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General Physiology and Biophysics

Volume 29, 2010, No. 3


  Why the xanthine derivatives are used to study of P‐glycoprotein‐mediated multidrug resistance in L1210/VCR line cells
Peter Dočolomanský 1), Viera Bohacova, Miroslav Barancik, Albert Breier

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

There is generally well known that various xanthines occur frequently in natural products, e.g. black coffee, black tea, green tea, natural dyes etc. Xanthine molecules are good tolerated and metabolised by organisms. Moreover, natural xanthines and/or sythesized xanthines may recall a lot positive affects (hemorheologic properties, anti‐inflammatory properties, tracheal smooth muscle relaxant, positive chronotropic and central nervous system‐stimulating, etc.) and may even induce a quantity of changes on the molecular level (inhibition of cyclic nucleotide phosphodiesterases, inhibition of the synthesis of tumor necrosis factor (TNF‐α), cellular Ca2+ homeostasis, etc.). In our previous paper we showed that some xanthine derivatives (pentoxifylline and its derivatives) depress P‐glycoprotein (P‐gp) mediated multidrug resistance of the mouse leukemic cells. Other authors, first of all Sadzuka and co‐workers, confirm this usefulness of long side substituted xanthines as biochemical modulators. However, the mechanism of molecular action of xanthine derivatives has not been clarified. One of the possible ways to chemosensitize the cancer cells is direct competiting in defence mechanism – inhibition of efflux pump (P‐gp). Interaction of xanthine derivatives with binding site of P‐gp is a question which could be solved by experiment; although, molecular modelling may clear up this matter. But, each dynamic and static program for molecular simulation of P‐gp action is dividing on input variable, considering mechanistic view of insight drug transport.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 215-221.

  Hypoxia causes connexin 43 internalization in neonatal rat ventricular myocytes
Asaf Danon 1), Naama Zeevi-Levin, Dani Pinkovich, Tomer Michaeli, Alexander Berkovich, Moshe Flugelman, Yonina Eldar, Michael Rosen, Ofer Binah

1)Department of Physiology, Ruth and Bruce Rappaport Faculty of Medicine, Technion‐Israel Institute of Technology, P.O.Box 9649, Haifa 31096, Israel.

Gap junctions produce low resistance pathways between cardiomyocytes and are major determinants of electrical conduction in the heart. Altered distribution and function of connexin 43 (Cx43), the major gap junctional protein in the ventricles, can slow conduction, and thus contribute to arrhythmogenesis in experimental models such as ischemic rat heart and pacing‐induced atrial fibrillation. The mechanisms underlying reduced gap junctional density and conductance during ischemia may involve decreased Cx43 synthesis, increased degradation and/or Cx43 migration into areas which do not contribute to intercellular communication. To test more rigorously the hypothesis that hypoxia resulting from ischemia causes Cx43 internalization, we infected neonatal rat ventricular myocytes (NRVM) with a Cx43‐GFP chimera, which enabled us to investigate by means of confocal microscopy the effect of hypoxia (1% O2 for 5 h) on Cx43 distribution in live cardiomyocytes. Importantly, this protocol permitted each culture to serve as its own control. To this end we used life confocal microscopy analysis to determine in the same pair of myocytes the effects of hypoxia on Cx43‐GFP distribution at the gap junctional (GJ) and non‐GJ areas. In support of this hypothesis, we found that compared to normoxia, 5 h of hypoxia reduced the Cx43‐GFP signal at the GJ areas (defined as the border area) and caused a corresponding increase in the Cx43‐GFP signal at the non‐border areas, thus providing an additional explanation for the intercellular uncoupling during ischemic conditions.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 222-233.

  A possible role of NF‐κB and HSP72 in skeletal muscle hypertrophy induced by heat stress in rats
Yoshitaka Ohno 1), Sumio Yamada, Takao Sugiura, Yoshinobu Ohira, Toshitada Yoshioka, Katsumasa Goto

1)Laboratory of Physiology, School of Health Sciences, Toyohashi SOZO University, 20-1 Matsushita, Ushikawa, Toyohashi, Aichi 440-8511, Japan.

Effects of heat stress on phosphorylated nuclear factor‐κB (phospho‐NF‐κB) and tumor necrosis factor α (TNFα) contents in skeletal muscles were studied. Male Wistar rats (7‐week‐old) were randomly assigned to control and heat‐stressed groups. Rats in heat‐stressed group were exposed to heat stress (42°C for 60 min) in an incubator without anesthesia. Soleus muscles were dissected and weighted 1, 3, and 7 days after the heat exposure. Significant increases in the wet weight and protein content of soleus were observed 7 days following the exposure (p < 0.05). Heat stress also induced the up‐regulation of heat shock protein 72 (HSP72), IκBα (inhibitor of NF‐κB) and the increase in the relative population of Pax7‐positive satellite cells to total muscle nuclei before the increase in muscle mass. The content levels of phospho‐NF‐κB and TNFα were significantly decreased 1 and 3 days after heat stress, respectively (p < 0.05). A negative correlation between HSP72 and phospho‐NF‐κB contents was observed 1 day after the heat exposure. These observations suggest that the decrease in NF‐κB signaling may play a part of a role in heat stress‐associated muscle hypertrophy.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 234-242.

  Three methods for estimation of changes in frequency characteristics of potentials elicited by long‐lasting (fatiguing) activity of isolated muscle fibres
Teodora Vukova 1), Vladimir Dimitrov, Nikolina Radicheva

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

The present study was assigned to compare the applicability and sensitivity of three different methods (discrete wavelet transforms (DWT), median frequency (MDF) – calculated on the basis of fast Fourier transform, and spectral indices) for analysis of frequency content changes in potentials produced by repetitive stimulation from isolated slow (SMFs)‐ and fast (FMFs)‐fatigable muscle fibers during uninterrupted activity. In order to affect fatigue, prior to starting the fatiguing stimulation (5 Hz), some of the fibres were exposed to microwave electromagnetic field and others were sham exposed. All the methods studied demonstrated that during a long‐lasting fibre activity, the shift of the potential frequency content to lower frequencies was a consequence of lowering in higher frequency components and increasing in low frequency ones. The changes were faster in FMFs than SMFs and stronger in sham exposed fibres vs. microwave exposed. The MDF alterations in both fibre types and protocols were quite similar to other methods characterizing either individual changes in low and high frequency scales of the signal (discrete wavelet transform – DWT) or changes in their spectral ratio (spectral indices). The spectral indices showed sensitivity to fatigue effect at the final stages that made their changes considerably non‐linear. DWT allowed processing the temporal occurrence of frequency content changes that corresponded to the alterations in the separate potential phases as well as earlier detection and precise evaluation of fatigue onset and progressing. All methods are applicable but DWT is a preferable for study of muscle fibre fatigue in clinical and experimental neurophysiology.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 243-254.

  Rate of oxidative modification of cytochrome c by hydrogen peroxide is modulated by Hofmeister anions
Nataša Tomášková 1), Lenka Varinská, Erik Sedlák

1)Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Košice, Slovakia.

Cytochrome c (cyt c) and other heme proteins are oxidatively modified in the presence of hydrogen peroxide in a concentration‐ and time‐dependent manner. Cyt c modification has been monitored by several spectral probes by absorption spectroscopy (at wavelengths 410 nm, 530 nm), and circular dichroism (222, 268, 288 and 417 nm). Kinetics monitored with these spectral probes indicates that the oxidative modification of cyt c: i) proceeds in the order: heme → aromatic amino acids → secondary structure, and ii) the rate of the oxidative modification is proportional to the protein flexibility. The flexibility of cyt c was modulated by anions of Hofmeister series (sulfate, chloride, perchlorate) (Varhač et al. 2009). A minimalist scheme of the interaction of cyt c with hydrogen peroxide can be described by two steps: 1) interaction of hydrogen peroxide with heme iron forming the postulated ferryl intermediate, 2a) oxidation of another molecule of hydrogen peroxide and 2b) parallel oxidation of close amino acid residue(s) and/or heme. The catalase activity of cyt c is independent from the presence of Hofmeister anions, which indicates that both steps (1 and 2a) in the catalase reaction are independent from the flexibility of the heme region of the protein matrix. On the other hand, the flexibility of the polypeptide chain of the protein modulates the rate of parallel oxidative modification of the heme and amino acid residues.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 255-265.

  Influence of sub‐chronic diabetes mellitus on functional properties of renal Na+,K+‐ATPase in both genders of rats
Veronika Javorková 1), Lucia Mézešová, Jana Vlkovičová, Norbert Vrbjar

1)Institute for Heart Research, Department of Biochemistry, Slovak Academy of Sciences, Dúbravská cesta 9, P.O.Box 104, 840 05 Bratislava 45, Slovakia.

For characterization of Na+,K+‐ATPase, a key enzyme involved in maintenance of intracellular sodium homeostasis, expression of α1 subunit and the ATP‐ and Na+‐binding properties were investigated by Western blot analysis and by enzyme kinetics, respectively. Previous studies documented time‐dependent alteration of properties of renal Na+,K+‐ATPase from its mobilization after 8 days to serious deteriorations after 16 weeks of diabetes in rats. Characterizing the critical period during development of the disease, when mobilization of Na+,K+‐ATPase observed in the acute phase turns to its damage, we examined the enzyme properties after 8 weeks lasting diabetes which was induced by a single intraperitoneal administration of streptozotocin in a dose of 65 mg·kg−1. The unchanged expression of Na+,K+‐ATPase α1‐subunit in both genders indicates that 8 weeks represent the time when the mobilization of enzyme synthesis observed previously in acute diabetes is lost. In this time the renal Na+,K+‐ATPase undergoes structural changes in the vicinity of Na+‐binding site resulting in worsened affinity to sodium in both genders as indicated by 13% and 18% increase of KNa value in female and male rats, respectively. However, gender specific was the diabetes‐induced decrease in affinity to ATP by 18% which occurred in female rats only.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 266-274.

  Exposure to fractionated dose of 60 Gy affects molecular response of HL‐60 cells to irradiation
Jiřina Vávrová 1), Aleš Tichý, Martina Řezáčová, Lenka Zárybnická, Zuzana Šinkorová, Jan Österreicher

1)Department of Radiobiology, Faculty of Health Sciences, Hradec Králové, University of Defence Brno, Czech Republic.

In this work we evaluated changes in molecular response of human promyelocyte leukemia cells HL‐60 and HL‐60‐IR cells (HL‐60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months) to irradiation by the dose of 2 and 8 Gy. Analysis of CD11b and apoptosis by flow‐cytometry revealed that on 3rd day after irradiation by 8 Gy the HL‐60‐IR are more resistant to radiation‐induced apoptosis and more differentiated (increase in CD11b in non‐apoptotic cells) than regular HL‐60. We found that both types of cells have high basal level of phosphorylated extracellular signal‐regulated kinases Erk1/2 . Irradiation induces decrease in Erk1/2 phosphorylation after 4 and 8 h in both cell types. However, in HL‐60‐IR cells Erk1/2 phosphorylation is restored faster than in HL‐60. Also it was found that in contrary to HL‐60 cells, the HL‐60‐IR cells react to 2 Gy irradiation by p53 independent increase in p21(WAF1/Cip1), and not by activation of checkpoint kinase Chk‐2. Therefore we concluded that relatively high dose of radiation (6 Gy) does not lead after 10 repetitive irradiations to eradication of HL‐60 cells, but instead increases their radioresistance, increases the ability to differentiate, alters MAPK response, increases amount of p21(WAF1/Cip1), and decreases induction of apoptosis by ionizing radiation. p21(WAF1/Cip1) might prevent apoptosis induction and trigger permanent cell‐cycle arrest, which can also contribute to regression of this leukaemia after therapy.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 275-281.

  An investigation of the heart rate, heart rate variability, cardiac ions, troponin‐I and CK‐MB in men exposed to 1.5 T constant magnetic fields
Cemil Sert 1), Zeynep Aktı, Öcal Sırmatel, Remzi Yılmaz

1)Department of Biophysics, Faculty of Medicine, Harran University, Şanlıurfa, Turkey.

The aim of this study was to investigate heart rate (HR), heart rate variability (HRV) and other cardiac parameters in individuals who were exposed to a high static magnetic field. 30 healthy male volunteers aged between 20–30 years were included. The searching was divided into three phases: pre‐magnetic field, during the magnetic field and post‐magnetic field. Every phase lasted 30 minutes. Pulse, systolic and diastolic blood pressure, respiration rate and elektrocardiography (ECG), recorded for 30 minutes, in all of the individuals were measured during three phases. The men were exposed to a 1.5 T static magnetic field. The levels of Na+, K+, Ca2+, Cl, CK‐MB, troponin‐I and HR and HRV parameters were investigated. There was an increase in the respiration rate, and no change in the systolic and diastolic blood pressure and pulse in the individuals exposed to the magnetic field. There was also an enhancement in the values of ions, CK‐MB and troponin‐I after exposure to the magnetic field. Heart rate parameters (minimum HR‐I, maximum HR‐I, average HR‐I) were decreased and rMSSD, pNN50, power, VLF, HF, LF values increased during the magnetic field.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 282-287.

  Effects of acute exposure to static magnetic field on ionic composition of rat spinal cord
Elferchichi Miryam 1), Lahbib Aida, Missaoui Samira, Sakly Mohsen, Abdelmelek Hafedh

1)Laboratoire de Physiologie Integrée, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisia.

The purpose of this study is to evaluate the effect of static magnetic fields (SMF) on ionic composition of rat spinal cord. Male Wistar rats were daily exposed to SMF of 128 mT, for 1 h/day during five consecutive days. Spinal cord samples were extracted, weighed and mixed in bidstilled water in order to be analyzed by inductively coupled plasma (ICP). Plasma ionic composition was also made. Acute exposure to SMF increased significantly the calcium (+68%, p < 0.05) and iron (+35%, p < 0.05) contents of rat spinal cord, whereas magnesium and copper levels remained unchanged. In plasma, SMF treatment failed to alter calcium concentration but decreased iron level (−17%, p < 0.05). These data indicate that SMF acute exposure can induce alterations of ionic composition in the rat spinal cord and the plasma electrolytes balance.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 288-294.

  A mechanistic interpretation of root transport of water
Grazyna Suchanek 1)

1)Independent Department of Environmental Protection, The Jan Kochanowski University of Humanities and Sciences, Świetokrzyska 15, 25-406 Kielce, Poland.

The present paper offers a mechanistic interpretation of filtration coefficients Lpr in isolated maize roots, measured with the use of the root pressure probe by Steudle and others (Steudle 1990, 1992; Steudle and Brickmann 1989; Steudle and Frensch 1989; Steudle and Jeschke 1983; Steudle et al. 1993) on the basis of the Kedem‐Katchalsky equations. Detailed investigations have been based on the mechanistic equations of solute and solvent membrane transport across porous membranes (Kargol 2001; Kargol and Kargol 2003a,b). It must be stressed that transport equations of both these (thermodynamic and mechanistic) formalisms are mutually compatible, which has been demonstrated for instance in the works Kargol and Kargol (2003) and Suchanek (2005, 2006).

General Physiology and Biophysics. Volume 29, 2010, No. 3: 295-301.

  Accelerated removal of deamidated proteins and endogenous electric fields: possible implications
Isabella Panfoli 1), Silvia Ravera, Daniela Calzia, Alessandro Morelli

1)Department of Biology, University of Genova, V.le Benedetto XV 3, 16132 Genova, Italy.

Changes in protein structure through the spontaneous deamidation of asparaginyl (Asn) and glutaminyl (Gln) residues have been observed in many proteins. Amide residues were supposed to serve as clocks for development and aging. Deamidated proteins are rapidly degraded by as yet unclear molecular mechanisms. Deamidation leads to elevation of the ratio of charged versus polar residues (CH‐PO) of a protein and to a decrease in its pI value. We had reported that those enzymes, characterized by a high CH‐PO, are prone to inactivation and loss of ordered structure by exposure to direct current from low voltage in solution. Nano‐local endogenous electric fields arise within cells. Endogenous currents may cause the unfolding of the products of deamidation at Asn. In turn, these unfolded proteins would be removed, likely by proteolysis.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 302-308.

  Drug‐induced changes in action potential duration are proportional to action potential duration in rat ventricular myocardium
László Bárándi 1), Gábor Harmati, Balázs Horváth, Norbert Szentandrássy, János Magyar, András Varró, Péter Nánási, Tamás Bányász

1)Department of Physiology, University of Debrecen, Nagyerdei krt 98, H-4012 Debrecen, Hungary.

Several cardioactive agents exhibit direct or reverse rate‐dependent effects on action potential duration (APD) depending on the experimental conditions. Recently, a new theory has been proposed, suggesting that the reverse rate‐dependent mode of drug‐action may be a common property of canine, rabbit, guinea pig and human cardiac tissues, and this phenomenon is based on the dependence of drug‐action on baseline APD. The aim of the present work was to examine the limitations of this hypothesis by studying the APD lengthening effect of K+ channel blockers and the APD shortening effect of Ca2+ channel blockers during the electrical restitution process of rat ventricular action potentials. Rat ventricular muscle was chosen because it has a set of ion currents markedly different from those of other species, its APD is shorter by one order of magnitude than that of the "plateau‐forming" larger mammals, and most importantly, its APD increases at higher heart rates – opposite to many other species. The restitution of APD was studied as a function of the diastolic interval, a parameter indicating the proximity of action potentials. It was found that drug‐induced APD changes in rat myocardium are proportional with the pre‐drug value of APD but not with the diastolic interval, indicating that not the proximity of consecutive action potentials, but the baseline APD itself may determine the magnitude of drug‐induced APD changes.

General Physiology and Biophysics. Volume 29, 2010, No. 3: 309-313.