Showing papers in "General Physiology and Biophysics in 2005"

Voltage-dependent calcium channels.

Abstract: Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA) Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)23 channels more effectively Furthermore, alpha2delta-2 may modulate the LVA Ca(v)31 channel Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized The gamma-2 subunit modulated both the non-neuronal Ca(v)12 channel and the neuronal Ca(v)21 channel The gamma-4 subunit affected only the Ca(v)21 channel The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)31 channel The Ca(v)12 channel is a major target for treatment of cardiovascular diseases We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism High-affinity inhibition is characterized by interaction with inactivated state of the channel Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit We have cloned and characterized murine neuronal LVA Ca(v)31 channel The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat The channel is insensitive to tetrodotoxin and DHPs The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+ The current through the Ca(v)31 channel inactivates faster with Ba2+ compared to Ca2+ Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current Prolonged depolarization does not immobilize charge movement of the Ca(v)31 channel

Controversy of free radical hypothesis: reactive oxygen species--cause or consequence of tissue injury?

Abstract: For a decade or two, the hypothesis of causality of various disorders by reactive oxygen species (ROS), due to their potentially harmful effect towards cellular constituents, is one of the most frequently cited in biomedical sciences In fact, the ROS-mediated alterations of biomacromolecules are considered to be essential events in the etiopathogenesis of those diseases where involvement of ROS has been indicated ROS easily react in vitro with most biological molecules, causing their degradation and destruction This may implicitly suggest that, when excessively produced in vivo, ROS are deleterious to integral components of the cell and cause their dysfunctions Some experimental data indicate that ROS-mediated lipid peroxidation, protein oxidation and oxidative alterations to nucleic acids are crucial events of unfavorable actions of ROS Yet the most convincing evidence, ie unambiguous inhibition of tissue injury by pretreatment with antioxidants, has not been provided On the contrary, there are quite a few papers reporting failure in applying antioxidants to heal those pathologies where the causal role of ROS was supposed Other papers reported serious complications arising from antioxidant therapy, which is quite in contradiction to its expected effect On the other hand, an increasing number of recent findings have provided evidence of a key role of ROS in both intracellular signaling and intercellular communication, processes involved in maintaining homeostasis Hence, some investigators consider excessive production of ROS to be rather a "smoke after the fire" than "a deleterious fire" itself, suggesting the occurrence of overproduced ROS as being the consequence of some primary damage The present paper aims at summarizing some pros and cons of various opinions with an attempt to help better understand the involvement of ROS in tissue injury

Antidiabetic effect of Scoparia dulcis: effect on lipid peroxidation in streptozotocin diabetes.

Abstract: Oxidative damage has been suggested to be a contributory factor in the development and complications of diabetes. The antioxidant effect of an aqueous extract of Scoparia dulcis, an indigenous plant used in Ayurvedic medicine in India was studied in rats with streptozotocin-induced diabetes. Oral administration of Scoparia dulcis plant extract (SPEt) (200 mg/kg body weight) for 3 weeks resulted in a significant reduction in blood glucose and an increase in plasma insulin. The aqueous extract also resulted in decreased free radical formation in tissues (liver and kidney) studied. The decrease in thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HPX) and increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione-S-transferase (GST) clearly show the antioxidant properties of SPEt in addition to its antidiabetic effect. The effect of SPEt at 200 mg/kg body weight was better than glibenclamide, a reference drug.

The role of NO in ischemia/reperfusion injury in isolated rat heart.

Abstract: 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 pre- conditioning, 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 µmol/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 signif- icantly 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

Interactions between tricyclic antidepressants and phospholipid bilayer membranes.

Abstract: Participation of electrostatic and other noncovalent interactions in the binding of tricyclic antidepressants (TCAs) to the lipid bilayers was estimated from pH-dependencies of imipramine, desipramine, amitriptyline and nortriptyline binding to the lipid bilayers prepared from different phospholipids, both electroneutral and acidic. The binding was studied using a radioligand binding assay. It was found that the membrane phospholipid composition and methylation of the acyl side chain of TCA has a decisive effect on participation of particular noncovalent interactions in the binding. Apparent high-affinity binding of TCAs to the phosphatidylcholine or phosphatidylethanolamine membranes are achieved mainly by incorporation of uncharged drug molecules into the hydrophobic core of the bilayers. Van der Waals forces and hydrophobic effect are responsible for this binding. Both charged and uncharged drug molecules bind to phosphatidylserine membranes, therefore coulomb- or ion-induced dipole interactions play a role in these binding. Different spatial distribution of charged residues within the interface causes different electrostatic interactions between charged TCAs and vesicles formed from phosphatidylserine and phosphatidylinositol. The data supports the hypothesis under which TCAs could have effect on affective disorders partially via binding to the lipid part of the membrane and following changes of lipid-protein interactions.

Changes in ultraweak photon emission and heart rate variability of epinephrine-injected rats.

Abstract: Ultraweak photons which are spontaneously emitted from a living body may be applicable as a non-invasive tool to characterize the physiological state of the living body. We investigated changes in the intensity of ultraweak photon emission, body temperature and the cardiovascular autonomic activity induced by epinephrine injection to rats. A high dose of epinephrine can make changes to the cardiovascular autonomic activity or body temperature. Photon emission of the dorsal part, rectal temperature and heart rate variability (HRV) were measured from eight Sprague-Dawley rats. The intensities of photon emissions for saline injections, which were used as a control, decreased from 13042+/-71 counts/min at the start of measurements to 8709+/-915 counts/min at 1 h after the injections. In the case with epinephrine injections, the intensity of photon emission reduced slowly from 13361+/-354 counts/min to 11040+/-433 counts/min. Rectal temperature increased in both saline- and epinephrine-injected rats, but one hour after the injections the temperature in the epinephrine case was slightly higher than that in the saline case. The standard deviation of the QRS wave complex interval (RR interval) increased from 1 to 4 (p<0.05) and the spectral ratio of the low frequency component to the high frequency component in the HRV data LF (0.19 approximately 0.74 Hz) / HF (0.78 approximately 2.50 Hz) decreased from 0.81 to 0.26 (p<0.05) in the case of epinephrine injection while no change was found in the case of saline injection. Thus, ultraweak photon emission was closely related to the cardiovascular autonomic activity.

Effect of ajmaline on action potential and ionic currents in rat ventricular myocytes.

Abstract: The effect of ajmaline on action potential (AP) and ionic current components has been investigated in right ventricular myocytes of rat at room temperature using the whole cell patch clamp technique. Ajmaline decreased the upstroke velocity ((dV/dt)max) of AP and the AP amplitude, increased the AP duration measured at 50 and 90% repolarization, and reversibly inhibited most components of membrane ionic current in a concentration-dependent manner. The following values of IC50 and of the Hill coefficient (nH) resulted from approximation of the measured data by the Hill formula: for fast sodium current (INa) IC50=27.8+/-1.14 micromol/l and nH=1.27+/-0.25 at holding potential -75 mV, IC50=47.2+/-1.16 micromol/l and nH=1.16+/-0.21 at holding potential -120 mV; for L-type calcium current (ICa-L) IC50=70.8+/-0.09 micromol/l and n(H)=0.99+/-0.09; for transient outward potassium current (Ito) IC50=25.9+/-2.91 micromol/l and nH=1.07+/-0.15; for ATP-sensitive potassium current (IK(ATP)) IC50=13.3+/-1.1 micromol/l and nH=1.16+/-0.15. The current measured at the end of 300 ms depolarizing impulse was composed of an ajmaline-insensitive component and a component inhibited with IC50=61.0+/-1.1 micromol/l and nH=0.91+/-0.08. At hyperpolarizing voltages, ajmaline at high concentration of 300 micromol/l reduced the inward moiety of time-independent potassium current (IK1) by 36%. The results indicate that the inhibition of INa causes both the decreased rate of rise of depolarizing phase and the lowered amplitude of AP. The inhibition of Ito is responsible for the ajmaline-induced AP prolongation.

Mitochondrial Alterations Induced by 532 nm Laser Irradiation

Abstract: Mitochondrial alterations were monitored after low power green laser (532 nm, 30 mW) irradiation in the case of whole cells (B-14) and isolated mitochondria (from Wistar rat heart). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay products were significantly higher (by 8%) in irradiated B-14 cells as compared to non-irradiated controls. Mitochondrial transmembrane potential of B-14 cells, measured by means of a fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), significantly increased (by 13%) after exposure to green laser irradiation. Another MTT assay was used for isolated mitochondria suspensions in order to examine the effect of green laser irradiation on stimulation of processes related to oxidative phosphorylation. It revealed 31.3%-increase in MTT assay products in irradiated mitochondria as compared to controls. Laser irradiation of isolated mitochondria suspension did not significantly change 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, indicating that mitochondrial membrane fluidity was not affected by laser light. Fluorescence emission spectra of irradiated as well as non-irradiated mitochondria suspensions showed fluorescence maximum at 635 nm, corresponding to emission of Protoporphyrin IX, which was significantly lower (by 20.7%) in irradiated sample.

Thioredoxin - Structural and Functional Complexity

Abstract: Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.

Ischemia-reperfusion injury--antiarrhythmic effect of melatonin associated with reduced recovering of contractility.

Abstract: The effect of melatonin on reperfusion arrhythmias and postischemic contractile dysfunction was studied in the isolated rat heart. 25 min global ischemia was induced and followed by 30 min of reperfusion. Melatonin (10 µmol/l) was present in the perfusion solution during the whole experiment. Experiment revealed protective effect of melatonin on reperfusion-induced arrhythmias - arrhythmia score was significantly lower as well as the total time of arrhythmias duration was significantly shorter in melatonin group than in controls. On the other hand, post- ischemic recovering of contractility was significantly reduced in melatonin group.

Effect of ajmaline on transient outward current in rat ventricular myocytes.

Abstract: The mechanism of ajmaline-induced inhibition of the transient outward current Ito has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of Ito in a concentration-dependent, but frequency- and use-independent manner. We conclude that in the rat right ventricular myocytes ajmaline is an open channel blocker of Ito with fast recovery from the block at resting voltage.

In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy.

Abstract: In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.

Effect of Long-Term Administration of Antidepressants on the Lipid Composition of Brain Plasma Membranes

Abstract: The connection between changes in lipid pattern in brain plasma mem- branes and long-term administration of therapeutically effective doses of antide- pressants has not been sufficiently demonstrated so far. Therefore, we analyzed effect of antidepressants that differ in pharmacological selectivity on membrane lipid composition in the rat brain tissue. Laboratory rats were given desipramine, maprotiline, citalopram, moclobemide or lithium for a 4-week period. We observed a significant decrease in phosphatidylethanolamine representation after adminis- tration of maprotiline, citalopram and moclobemide when compared with controls. Membrane cholesterol content was decreased after desipramine administration and increased after citalopram or lithium treatment. Electroneutral phospholipids were decreased after the administration of all tested antidepressants except for de- sipramine. Decrease in phosphatidylserine was found following long-term adminis- tration of maprotiline or desipramine; relative representation of phosphatidylinosi- tol was reduced after lithium treatment. Statistically significant negative correlation between cholesterol and electroneutral phospholipids was discovered. Membrane microviscosity evaluated by fluorescence anisotropy of membrane probes was only slightly decreased after desipramine and increased after citalopram administration. Hypothesis was supported that changes in brain neurotransmission produced by antidepressants could be, at least partially, associated with adaptive changes in membrane cholesterol and phospholipids.

Effects of vitamin C on liver enzymes and biochemical parameters in rats anesthetized with halothane.

Abstract: Halothane is an important human and veterinary anesthetic, which produces free radicals during biotransformation. Occasionally, these free radicals may cause hepatic injury, especially in case of multiple halothane exposures over short periods. Vitamin C may protect cellular lipids and lipoproteins against oxidative damage by the free radicals. This study investigated the effects of vitamin C on liver enzymes and other biochemical parameters in rats anesthetized with halothane. One group of rats was used as a control, and saline (0.9% NaCl) was injected intraperitoneally into these animals as a placebo. The second group of rats was used as an anesthesia control group and was only anesthetized by halothane for 2 h. The third group was anesthetized by halothane and injected vitamin C intraperitoneally. Activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase enzymes were significantly increased (p < 0.05, p < 0.01, p < 0.05, respectively) by halothane anesthesia, but decreased (p < 0.05, p < 0.05, p < 0.05, respectively) with administration of vitamin C. Concentrations of triglycerides, cholesterol, total bilirubin and creatinine were statistically affected (p < 0.05, p < 0.01, p < 0.05, and p < 0.01, respectively) by injection of vitamin C. Values of erythrocyte counts, packet cell volumes, hemoglobin concentration, leukocyte counts, rates of neutrophils and lymphocytes were significantly affected (p < 0.01, p < 0.05, p < 0.05, p < 0.01, p < 0.001 and p < 0.01, respectively) by halothane anesthesia. The values of erythrocyte counts, leukocyte counts, neutrophil and lymphocyte rates were significantly decreased (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.01, respectively) with administration of vitamin C. Based upon these results, vitamin C may play an important role in the prevention of hepatic cellular injury inflicted by halothane anesthesia.

Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats.

Abstract: The effect of chronic exposure to light of adult Wistar rats on growth and function of adrenal zona glomerulosa (ZG) and zona fasciculata (ZF) were examined. The females were exposed to continuous light of 600 lux for 95 days, starting on day 30 of age. The controls were kept under a 12:12 h light-dark cycle, at ambient temperature. The rats were sacrificed by decapitation and the left adrenal gland of each animal was dissected out and prepared for morphometric analyses. In animals exposed to chronic lighting, the absolute and relative volume of ZG were insignificantly increased by 5% (p>0.05) compared to controls. The volume of ZG cells and their nuclei were insignificantly changed by 1% (p>0.05) in comparison with corresponding controls. The absolute and relative volume of ZF were significantly increased (by 14 and 9%, respectively; p<0.05), as compared to controls. The volume of ZF cells and their nuclei were significantly increased (by 12 and 9%, respectively; p<0.05). Serum concentration of corticosterone was also significantly (p<0.05) increased by 13% in light-exposed group in comparison with control rats. These findings suggest that continuous exposure of female rats to constant light increased growth and secretory activity of ZF cells.

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus.

Abstract: Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.

Effects of stress and of amphetamine on passive avoidance conditioning in rats.

Abstract: This study examined the effects of immobilization stress combined with water immersion (ICS) and/or amphetamine (AM) on different memory phases in the passive avoidance task in rats. The performance of rats was evaluated in the retention tests 24 and 48 h after a single acquisition trial. ICS exposure lasting 1 h impaired retention of the learned avoidance response if applied 2 to 4 h before or immediately after training. The stressor did not affect retrieval if presented 5 or 2 h before the retention test. AM was used i.p. at the dose of 8 or 1 mg/kg. Neither 8 mg AM administered 4 h before nor 8 or 1 mg doses given after training did not impair the retention performance in unstressed rats. The 1 mg AM prevented the impairment of retention in animals exposed to the stressor 3 or 4 h before training but had no effect when the stronger impairment was induced by ICS 2 h before training. However, when given 1 h before retention testing, 1 mg AM attenuated even the severe impairment induced by the pre-training stressor exposure. Our results suggest that ICS impairs primarily the early phase of memory consolidation and a low dose of AM can prevent this effect.

ESR and monolayer study of the localization of coenzyme Q10 in artificial membranes.

Abstract: 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 hy- drophilic 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 pres- ence 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.

Effect of pharmacologically selective antidepressants on serotonin uptake in rat platelets.

Abstract: We tested a hypothesis that a long-term administration of antidepres- sants acting through different primary biochemical mechanisms is associated with changes in the platelet serotonin (5-hydroxytryptamine, 5-HT) transport. Labo- ratory rats were administered norepinephrine reuptake inhibitors (desipramine, maprotiline), selective 5-HT reuptake inhibitor (citalopram), reversible monoamine oxidase inhibitor (moclobemide), and lithium (inositol monophosphatase inhibitor among others) during a 4-week period. Apparent kinetic parameters of platelet 5-HT transport were analyzed. Significant decrease in apparent Michaelis constant (KM) was found after the administration of all tested antidepressants except for desipramine. There was certain increase in maximal velocity (Vmax) values follow- ing the administration of desipramine, maprotiline, and citalopram; however, the all Vmax changes were not significant. Vmax/KM ratio representing limiting perme- ability at low extracellular concentrations of 5-HT was systematically increased in all the tested drugs, but significant changes were occurred only in maprotiline- and citalopram-treated rats. Adaptive changes in platelet 5-HT transport induced by citalopram were opposite to the acute inhibitory effect of this drug on 5-HT trans- porter activity. An increase in limiting membrane permeability for 5-HT could be included in the common adaptive effect of the long-term administration of antide- pressants that differ in pharmacologic selectivity.

Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences.

Abstract: Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.

The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart.

Abstract: The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by N ω -nitro-L- arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihy- droguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n = 18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during con- trol conditions, in the presence of NO synthesis inhibitor L-NAME (30 µmol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered sep- arately or in combination. CF varied in autoregulatory range from 4.12 ± 0.26 ml/min/g wt at 50 cm H2 Ot o 5.22± 0.26 ml/min/g wt at 90 cm H2O. In au- toregulatory range, nitrite outflow varied from 2.05 ± 0.17 nmol/min/g wt at 50 cm H2 Ot o 2.52± 0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22 ± 0.12 ml/min/g wt and 2.90 ± 0.25 ml/min/g wt, respec- tively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56 ± 0.11 nmol/min/g wt at 40 cm H2 Ot o 1.45± 0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42 % at 50 cm H2O to 12 % at 90 cm H2O, respectively) with no significant changes in nitrite out- flow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34 % at 50 cm H2O to 50 % at 90 cm H2O, respec- tively) with parallel changes in nitrite outflow (from 40 % at 50 cm H2 Ot o 51 % at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA)

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats.

Abstract: Neonatal STZ (nSTZ) treatment results in damage of pancreatic B- cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secre- tion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 µg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro .T he ex- pression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glu- cose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.

Effect of two distinct stressors on gene expression of the type 1 IP3 receptors.

Abstract: Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers, which triggers calcium release from intracellular pools via IP3 receptors. Previously we have shown that single immobilization stress increased gene expression of both, the type 1 and type 2 IP3 receptors (IP3R1 and IP3R2, respectively). In this study we evaluated whether long-term exposure to softer stressor (cold exposure to 4 ◦ C) can affect the response to single immobilization stress. We examined modulation of the type 1I P3 receptor gene expression by each stressor separately, and then in their combination. Rats were immobilized for 30 and 120 min and were decapitated immediately or 3 h after immobilization. Cold stress was performed by exposure of animals to 4 ◦ C temperature for 1, 7 and 28 days. To determine the effect of both stressors in combination, animals exposed to cold for 28 days were afterwards exposed to immobilization for 120 min and decapitated 3 h after the end of stressful stimulus. Our results verify that single immobilization increases the IP3R1 gene expres- sion in left atria of rat heart, while cold stress elevates the level of gene expression only after the exposure to cold for 7 days. The exposure to cold for 28 days did not increase the gene expression of the type 1 IP3 receptor compared to control. Application of both stressors (28 days of cold exposure followed by 120 min of immobilization with subsequent 3 h rest) showed the tendency of increased IP3R1 gene expression compared to absolute, nonstressed control, but level of the type 1 IP3 receptor mRNA was significantly lower compared to mRNA levels of solely im- mobilized animals. Thus, cold exposure affects the response of the gene expression of the type 1I P3 receptor to immobilization stress.

Relevance of ventricular electrical dispersion to arrhythmogenesis in ischemic myocardium--a simulation study.

Abstract: 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 + -Ca 2+ 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.

On the Derivation of the Kargol's Mechanistic Transport Equations from the Kedem-Katchalsky Phenomenological Equations

Abstract: In the present article, it was demonstrated that - by starting from the so-called adjusted Kedem-Katchalsky (KK) phenomenological equations (Sucha- nek et al. 2004), i.e. the equations: Jv = Lp∆P − LpD∆Π JD = −LDp∆P + LD∆Π it is possible to derive practical transport equations (for the volume flow and the solute flow) in the form of the Kargol's mechanistic transport equations (Kargol and Kargol 2000, 2001, 2003a,b,c, 2004; Kargol 2002). On this basis, it has been found that the KK thermodynamic formalism for membrane transport (practical equations) is in general identical with the mecha- nistic equations for membrane transport.

Binding of a 23 kD endonuclease to the rat liver nuclear matrix.

Abstract: In a previous paper we have described a 23 kD nuclear endonuclease (p23) that was mostly found to exist in a state of association with the isolated rat hepatocyte nuclear matrix. To investigate the nature of this interaction, the nuclear matrix was prepared using different procedures and examined for the presence/absence of the enzyme by activity gel analysis. Treatment of isolated nuclei with sodium tetrathionate (NaTT), a sulfhydryl-cross-linking agent, led to the complete recovery of p23 in the nuclear matrix, whereas incubation of nuclei with dithiothreitol (DTT), a sulfhydryl-reducing agent, led to its complete solubilization and resulting absence from the nuclear matrix. Exposure of the isolated nuclear matrix to DTT in high-ionic strength buffer, a procedure that promotes the solubilization of the internal nuclear matrix, caused the nearly complete solubilization of p23. It was concluded that disulfide bonds play an essential role in the association of p23 with the nuclear matrix and that p23 is mostly localized in the nuclear matrix interior.

Identification of all alpha1-adrenoceptor subtypes in rat lung.

Abstract: The function of lung tissue is regulated via a release of neurotransmitters from autonomic nerves. The neurotransmitters of sympathetic nervous system, adrenaline and noradrenaline, activate both alpha-adrenoceptors and beta-adrenoceptors. Although the function and expression of beta-adrenoceptors can be considered major, some doubts exist about the function and expression of alpha1-adrenoceptor subtypes in the lung tissue. Therefore a set of competition binding experiments was employed in order to discriminate between the alpha1-adrenoceptor binding site subtypes in the rat lung. We identified three subpopulations of alpha1-adrenoceptor binding sites in the rat lung (alpha1A, alpha1B and alpha1D).

Tapering of human nerve fibres.

Abstract: 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 alpha-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 gamma-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 microm would be reduced to approximately 9.8 microm 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 alpha1-motoneuron (FF) axons increased from the thoracic towards the lumbo-sacral region before decreasing again in the lower sacral region. Usually no alpha1-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 microm. The unmyelinated fibres with diameters around 0.25 microm 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.

Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics.

Abstract: 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.

Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells.

Abstract: 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 re- vealed that only a higher concentration of pyrithiamine, which accelerates the in- tracellular 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.