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General Physiology and Biophysics

Volume 28, 2009, No. 3


  Architectural and functional remodeling of cardiac and skeletal muscle cells in mice lacking specific isoenzymes of creatine kinase
Lucia Tylková 1)

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

Muscle is the major consumer of fuels and ATP in the body. Mitochondria and glycolytic complexes serve as the main energy production locations, while the highest energy demands are associated with the sarcoplasmic reticulum, myofibrillar compartments and plasma membrane. Creatine kinase (CK) is a dimeric protein, which is deeply involved in the production of high energy storage compounds. This enzyme reversibly phosphorylates creatine (Cr) to phosphocreatine (PCr), and it is also highly adapted to specialized muscle function. To date, four major isoenzymes of CK have been identified, two of which occur in the cytosol and two in mitochondria. Disruption of the phosphotransfer system induced by an absence of either the sarcomeric mitochondrial CK or cytosolic CK or both isoenzymes of CK (CK–/–) in muscle cells leads to morphological and functional adaptations towards preservation of muscle contractile abilities. Remodeling of the cell ultrastructure observed in CK–/– cardiomyocytes and glycolytic fibers was associated with direct transfer of energy from places of energy production to locations of energy utilization. This direct interaction among the organelles can maintain a high ATP/ADP ratio near the cellular ATPases when CK is not functionally active. This review summarizes the function and role of CK across different muscle cells in knockout mice.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 219-224.

  Effect of 7-nitroindazole on the expression of intracellular calcium channels in the kidney of spontaneously hypertensive rats
Barbora Sedlakova 1), Sona Cacanyiova, Karol Ondrias, Frantisek Kristek, Olga Krizanova

1)Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia.

The spontaneously hypertensive rats (SHR) were fed with nitric oxide synthase (NOS) blocker 7-nitroindazole (7-NI, 10 mg/kg/day) for 6 weeks and an expression of intracellular calcium channels, SERCA and proapoptotic agents was evaluated in kidney. Treatment of rats with 7-NI resulted in a significant increase in mRNA and protein levels of the IP3 receptors type 1 and type 2, while mRNA levels of the IP3 receptor type 3 remained unchanged. The mRNA of other intracellular calcium channels, ryanodine receptors type 1 and type 2 was also upregulated by 7-NI treatment. Gene expression of the SERCA2a, calcium pump responsible for loading intracellular stores with calcium, revealed increased gene expression due to 7-NI as well. Interestingly, proapoptotic agents caspase 3 and Bax were also upregulated by the 7-NI treatment. These results may indicate that nNOS blocker 7-NI modifies intracellular calcium transport system, which may have impact on altered calcium handling and regulation of various metabolic pathways.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 225-232.

  D-pinitol attenuates the impaired activities of hepatic key enzymes in carbohydrate metabolism of streptozotocin-induced diabetic rats
Selvaraj Sivakumar 1), Sorimuthu Subramanian

1)Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, Tamilnadu, India.

During diabetes mellitus, endogenous hepatic glucose production is increased as a result of impaired activities of the key enzymes of carbohydrate metabolism, which leads to the condition known as hyperglycemia. D-pinitol, a bioactive constituent isolated from soybeans, has been shown to reduce hyperglycemia in experimental diabetes. We therefore designed this study to investigate the effect of oral administration of D-pinitol (50 mg/kg b. w. for 30 days) on the activities of key enzymes in carbohydrate and glycogen metabolism in the liver tissues of streptozotocin-induced diabetic rats. The efficacy was compared with glyclazide, a standard hypoglycemic drug. Oral administration of D-pinitol to diabetic group of rats showed a marked decrease in the levels of blood glucose, glycosylated hemoglobin and an increase in plasma insulin and body weight. The activities of the hepatic enzymes such as hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glycogen synthase and hepatic glycogen content were significantly (p < 0.05) increased whereas the activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, lactate dehydrogenase and glycogen phosphorylase were significantly (p < 0.05) decreased in diabetic rats treated with D-pinitol. The results suggest that alterations in the activities of key metabolic enzymes of carbohydrate metabolism could be one of the biochemical rationale by which D-pinitol attenuates the hyperglycemic effect in diabetic rats.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 233-241.

  Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain
Jozef Burda 1), Mariluz Viadel, Viera Danielisova, Miroslava Nemethova, Carmina Montoliu, Vicente Felipo

1)Institute of Neurobiology, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.

The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 242-248.

  Haloperidol moderately inhibits cardiovascular L-type calcium current
Bohumila Tarabová 1), Marie Novakova, Ľubica Lacinová

1)Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia.

Effects of haloperidol on L-type CaV1.2 channel were studied. Calcium current was measured in whole cell patch-clamp using calcium as a charge carrier. Inhibition by haloperidol was investigated in CaV1.2 channel natively expressed in rat cardiac myocytes and recombinant cardiac (CaV1.2a) and vascular (CaV1.2b) splice variants of the channel expressed in HEK 293 cells. Haloperidol inhibited L-type calcium current in a concentration-dependent manner with a threshold of 1 nmol/l. 1 µmol/l haloperidol inhibited 20.6 ± 3.6% of calcium current amplitude in cardiomyocytes, 25.4 ± 2.6% of current amplitude through the CaV1.2b channel and 28.0 ± 2.7% of current through the CaV1.2a channel. Inhibition was not accompanied by alteration of current waveform or by shift of current-voltage relation. In a current clamp haloperidol suppressed action potential generation. 1 µmol/l of the drug shortened the action potential duration in part of the cells and suppressed fully action potential in other cells. Moderate inhibition of the L-type calcium channels by haloperidol might cause shortening of action potential. Complete abolishment of action potential must have been mediated by inhibition of another, likely sodium channel.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 249-259.

  Effects of static magnetic field exposure on antioxidative enzymes activity and DNA in rat brain
Salem Amara 1), Thierry Douki, Catherine Garel, Alain Favier, Mohsen Sakly, Khémais Rhouma, Hafedh Abdelmelek

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

The present study was undertaken in order to investigate the effects of static magnetic field (SMF) exposure on the antioxidative enzymes activity, malondialdehyde (MDA) concentration and DNA oxidation in male rat brain. The exposure of rats to SMF (128 mT, 1 h/day during 30 consecutive days) decreased the glutathione peroxidase (GPx; −39%, p < 0.05), CuZn superoxide dismutase (CuZn-SOD; −35%, p < 0.05) and catalase (−59%, p < 0.05) activities in frontal cortex. The same treatment decreased the CuZn-SOD (−51%, p < 0.05) and Mn-SOD (−13%, p < 0.05) activities in hippocampus. However, the glutathione levels remained unchanged in the both brain structures. In the hippocampus, SMF exposure increased MDA concentration (+32%, p < 0.05). Interestingly, exposed-rats to SMF displayed a significant increase of metallothioneins level in frontal cortex (+100%, p < 0.05), while the 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) concentration remained unaffected, indicating the absence of DNA oxidation. Our results indicated that sub-chronic exposure to SMF induced oxidative stress in rat hippocampus and frontal cortex. Metallothionein induction protected probably DNA against oxidative damage.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 260-265.

  Dietary supplementation with arachidonic acid but not eicosapentaenoic or docosahexaenoic acids alter lipids metabolism in C57BL/6J mice
Sameh Magdeldin 1), Yaser Elewa, Takako Ikeda, Junko Ikei, Ying Zhang, Bo Xu, Masaaki Nameta, Hidehiko Fujinaka, Yutaka Yoshida, Eishin Yaoita, Tadashi Yamamoto

1)Department of Structural Pathology Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Japan.

In order to investigate the effects of dietary supplementation rich in omega 3 and omega 6 fatty acids, we set up an experiment of twenty four C57BL/6J male mice segregated into 3 groups: normal diet (ND), omega 3 polyunsaturated fatty acid (n-3 PUFA,) and omega 6 (n-6 PUFA). At the end of the experiment that lasted for 1 month, food consumption of ND and n-3 PUFA were similar while it decreased in n-6 PUFA group. Total cholesterol, triglycerides, free fatty acids, and phospholipids profiles were increased in n-6 PUFA. LDL decreased in n-3 PUFA while increased in n-6 PUFA fed mice comparing to control group. On the other hand, there was no difference between treatments in HDL and glucose levels. Expression of leptin (ob) gene transcripts in epididymal fat were significantly elevated in n-6 PUFA mice compared to ND and n-3 PUFA groups while hypothalamic ob receptor A (obRa) mRNA did not changed in response to diet regimes. Transmission and scanning electron microscopy showed different degrees in fatty changes in the liver of both PUFA groups including lipid droplet infiltration and Ito cells with over accumulated lipids. In conclusion, under PUFA dietary supplementation, the hyperlipidemic status and elevated ob expression of n-6 PUFA but not n-3 PUFA fed mice suggests altered lipid metabolism between PUFA groups and/or different endocrine involvement. Moreover, the coincidently structural changes observed in liver of this group direct us to call for further studies to investigate the anti-obesity effect and safety of these PUFA under high supplementation condition.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 266-275.

  Temporal analysis of electroretinographic responses in fishes with rod-dominated and mixed rod-cone retina
Milena Milošević 1), Željka Višnjić-Jeftić, Ilija Damjanović, Miroslav Nikčević, Pavle Andjus, Zoran Gačić

1)School of Biology, University of Belgrade, Studentski trg 3, 11001 Belgrade, Serbia.

Photoreceptor content of fish retinas could be accessed by comparative electroretinographic (ERG) studies using flickering light stimuli that could separate rod-mediated vision where critical flicker frequency (CFF, frequency when the eye loses its ability to resolve individual light pulses) is usually less than 15 Hz from cone-mediated vision. Four fish species inhabiting different photic environments (small-spotted dogfish shark – Scyliorhinus canicula, eel – Anguilla anguilla, painted comber – Serranus scriba, Prussian carp – Carassius gibelio) were investigated. Dogfish shark b-wave amplitudes significantly decreased at low frequency of stimulation and CFF was reached at 3.2 Hz. A similar effect on the b-wave amplitude was observed in the eel, but CFF occurred at around 20 Hz. Conversely, b-waves of painted comber and Prussian carp remained unaltered under intermittent low-frequency stimulation, and CFFs were around 25 and 30 Hz, respectively. Additional support in accessing the receptor content of fish retinas was given by the characterization of the OFF-response (d-wave) after light adaptation. Monotonous time course of the b-wave dark adaptation indicated a rod dominated retina of the dogfish shark. Observed results indicate that the dogfish shark possesses preponderantly rod retina, that of the eel is rod-dominated, while Prussian carp and painted comber have cone-rich retinae.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 276-282.

  Fluorescence approach to evaluating conformational changes upon binding of β-spectrin ankyrin-binding domain mutants with the lipid bilayer
Grzegorz Pazdzior 1), Anna Chorzalska, Aleksander Czogalla, Tomasz Borowik, Aleksander Sikorski, Marek Langner

1)Institute of Biomedical Engineering and Instrumentation, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland.

The major component of the cell membrane skeleton, spectrin, is anchored in the cell membrane via interactions with membrane proteins. It has been previously shown that both erythroid and non-erythroid spectrin interact directly with membrane phospholipids (mainly aminophospholipids). One of the binding sites responsible for these interactions is located in the ankyrin-binding domain. In the present study, in order to better understand the character of binding, a more detailed investigation of the interactions between the β-spectrin fragments corresponding to the truncated mutants of the ankyrin-binding domain (Frag1 and Frag3) and liposomes of different compositions were carried out. The obtained results suggest that the binding of both spectrin fragments with liposomes induces conformational changes within the protein. Analysis of the changes in intrinsic tryptophan fluorescence spectra upon binding with liposomes, together with quenching studies (from the water and membrane hydrocarbon environment), allows for qualitative description of changes in proteins conformation. Our results suggest that the largest conformational changes occur for Frag1 bound to PC : PE (2 : 3) liposomes what is consistent with previous studies on monolayers. They are also in good agreement with those obtained previously for native erythroid and nonerythroid spectrin molecules.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 283-293.

  Modulation of cell proliferation and differentiation of human lung carcinoma cells by the interferon-alpha
Daniela Krejčová 1), Jiřina Procházková, Lukáš Kubala, Jiří Pacherník

1)Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.

Treatments of non-small cell lung cancer (NSCLC), the most common form of lung cancer, still remain poor. Interferon alpha (IFN-α), an important physiological immunomodulator, possesses direct cytotoxic and cytostatic effects on tumour cells, antiangiogenic effects, and activates anti-tumour immunity. Recently, the IFN-α oncologic indications have included melanoma, renal carcinoma, and different types of leukaemia. However, the application of IFN-α in therapy of lung cancer has not been validated yet. Herein the human lung carcinoma cell line A549, a model of NSCLC in vitro, was used to pursue the effect of IFN-α on A549 cell proliferation and differentiation together with the effect on protein expression and activity of three ATP-transporters mediating multi-drug resistance (MDR). IFN-α significantly inhibited the proliferation of A549 cells which was not connected with arrest in a particular cell cycle phase. Further, IFN-α-mediated differentiation of A549 was observed based on an increase in alkaline phosphatase activity. Simultaneously, IFN-α increased the expression and activity of ATP-transporters mediating MDR. Thus, the IFN-α down-regulation of NSCLC cell proliferation was accompanied by a potential of cells to exclude potential therapeutic substances such as chemotherapeutic agents. These effects could have a significant impact on considerations of IFN-α as a therapeutic agent for NSCLC.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 294-301.

  Influence of decavanadate on rat synaptic plasma membrane ATPases activity
Danijela Krstić 1), Mirjana Čolović, Nada Bošnjaković-Pavlović, Anne Spasojević-De Bire, Vesna Vasić

1)Institute of Medicinal Chemistry, University School of Medicine, Višegradska 26, 11 000 Belgrade, Serbia.

The in vitro influence of decameric vanadate species on Na+/K+-ATPase, plasma membrane Ca2+-ATPase (PMCA)-calcium pump and ecto-ATPase activity, using rat synaptic plasma membrane (SPM) as model system was investigated, whereas the commercial porcine cerebral cortex Na+/K+-ATPase served as a reference. The thermal behaviour of the synthesized decavanadate (V10) has been studied by differential scanning calorimetry and thermogravimetric analysis, while the type of polyvanadate anion was identified using the IR spectroscopy. The concentration-dependent responses to V10 of all enzymes were obtained. The half-maximum inhibitory concentration (IC50) of the enzyme activity was achieved at (4.74 ± 1.15) × 10−7 mol/l for SPM Na+/K+-ATPase, (1.30 ± 0.10) × 10−6 mol/l for commercial Na+/K+-ATPase and (3.13 ± 1.70) × 10−8 mol/l for Ca2+-ATPase, while ecto-ATPase is significantly less sensitive toward V10 (IC50 = (1.05 ± 0.10) × 10−4 mol/l) than investigated P-type ATPases. Kinetic analysis showed that V10 inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity and apparent affinity for ATP (increasing Km value), implying a mixed mode of interaction between V10 and P-type ATPases.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 302-308.

  Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers
Věra Štětinová 1), Libuše Smetanová, Dagmar Kholová, Zbyněk Svoboda, Jaroslav Květina

1)Institute of Experimental Biopharmaceutics, Joint Research Centre of the Academy of Sciences of the Czech Republic and PRO.MED.CS Praha a.s., Heyrovského 1207, Hradec Králové 500 03, Czech Republic.

This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 µmol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (Papp = 45 × 10−6 cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 309-315.

  Activation of MAPKs influences the expression of drug-metabolizing enzymes in primary human hepatocytes
Petr Bachleda 1), Radim Vrzal, Zdeněk Dvořák

1)2nd Department of Surgery, University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic.

We examined the effects of model activators of mitogen-activated protein kinases (MAPKs) on basal and rifampicin-, phenobarbital- and dioxin-inducible expression of phase I and phase II biotransformation enzymes in primary human hepatocytes. Cells were treated for 24 h with sorbitol (SOR), anisomycin (ANI) and epidermal growth factor (EGF) in the presence or absence of inducers. The levels of CYP1A1, CYP1A2, CYP2B6, CYP3A4, UGT1A1, UGT2B17, SULT1A1, SULT2A1, SULT1B2, GSTA1, GSTA2 mRNAs were determined. SOR and EGF inhibited the expression of the tested genes, while ANI had no effect. We conclude that MAPKs play important role in the transcriptional regulation of drug-metabolizing enzymes.

General Physiology and Biophysics. Volume 28, 2009, No. 3: 316-320.