different from those in control animals. The volume fraction was increased only in the vicinity of the wound, in the region with severe astrogliosis. Both ADC_TMA and ADC_W were significantly decreased after lesion in the vicinity of the wound as well as distant from the wound.

We conclude that an increase in diffusion barriers, manifested by ADC_TMA and ADC_W decreases, occurs in the entire ipsilateral cortex. These changes are related to astrogliosis and to an accumulation of extracellular matrix molecules. There is a good correlation between MR and TMA diffusion measurements.

Vyklick_ L, Jr.¹, Abdrachmanova G¹, Chodounská H²

In motoneurons glutamate receptors mediate excitatory synaptic currents, elicit a locomotor-like pattern of activity and were suggested to be involved in the developmentally regulated death induced by peripheral nerve injury. The aim of the present study was to characterize effect of neurosteroids on NMDA receptors and excitatory synaptic transmission in rat motoneurons.

Motoneurons were labelled by fluorescent dyes injected into the anterior tibialis muscle and visualized in slices prepared from 4 to 14-day-old animals using fluorescence/IR/DIC microscopy. Patch clamp technique was used to record responses induced by glutamate receptor activation.

Responses induced by NMDA in outside-out patches were in the presence of 20-oxopregn-5-en-3β-yl sulphate (PS) potentiated by 79%, while in the presence of 20-oxo-5β-pregnan-3α-yl sulfate (3α5βS) diminished by 66%, respectively. The mean current transferred by NMDA receptor channel openings to individual conductance levels indicated that in the presence of PS the current induced by 55 pS openings was significantly increased while in the presence of 3α5βS the current induced by 55 pS and 70 pS openings was significantly decreased. The amplitude of NMDA receptor-mediated EPSCs was potentiated in the presence of PS by 54% and the deactivation slowed. Neither the amplitude nor the kinetics of NMDA receptor-mediated EPSCs was significantly changed in the presence of 3α5βS.

The results of our experiments indicate a subunit specific effect of neurosteroids on NMDA receptors in motoneurons.

The aim of this study was to examine the effect of H₁-, H₂- and H₃-antagonists administration on histamine evoked gastroprotection against stress induced gastric ulcers in rats.

Male Wistar rats were exposed to water immersion and restrain stress (WRS) for 3.5 h. Before WRS rats were pretreated with: saline (control), histamine; pyrilamine (H₁-antagonist), ranitidine (H₂-antagonist), or thioperamide (H₃-antagonist) alone or in combination.

The control ulcer area was markedly reduced by histamine and ranitidine. Pyrilamine and thiperamide given alone did not affect gastric ulceration evoked by WRS. Pyrilamine in combination with histamine caused an increase in gastric ulcer area above value observed in saline treated rats. Pretreatment with thioperamide before histamine administration abolished histamine evoked reduction in gastric ulcer area. Treatment with histamine alone increased gastric blood flow (GBF). Administration of pyrilamine or thioperamide given alone reduced GBF by 22 or 18 %, respectively. Pretreatment with pyrilamine or thioperamide before histamine administration abolished histamine evoked an increase in GBF. Similar alterations were observed in mucosal DNA synthesis. Plasma IL-1b was significantly reduced after administration of histamine or ranitidine given alone or in their combination.

Melatonin (MEL), the main endocrine product of the pineal gland, has been claimed to exhibit distinct antioxidant features in vitro as well as in vivo, which has been considered to be part of its physiological as well as pharmacological effects. Since reactive oxygen species (ROS) are known to influence a variety of biologic processes and to constitute a major signal for programmed cell death in normal and tumor cells, we were interested whether MEL may interfere with intracellular ROS formation in lymphoid cells, e.g. in peripheral blood lymphocytes (PBL) as well as in Jurkat cells, a human leukemic T-lymphoblastic cell line. While in human PBL pharmacological concentrations of MEL exhibited antioxidant properties against t-butylated-hydroperoxide- and diamide-induced ROS-formation and subsequently decreased the percentage of cells undergoing ROS-induced cell death, in resting Jurkat cells MEL exhibited prooxidant activity, as was shown by increased intracellular oxidation of ROS-sensitive fluorescent dyes. Furthermore, enhanced ROS formation by MEL led to a promotion of fas- and ROS-induced cell death in this leukemic cell line. The cell death promoting effect of MEL was dependent on intracellular glutathione concentration as well as inhibited by addition of trolox, a water-soluble analogue of alpha-tocopherol, and glutathione indicating that this effect of MEL is indeed mediated by formation of intracellular ROS. These results give evidence that MEL may be a modulator of the cellular redox status in both ways in lymphoid cells, and is not necessarily protecting against oxidative hazard.

Yamamotová A¹, Papežová H², Vurmová I², Kočová M^3 
1Charles University, 3^rd Medical Faculty, Dept. of Physiology, ²Dept. of Psychiatry, 1^st Medical Faculty and ³Faculty of Natural Sciences, Prague, Czech Republic

Patients with anorexia and bulimia nervosa have decreased sensitivity to pain and parallel changed perception of their body size (they feel more fatty than they are in reality). The aim of our study was to compare and correlate the thermal pain threshold measured under the rest and stress conditions with the score of Body Attitude Test (BAT). The self-report questionnaire BAT has been developed for female patients suffering from eating disorders (Probst et al, 1995). It comprises three subscales, BAT-1: negative appreciation of body size, BAT-2: lack of familiarity with one’s own body (dissociation), BAT-3: general body dissatisfaction.

The pain threshold and body perception were tested in 20 patients hospitalized with eating disorders and 11 healthy women. The four-minute rest period was followed by mental arithmetic task (MAT - subtraction 7 from 700), alimentary test (AT - consumption of sweet biscuits) and cold pressor test (CPT) separated by rest periods. BAT was administered at the beginning of the experiment; subjective unpleasantness of three stress trials was assessed using visual analogue scale.

The pain threshold during MAT and CPT correlated positively with BAT-2. Perceived unpleasantness of AT correlated positively with BAT-1 and BAT-2. Unpleasantness of AT correlated positively with the pain threshold during the AT test and rest conditions preceding and following AT. No such interrelations were found in healthy control women.

We suggest that the pain threshold might be used as a marker of dissociation and illness severity. Unpleasantness during AT could measure the intensity of diagnose-specific stressor.

Zachařová G¹, Smerdu V², Semelová V¹, Mráčková K¹, Soukup T¹

The soleus muscle from young 2 to 4-week-old inbred Lewis rats was isotransplanted into the fast host extensor digitorum longus (EDL) muscle of adult rats of the same strain. The regeneration of grafts proceeded under different thyroid status in order to analyze the effect of decreased and increased levels of thyroid hormones on grafted, host and control muscles. Two to 4 months after the transplantation, the muscle fibre mean cross-sectional area (MA) was measured using the C.A.S.T. Grid system based on 2-D stereological methods (1). Irrespective of thyroid status the total fibre MA in regenerated slow soleus grafts was markedly decreased (it attained only slightly more than one third of that of the host muscles) and the difference between MA of type 1 and type 2 fibres was not as pronounced as in host and control muscles. Under euthyroid conditions, the fibre type composition of the slow soleus graft resembled the host fast EDL muscle. Slow fibres in the grafts regenerated in hyperthyroid animals were further suppressed and MA of type 2A fibres was affected more than that of type 2B fibres. In the grafts of hypothyroid host rats, the number of slow fibres was substantially increased and, compared to euthyroid grafts, the most affected was the MA of fast 2B fibres.

(1) Zachařová G., Kubínová L. (1995) J. Muscle Res. Cell Motil. 16: 295-302.

Zahradníková A, Zahradník I

The molecular basis of activation of the tetrameric ryanodine receptor (RyR) channel by calcium has been unclear until recently. Li and Chen [1] now described a RyR2 mutant, E3987A, which provided five gradually decreasing levels of calcium sensitivity on co-expression of native and mutant mRNA.

Here we report calculations on the Ca²⁺ sensitivity of all five tetramer variants that may arise from a mixture of the wild type and mutant monomers. We adopted our extended minimal gating model [2] to generate gating schemes of the tetramer variants. The E3987A mutation in individual monomers was represented by a 500x decrease of the calcium-binding rate constant.

The predictions of the derived models were in very good agreement with the experimental data of Li and Chen [1]. Specifically, the models reproduced very well both, the shift of the calcium sensitivity with the number of mutated monomers in the RyR channel, and the selective decrease of the Hill slope for the RyR channel containing only one mutated monomer.

The demonstrated consistency of the models developed from our functional studies [2] with the structural studies of Li and Chen [1] may be considered as evidence that each RyR channel monomer has a separate Ca-binding site encompassing E3987.

[1] Li and Chen, J. Gen. Physiol. 118: 33-44, 2001 
[2] Zahradníková et al., J. Gen. Physiol. 114: 787-798, 1999

Závodná E, Nováková Z, Honzíková N

The aim of the present study was to determine the interrelationship among the baroreflex sensitivity (BRS), the variability in systolic blood pressure (SBP) and pulse intervals (PI) at the frequency of 0.1 Hz.

Blood pressure was recorded in 228 children (11-15 years of age) for 5 min (Finapres, metronome controlled breathing at a frequency of 0.33 Hz). BRS was determined by a spectral method. The power spectra of SBP (in mmHg²/Hz) and PI (in ms²/Hz) were calculated. Children were divided into groups according to the spectral power at the frequency of 0.1 Hz. The following limits of power were used: high — hSBPÒ100, hPIÒ12500; middle —100ÒmSBPÒ55, 12500ÒmPIÒ5400; low — lSBP<55, lPI<5400. We analysed the relationships between the groups A(hSBP, hPI), B(mSBP, hPI), C(lSBP, hPI), D(hSBP, mPI), E (mSBP, mPI), F(lSBP, mPI), G(hSBP, lPI), H(mSBP, lPI), I(lSBP, lPI).

BRS increased in the following sequence of groups (**significant differences between adjacent groups, p<0.01): G H**D**I**E A**B**F**C. The variability of PI increased with increasing BRS in each triplet of groups with the identical SBP-variability. This relationship was shifted to the higher values of PI variability at the same BRS with increasing SBP variability.

The study proved that PI variability positively correlates with BRS and has a damping effect on SBP variability. Our analysis disclosed an additional effect of a primary variability of SBP on this relationship. The sequence of groups showed that BRS is more closely related to the variability in PI than in SBP.

In the past years the awareness is raised about dangers of endocrine-disrupting chemicals occuring in our environment. Human, fish and wildlife animals consume food and water containing environmental toxicants that behave like hormones and have the ability to cause effects ranging from sterility and abnormal sex differentiation to cancer. There is a great effort to control and eliminate these suspected compounds that exist as environmental contaminants or food additives.

Series of studies were performed in our Department to examine the possible estrogenic effects of selected environmental chemicals. Simple screening tests were used: a) time of vaginal opening in the neonatal rats, and b) uterine epithelial cellular hyperthrophy.

The results of our studies indicated estrogenic effects of insecticide Fosmet, herbicides Acetochlor, Atrazine, Chloridazon, Simazine, non-ionic surfactans Tween 80 and chemical sterilant formaldehyde.

On the base of our experience and recent knowledge about the possible risk of xenoestrogens we think that the test for estrogenicity is becoming a critical screening tool for determination the potential health consequences of novel chemicals. If xenoestrogens do play a role in breast cancer, reduction in exposure will provide an opportunity for primary prevention .

Zeman M¹, Herichová I¹, Nosáľová V², Zakálová M¹, Vician M^3 
1Dept Anim Physiol & Ethol, Comenius Univ Bratislava, Slovakia ²Inst Exp Pharmacol SASci, Bratislava, Slovakia ³First Surg Dept, Med Fac & Hosp, Comenius Univ Bratislava, Slovakia

Melatonin is a hormone synthesised predominantly in the pineal gland and involved in a control of circadian rhythms. Recent data suggest a possibility of extrapineal melatonin production and a broader spectrum of its action. Among others gastrointestinal tract (GIT) has been suggested as a possible target for melatonin action and a place of its production.

The aim of our study was to investigate if melatonin concentrations in gut parallel those in the pineal gland and circulation and if melatonin can exhibit protective effects in GIT.

Melatonin concentrations in gut tissue were substantially higher than those in circulation. In rats, melatonin levels in the pineal gland and plasma exhibit an expected daily rhythm with high levels found during the darktime. However, no such rhythm was found in colon and a high melatonin content was measured in this part of the gut also during a day. This finding is important in a context of reported ability of melatonin to scavenge reactive oxygen compounds (ROC) and low antioxidant capacity of colon. Exogenous melatonin exhibited protective effects against colitis induced in rats by intraluminal instillation of acetic acid. It is of interest that natural antioxidant pleuran (β-glucan) has a powerful protective effect against experimental colitis and increased pineal melatonin concentrations.

Summarising, melatonin can play a role in GIT function and may act through scavenging of ROC, relaxation of local circulation or other still unknown mechanisms.

Zemková H , Vaněček J

Melatonin by binding to its receptor(s) coupled to both pertussis toxin (PTX)-insensitive and PTX-sensitive G protein, has both potentiating and inhibitory effects on gonadotropin-releasing hormone (GnRH)-stimulated [Ca²⁺]_i increases in neonatal rat gonadotropes. The potentiating effect of melatonin is apparently due to stimulation of PLC activity but the mechanism of melatonin-induced inhibition is much less understood. We examined the effect of melatonin on extracellular Ca²⁺ entry stimulated during the latency period of GnRH-induced [Ca²⁺]_i increases and on Ca²⁺ entry during the restoration of intracellular Ca²⁺ stores depleted by repeated GnRH applications. To monitor the GnRH stimulated [Ca²⁺]_i changes, we used nystatin-perforated patch recording of a Ca²⁺-activated potassium current (I_K(Ca)) in acutely cultured neonatal rat gonadotropes.

Melatonin (1 nM) prolonged the latency of GnRH
(10 nM)-induced I_K(Ca) similarly as nifedipine or extracellular Ca²⁺ omission. When two pulses of GnRH (each 20 s) were given, the response to the second pulse elicited full recovery when the interval between pulses was at least 30s. Ca²⁺-free prolonged or abolished the recovery, but melatonin had no effect.

This indicates that melatonin inhibits GnRH stimulated Ca²⁺ entry which is required to coactivate Ca²⁺ release from IP₃-sensitive stores during latency, but it has no effect on Ca²⁺ entry induced by depletion of intracellular Ca²⁺ stores. Melatonin exerts this inhibitory effect most probably by inhibiting cAMP-dependent phosphorylation of L-type voltage-dependent Ca²⁺ channels.

Ziegelhšffer A.¹, Waczulíková I.² Ravingerová T.¹, Ziegelhšffer-Mihalovičová B.¹, Nagyová K. ², Styk, J. ¹

Introduction: Heart of rats with diabetes mellitus (DM) are characterized by energy demands exceeding their energy production, but they might also exhibit decreased vulnerability to ischemia and calcium overload. This indicates adaptation in cardiac energetics (CE), where energy trasport is not rate-limiting. This study was designed to elucidate the functional significance of the DM-induced adaptation in CE.

Methods: After week of streptozotocin-induced DM (45 mg/kg i.v.) the hearts of male diabetic and age-matched control rats (C) were isolated and relevant parameters evaluated.

Results: In C hearts, increasing Ca²⁺ induced both, positive inotropic response and elevated mitochondrial contact sites (MiCS) formation. In DM hearts, basic MiCS formation was already comparable with that induced by elevated Ca²⁺ in C hearts and could not be further stimulated by Ca²⁺. In DM rats, arrhythmia profile was similar to that in C, and the incidence of tachyarrhythmias and their severity were not enhanced. The infarct size was smaller in the DM than in C hearts.

Conclusions: Ca-signaling represents the link between the energy delivery from mitochondria (via MiCS) and energy requirements of the heart. In DM hearts, the energy transport via MiCS is elevated to maximum. This contributes to increased resistance of DM hearts to irreversible cell damage.

Žila I, Brozmanová A, Javorka M, Čalkovská A, Javorka K

Assessment of chemoreflexes can provide important information about respiratory control system in pathological conditions causing respiratory instability. The aim of study was to investigate the contribution of chemical control mechanisms in the development of respiratory changes during hyperthermia.

Material and methods: The experiments were carried out on 14 adult rabbits under general anaesthesia. Hypercapnic (HCVR) and hypoxic ventilatory responses (HVR) were estimated during body surface heating. HCVR: animals breathed gas mixture of 40% O2, balanced with N2. For continuous rise of ETCO2 CO2 was added. CO2 sensitivity was estimated as a slope of ventilation-ETCO2 regression line. HVR: 4 episodes of hypoxia were performed using gas mixtures containing 11%, 9%, 7%, 5% O2.

Results: HCVR: CO2 sensitivity in normothermia was 115 ± 22 ml.min^-1.kPa^-1 (mean ± SEM). During overheating the sensitivity was significantly increased - at 42^oC it was 162 ± 20 ml.min^-1.kPa^-1. HVR: gradual decrease of FiO2 during overheating caused rise of ventilation lesser than in normothermia. The only significant change was found between ventilation at 42^oC compared to the ventilation in normothermia during episodes of hypoxia. Changes of VT were similar to those in ventilation. The increase in frequency of breathing was less prominent at 39.5-40.5^oC, hovewer at 42^oC the decrease in frequency was observed.

Conclusion: During hyperthermia HCVR and CO2 sensitivity were augmented and HVR was attenuated.

Zlámal M, Hodyc D, Hampl V, Herget J

We conclude that vascular injury in early stages of HPH results in the increase in the basal tonus of vascular smooth muscle in pulmonary arterial segment.

The study was supported by grant GACR 305/97/S070 

1.

217  (abstract received after the deadline)

Mlcek M, Novák V, Kittnar O

Institute of Physiology, Charles University - 1^st Medical Faculty, Prague, Czech Republic

Integrating our recent model of excitation-contraction coupling and model of core calcium dynamics in cardiomyocyte (comprising plasma membrane processes, sarcoplasmic reticulum, mitochondria and sarcomere compartments), a single, more complex one was created. The time-course of action potential obtained experimentally was used as the input. The model helps to identify the contribution of different mechanisms to the functionality of the system by switching on and off its individual components, which necessarily interact in a common timeline. This time-dependent interaction can give more accurate understanding complex biological processes. Previous simulation results studying the possible role of the sarco-endplasmic pump dysfunction in the chronical heat failure were compared to the results obtained from the new one. It was found that the obvious effect of the pump dysfunction reported earlier (increase in resting [Ca2+]i and prolonged [Ca2+]i decline) was maintained with similar trends. Lately incorporated calcium handling mechanisms contribute mostly in shortening the time intervals during which the altered sarcoplasmic calcium levels (compared to physiological situation) were observed. Thus reduced maximal concentration of cross-bridges (reflected by systolic function) was observed already in 1Hz stimulation frequency, while the increased minima of cross-bridge concentrations (impaired diastolic function) could be expressed more (or only) in higher heart rates. In this case, an increased heart rate could worsen the situation of a failing heart not only by generally increasing metabolic demands but also unmasking certain aspects of compromised calcium-handling situation.

In this work we present the experimental data about neuronal organization of a system "input - output" parietal cortex. The electrophyziological investigation of neurons forming an exit parietal cortex neurons in ipsilateral motor, homotopic site of other hemisphere, nucleus of the pons bridge, pyramidal tract and back brain are reduced. Here we discuss neuronal organization and functional properties of associative connections between parietal and motor zones, and also singularity of interaction of signals from parietal and motor zones among themselves and with from periferal of a nerve on interneurons cervical of a department of spinal cord. The special attention is given to the analysis of a convergence of inputs from peripheral receptors and cortex of representations somatic, visual and acoustical sensory systems on intrneurons and neurons of an output parietal cortex. The dates on a role of neurons of different functional groups of area 5 are represented during preparation, start and monitoring of arbitrary driving.

Key word: parietal cortex, neurons, potentials, afferent and efferent connection

219  (abstract received after the deadline)

Kolchiba EB¹, Ladutin VV²

The influence of different doses irradiation at the Mg,Ca-ATPase activity at the plasmatic membrane of rat’s brain bark and cere-bellum has been investigated. Investigation’s doses: 0.31 C/kg and 9.288 C/kg. Exposition — 1, 3, 6, 24 and 48 hours after action of ionizing radiation. The Mg,Ca-ATPase activity has been determined at the incubation surroundings.

Dose 0.31 C/kg: head brain,s bark: 1hours — decrease of activity(8%), 3hours — recovery to control’s level , 24hours — fall of ATPase activity(12%), 48 hours — recovery to 94% relatively control’s level.

Dose 3.288 C/kg, brain’s bark and cerebellum: 1hour — fall of ATPase activity at the brain’s bark (to 67% relatively control), at the cerebellum to 81%. 3hours — Increase activity at the brain’s bark to 96% and cerebellum to 145%. 6huors — fall of activity at the brain’s bark to 64% and at the cerebellum to 47% relatively control’s level.