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General Physiology and Biophysics

Volume 24, 2005, No. 4


  Relevance of ventricular electrical dispersion to arrhythmogenesis in ischemic myocardium – a simulation study
H Zhang 1), Z Zhang, L Yang, Y Jin, Y Huang

1)The Key Laboratory of Biomedical Information Engineering of the Ministry of Education of China, Institute of Biomedical Engineering, School of Life Science and Technology, Xi'an Jiaotong University, 28 West Xianning Road, 710049 Xi'an, P. R. China.

A computer simulation method was used to study the possible role of electrical dispersion induced by regional ischemia in the mechanisms underlying cardiac arrhythmias. Ischemic cells were simulated by considering the three major component conditions of acute ischemia (elevated extracellular K+ concentration, acidosis and anoxia) at the level of ionic currents and ionic concentrations. An ischemic area was introduced into a homogeneous healthy tissue to create a localized inhomogeneity. The constructed models were solved using the operator splitting and adaptive time step methods. The numerical experiments showed that action potential durations (APDs) of ischemic cells did not change with beats of shorter or longer cycle length. The smaller percentage increase of slow component of the delayed rectifier K+ current, Iks, and smaller outward Na+-Ca2+ exchange current were found to be the ionic mechanisms underlying the decreased rate dependence in ischemic cells. The results suggest that ischemia flattens the APD restitution curve; however, the dispersion of refractory period can be greatly increased by a premature beat in the constructed inhomogeneous sheet. This demonstrates that the dispersion of refractoriness rather than APD by a premature beat contributes to reentrant tachyarrhythmias in the locally ischemic tissue.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 365-380.

  Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics
M Vydevska-Chichova 1), K Mileva, R Todorova, M Dimitrova, N Radicheva

1)Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., bl. 21, Sofia 1113, Bulgaria.

Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiological characteristics. Based on the relative mATPase reactivity, the fibres of the studied muscle were classified as one of five different types (1-2, 2, 2-3, 3 and tonic). Smaller sized fibres (tonic and type 3) expressed higher succinate dehydrogenase activity than larger sized fibres (type 1-2, 2), which is related to the fatigue resistance. The differences between fatigue development in SMFs and FMFs during continuous activity were associated with fibre-type specific mATPase and succinate dehydrogenase activity.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 381-396.

  The ACAT inhibitor VULM1457 significantly reduced production and secretion of adrenomedullin (AM) and down-regulated AM receptors on human hepatoblastic cells
J Dřímal 1), V Fáberová, L Schmidtová, M Bednáriková, J Dřímal, D Dřímal

1)Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava 4, Slovakia.

Acyl-CoA:cholesterol acyltransferase (ACAT) is an important enzyme in the pathways of cholesterol esterification. It has been shown that new ACAT inhibitor 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM1457) significantly reduced atherogenic activity in animal experimental atherosclerosis. Proliferative hormone adrenomedullin (AM) has been shown to be released in response to hypoxia, however, its role in cellular protection has remained elusive. The effect of increased local production of AM in cells and resultant down-regulation of AM receptors has not been investigated yet. We hypothesized that increased expression of AM in hypoxic cells was the result of excessive AM production with resultant AM receptor down-regulation, surface-membrane protein degradation and that the new specific ACAT inhibitor would reduce AM induction in hypoxia and thus proliferation of cells. In order to investigate specific cellular AM signaling and protection induced by VULM1457, we characterized specific surface-membrane [125I]AM receptors expressed on cells, evaluated AM secretion (RIA assays), AM mRNA expression in cultured cells (RT-PCR analysis) and proliferation (incorporation of [3H]thymidine) in control, hypoxic and metabolically stressed human hepatoblastoma cell lines exposed to gradually increasing concentrations of VULM1457. The new ACAT inhibitor VULM1457 in concentration 0.03 and 0.1 µmol/l significantly down-regulated specific AM receptors on HepG2 cells, reduced AM secretion of HepG2 cells exposed to hypoxia. These results suggest that VULM1457, as new member of ACAT family of inhibitors could negatively regulate cell proliferation induced by AM, which may correlate with down-regulation of membrane-bound AM receptors on HepG2 cells, and moreover, with the induction and expression of AM in hypoxia.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 397-409.

  The role of NO in ischemia/reperfusion injury in isolated rat heart
E Andelová 1), M Barteková, D Pancza, J Styk, T Ravingerová

1)Institute for Heart Research, Slovak Academy of Sciences, Dúbravská cesta 9, 840 05 Bratislava 45, Slovakia.

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dtmax (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 ± 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 ± 5.7 %, p < 0.05). Recovery of both CF and dP/dtmax after TI was also significantly higher in the adapted hearts (101.5 ± 5.8 % and 83.64 ± 3.92 % ) as compared with the controls (71.9 ± 6.3 % and 35.7 ± 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 ± 3.1 %; dP/dtmax 67.5 ± 5.92 %) and increased CF to 82.4 ± 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 ± 3.1 %; CF 70.3 ± 3.4 % and dP/dtmax 43.25 ± 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) – 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 411-426.

  Tapering of human nerve fibres
G Schalow 1)

1)Institute of Exercise Biology and Physiotherapy, Centre of Behavioral and Health Sciences, University of Tartu, 5 Jakobi Street, Tartu 51014, Estonia.

To determine the tapering of human nerve fibres, rostral and caudal root pieces of cauda equina nerve roots were removed and nerve fibre diameter distributions were constructed for 4 myelin sheath thickness ranges for the two sites, and compared with each other. The reduction of the group diameter in the different α-motoneuron groups was 0.2 % per 13 cm. Accounting for systematic errors, there may be even less tapering. An identified single nerve fibre showed no tapering. Further, there is indication that γ-motoneurons, preganglionic sympathetic and parasympathetic fibres and skin afferents also reduce their fibre diameter by 0.2 % per 13 cm or less. Consequently, a nerve fibre with a diameter of 10 µm would be reduced to approximately 9.8 µm at 1m from the cell soma. Preganglionic parasympathetic fibres were found to be represented in roots S1 to S5. At similar distances from the spinal cord, the mean diameter of ventral root α1-motoneuron (FF) axons increased from the thoracic towards the lumbo-sacral region before decreasing again in the lower sacral region. Usually no α1-motoneuron axons were found in S5 roots. The diameter distribution of unmyelinated nerve fibres of a ventral S5 root showed three peaks at 0.25, 0.95 and 1.2 µm. The unmyelinated fibres with diameters around 0.25 µm may represent parasympathetic fibres. In six selected areas of the ventral S5 root, 6.6 times more unmyelinated nerve fibres than myelinated fibres were found on the average.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 427-448.

  ESR and monolayer study of the localization of coenzyme Q10 in artificial membranes
M Grzybek 1), K Stebelska, P Wyrozumska, P Grieb, M Langner, A Jaszewski, A Jezierski, A Sikorski

1)Institute of Biochemistry and Molecular Biology, University of Wroc&lslash;aw, Przybyszewskiego 63, 51-148 Wroc&lslash;aw, Poland.

The data obtained from the ESR experiments show a complex, depth dependent effect of CoQ10 on the lipid molecules mobility in the bilayer. These effects depend both on its concentration and the temperature. CoQ10 disturbs not only the hydrophobic core of the membrane but also the region close to the hydrophilic headgroups of phospholipids. Both these effects could be explained by the fact that the high hydrophobicity of CoQ10 causes the molecules to position itself in the interior of the bilayer, but at the same time its water seeking headgroup is located close to the region of the polar headgrops of membrane lipids. The presence of CoQ10 in the hydrophobic core has further implications on the properties of membrane intrinsic domain. Results of monolayer experiments indicate that CoQ10 may form aggregates when mixed with PC molecules in the lipid hydrocarbon chain-length dependent manner. CoQ10 is not fully miscible with DMPC or DPPC but it is well miscible with the long-chain DSPC molecules. Our suggestion is that CoQ10 when present in long-chain phospholipid bilayer, interacts with saturated fatty acyl-chains and adapt the structure which allows such interactions: either parallel to the saturated acyl chains or “pseudo-ring” conformation resembling sterol structure.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 449-460.

  Prolongation of pentoxifylline aliphatic side chain positively affects the reversal of P-glycoprotein-mediated multidrug resistance in L1210/VCR line cells
P Dočolomanský 1), I Fialová, M Barančík, A Rybár, A Breier

1)Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava 37, Slovakia.

We reported previously that derivatives of pentoxifylline (PTX) reverse multidrug resistance (MDR) in P-glycoprotein (P-gp) positive L1210/VCR cells. Based on the results of a recent study using 25 N-alkylated methylxanthines with carbohydrate side-chains of various lengths, we formulated the following design criteria for a methylxanthine molecule to effectively reverse P-gp mediated MDR: i) a massive substituent at the N1 position is crucial for MDR reversal potency; ii) elongation of the substituents at the N3 and N7 positions (from methyl to propyl) increases the efficacy of a xanthine to reverse MDR; iii) elongation of the substituent at the C8 position (from H to propyl) decreases the efficacy of a xanthine to reverse MDR. Based on these criteria, we synthesized and tested for potency to reverse MDR a new PTX derivative, 1-(10-undecylenyl)-3-heptyl-7-methyl xanthine (PTX-UHM), with prolonged substituents at the N1 and N3 positions. The derivative was obtained by alkylation of 3-heptyl-7-methyl xanthine with 1-methylsulfonyloxy-10-undecylenyl. NMR and IR structural analyses proved the identity of the product. Cytotoxicity study showed that PTX-UHM is only slightly more toxic to L1210/VCR cells than PTX. We found that both PTX-UHM and PTX were able to reverse vincristine resistance of L1210/VCR cells, yet PTX-UHM was significantly more efficient in the reversal than PTX.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 461-466.

  Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells
D Ham 1), B Karska-Wysocki

1)Institute of Preventive Medicine, Faculty of Medicine, Masaryk University, Sladkého 13, 639 00 Brno, Czech Republic.

Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.

General Physiology and Biophysics. Volume 24, 2005, No. 4: 467-474.