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Zoznam článkov

General Physiology and Biophysics

Volume 24, 2005, No. 3


  Controversy of free radical hypothesis: reactive oxygen species – cause or consequence of tissue injury?
I Juránek 1), Š Bezek

1)Ivo Juránek, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovakia.

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, i.e. 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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 263-278.

  pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus
G Žoldák 1), A Musatov, M Stupák, M Sprinzl, E Sedlák

1)Department of Biochemistry, Faculty of Science P. J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovakia.

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 ∼ 6.0 and is localized in proximity (<10 Å) 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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 279-298.

  Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats
V Milošević 1), S Trifunović, M Sekulić, B Šošić-Jurjević, B Filipović, N Negić, N Nestorović, M Manojlović Stojanoski, V Starčević

1)Institute for Biological Research “Siniša Stankovi&cacute, ”, Despota Stefana 142, 11060 Belgrade, Serbia and Montenegro.

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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 299-309.

  Effect of ajmaline on action potential and ionic currents in rat ventricular myocytes
M Bébarová 1), P Matejovič, M Pásek, M Šimurdová, J Šimurda

1)Department of Physiology, Faculty of Medicine, Masaryk University, Komenského nám. 2, 662 43 Brno, Czech Republic.

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 µmol/l and nH = 1.27 ± 0.25 at holding potential –75 mV, IC50 = 47.2 ± 1.16 µmol/l and nH = 1.16 ± 0.21 at holding potential -120 mV; for L-type calcium current (ICa-L) IC50 = 70.8 ± 0.09 µmol/l and nH = 0.99 ± 0.09; for transient outward potassium current (Ito) IC50 = 25.9 ± 2.91 µmol/l and nH = 1.07 ± 0.15; for ATP-sensitive potassium current (IK(ATP)) IC50 = 13.3 ± 1.1 µmol/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 µmol/l and nH = 0.91 ± 0.08. At hyperpolarizing voltages, ajmaline at high concentration of 300 µmol/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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 311-325.

  In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy
K Likavčanová 1), D Dobrota, T Liptaj, N Prónayová, V Mlynárik, V Belan, M Galanda, A Béreš, J De Riggo

1)Department of Biochemistry, Jessenius Faculty of Medicine, Malá Hora 4, 037 54 Martin, Slovakia.

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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 327-335.

  Computer-aided formation of the whole-cell patch-clamp recording configuration
P Novák 1), I Zahradník

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

The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 337-348.

  Identification of all α1-adrenoceptor subtypes in rat lung
M Nováková 1), J Mysliveček

1)Institute of Physiology, 1st Faculty of Medicine, Charles University, Albertov 5, 128 00 Prague, Czech Republic.

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 α-adrenoceptors and β-adrenoceptors. Although the function and expression of β-adrenoceptors can be considered major, some doubts exist about the function and expression of α1-adrenoceptor subtypes in the lung tissue. Therefore a set of competition binding experiments was employed in order to discriminate between the α1-adrenoceptor binding site subtypes in the rat lung. We identified three subpopulations of α1-adrenoceptor binding sites in the rat lung (α1A, α1B and α1D).

General Physiology and Biophysics. Volume 24, 2005, No. 3: 349-353.

  Ischemia-reperfusion injury – antiarrhythmic effect of melatonin associated with reduced recovering of contractility
R VaŽan 1), D Pancza, I Béder, J Styk

1)Department of Physiology, Faculty of Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava 1, Slovakia.

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.

General Physiology and Biophysics. Volume 24, 2005, No. 3: 355-359.