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General Physiology and Biophysics


Volume 33, 2014, No. 1

Content:


  Cooperative gating between ion channels
Kee-Hyun Choi 1)

1)Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea. keehyun@kist.re.kr.


Cooperative gating between ion channels, i.e. the gating of one channel directly coupled to the gating of neighboring channels, has been observed in diverse channel types at the single-channel level. Positively coupled gating could enhance channel-mediated signaling while negative coupling may effectively reduce channel gating noise. Indeed, the physiological significance of cooperative channel gating in signal transduction has been recognized in several in vivo studies. Moreover, coupled gating of ion channels was reported to be associated with some human disease states. In this review, physiological roles for channel cooperativity and channel clustering observed in vitro and in vivo are introduced, and stimulation-induced channel clustering and direct channel cross linking are suggested as the physical mechanisms of channel assembly. Along with physical clustering, several molecular mechanisms proposed as the molecular basis for functional coupling of neighboring channels are covered: permeant ions as a channel coupling mediator, concerted channel activation through the membrane, and allosteric mechanisms. Also, single-channel analysis methods for cooperative gating such as the binomial analysis, the variance analysis, the conditional dwell time density analysis, and the maximum likelihood fitting analysis are reviewed and discussed.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 1-12.

 
  Quantitative estimation of transmembrane ion transport in rat renal collecting duct principal cells
Alexander Ilyaskin 1), Denis Karpov, Dmitriy Medvedev, Alexander Ershov, Galina Baturina , Liubov Katkova, Evgeniy Solenov

1)Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia. ilyaskin@bionet.nsc.ru.


Kidney collecting duct principal cells play a key role in regulated tubular reabsorption of water and sodium and secretion of potassium. The importance of this function for the maintenance of the osmotic homeostasis of the whole organism motivates extensive study of the ion transport properties of collecting duct principal cells. We performed experimental measurements of cell volume and intracellular sodium concentration in rat renal collecting duct principal cells from the outer medulla (OMCD) and used a mathematical model describing transmembrane ion fluxes to analyze the experimental data. The sodium and chloride concentrations ([Na+]in = 37.3 ± 3.3 mM, [Cl-]in = 32.2 ± 4.0 mM) in OMCD cells were quantitatively estimated. Correspondence between the experimentally measured cell physiological characteristics and the values of model permeability parameters was established. Plasma membrane permeabilities and the rates of transmembrane fluxes for sodium, potassium and chloride ions were estimated on the basis of ion substitution experiments and model predictions. In particular, calculated sodium (PNa), potassium (PK) and chloride (PCl) permeabilities were equal to 3.2 × 10-6 cm/s, 1.0 × 10-5 cm/s and 3.0×10-6 cm/s, respectively. This approach sets grounds for utilization of experimental measurements of intracellular sodium concentration and cell volume to quantify the ion permeabilities of OMCD principal cells and aids us in understanding the physiology of the adjustment of renal sodium and potassium excretion.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 13-28.

 
  Increased firing frequency of spontaneous action potentials in cerebellar Purkinje neurons of db/db mice results from altered auto-rhythmicity and diminished GABAergic tonic inhibition
María Forero-Vivas 1), Arturo Hernández-Cruz

1)Chemistry Department, Faculty of Science, Antonio Nariño University, Bogotá, Colombia.


The hormone leptin, by binding to hypothalamic receptors, suppresses food intake and decreases body adiposity. Leptin receptors are also widely expressed in extra-hypothalamic areas such as hippocampus, amygdala and cerebellum, where leptin modulates synaptic transmission. Here we show that a defective leptin receptor affects the electrophysiological properties of cerebellar Purkinje neurons (PNs). PNs from (db/db) mice recorded in cerebellar slices display a higher firing rate of spontaneous action potentials than PNs from wild type (WT) mice. Blockade of GABAergic tonic inhibition with bicuculline in WT mice changes the firing pattern from continuous, uninterrupted spiking into bursting firing, but bicuculline does not produce these alterations in db/db neurons, suggesting that they receive a weaker GABAergic inhibitory input. Our results also show that the intrinsic firing properties (auto-rhythmicity) of WT and db/db PNs are different. Tonic firing of PNs, the only efferent output from the cerebellar cortex, is a persistent signal to downstream cerebellar targets. The significance of leptin modulation of PNs spontaneous firing is not known. Also, it is not clear if the increased excitability of cerebellar PNs in db/db mice results from hyperglycemia or from the lack of leptin signaling, since both conditions coexist in the db/db strain.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 29-41.

 
  Effect of antioxidants on functional recovery after in vitro-induced ischemia and long-term potentiation recorded in the pyramidal layer of the CA1 area of rat hippocampus
Zdenka Gasparova 1), Veronika Stara, Svorad Štolc

1)Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic. zdenka.gasparova@savba.sk.


Ischemic stroke is one of the leading causes of cognitive impairment. Antioxidants may be beneficial in brain diseases in which oxidative stress can be assumed. The effect of two antioxidants, stobadine and its new derivative coded SMe1EC2, was studied on post-ischemic functional recovery in the hippocampus of young and 18-month-old rats. The synaptic transmission was apparently absent after 6-min hypoxia/hypoglycemia in both age groups. Re-oxygenation resulted in negligible functional recovery in untreated slices, yet the presence of pyridoindoles tested elicited improved recovery upon re-oxygenation. SMe1EC2 was found more effective in post-ischemic functional recovery and was further tested in the hippocampus of 15-month-old rats in long-term potentiation (LTP) experiments, a synaptic model of learning and memory mechanisms. In slices of aged rats, 3.5-min hypoxia/hypoglycemia resulted in depression of the LTP induction phase (immediately after high frequency stimulation) and this was prevented in the presence of SMe1EC2 (3 µmol/l). Upon “normoxia”, marked amelioration of LTP was recorded in the presence of the antioxidant in about 1.5 order lower concentration. These results suggest a possible application of the pyridoindole in the management of brain ischemia and cognitive impairment.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 43-52.

 
  Cisplatin enhances the efficacy of 5-Aminolevulinic acid mediated photodynamic therapy in human head and neck squamous cell carcinoma
Jin-Chul Ahn 1), Raktim Biswas, Arindam Mondal, Young-Ki Lee, Phil-Sang Chung

1)Medical Laser Research Center, College of Medicine, Dankook University, Cheonan, Chungnam, Korea. pschung@dankook.ac.kr.


Photodynamic therapy (PDT) has become a promising option for the treatment of head and neck, and other forms of cancer. 5-Aminolevulinic acid (ALA) is one of the popular photosensitizers used in PDT. It is a heme precursor and is converted to a photosensitizer protophorphyrin IX. In this present study, the combination of anticancer drug cisplatin (CDDP) and ALA mediated PDT was used to study the cytotoxicity in vitro as well as in vivo. Human head and neck cancer cells AMC-HN3 were treated with cisplatin and ALA-mediated PDT individually, and also in combination. Several approaches like confocal microscopic study, cytotoxicity assay etc. have been performed to study the intracellular accumulation of protophorphyrin IX in cells and its effectiveness in PDT, when treated in combination with chemotherapy drug, cisplatin (CDDP). The combination of treatments efficacy was also studied in tumor xenograft model. Compared to the individual treatments, combination of CDDP and PDT was found to be more cytotoxic in AMC-HN3, and also more effective in reducing the tumor volume in mice xenograft. Thus, with the combined therapy, not only the efficacy of treatment can be enhanced, but the doses of the drugs can also be lowered. This in turn can reduce the side effects of the chemotherapy drugs. Therefore, this study may lead to a potential drug-PDT combination that may be a useful treatment modality for human head and neck cancer.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 53-62.

 
  Photon emission from melanoma cells during brief stimulation by patterned magnetic fields: Is the source coupled to rotational diffusion within the membrane?
Blake Dotta 1), Robert Lafrenie, Lukasz Karbowski, Michael Persinger

1)Biomolecular Sciences Program, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada. mpersinger@laurentian.ca..


If parameters for lateral diffusion of lipids within membranes are macroscopic metaphors of the angular magnetic moment of the Bohr magneton then the energy emission should be within the visible wavelength for applied ~1 µT magnetic fields. Single or paired digital photomultiplier tubes (PMTs) were placed near dishes of ~1 million B16 mouse melanoma cells that had been removed from incubation. In very dark conditions (10-11 W∙m-2) different averaged (RMS) intensities between 5 nT and 3.5 µT were applied randomly in 4 min increments. Numbers of photons were recorded directly over or beside the cell dishes by PMTs placed in pairs within various planes. Spectral analyses were completed for photon power density. The peak photon emissions occurred around 1 µT as predicted by the equation. Spectra analyses showed reliable discrete peaks between 0.9 and 1.8 µT but not for lesser or greater intensities; these peak frequencies corresponded to the energy difference of the orbital-spin magnetic moment of the electron within the applied range of magnetic field intensities and the standard solution for Rydberg atoms. Numbers of photons from cooling cells can be modified by applying specific intensities of temporally patterned magnetic fields. There may be a type of “cellular” magnetic moment that, when stimulated by intensity-tuned magnetic fields, results in photon emissions whose peak frequencies reflect predicted energies for fundamental orbital/spin properties of the electron and atomic aggregates with large principal quantum numbers.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 63-73.

 
  A physiological view and structures of mean residence times
Mária Ďurišová 1)

1)Department of Pharmacology of Inflammation, Institute of Experimental Pharmacology and Toxicology, Dúbravská cesta 9, 841 04 Bratislava, Slovak Academy of Sciences, Slovak Republic. Maria.Durisova@savba.sk.


The author’s previous theoretical study described the determination of a physiologically realistic structure of a mean residence time of an intravenously administered drug (Ďurišová 2012). This study continues previous work and the aim is to determine physiologically realistic structures of mean residence times of a drug administered either orally (MRTpo) or intramuscularly (MRTim). The determinations are based on the following assumption: a cardiopulmonary, portal, portal-venous, hepatic-portal subsystem, and a subsystem that mathematically represents non-eliminating tissues can be considered to be most important in terms of their impact on MRTpo and MRTim. If drug fate and disposition is a linear process, the used method allows developing mathematical models without any prior knowledge or hypothesis concerning drug fate and disposition. This is a great advantage of the method considered here when compared with compartment methods. The research presented in this study was aimed at contributing to the knowledge base of physiological origins of mean residence times.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 75-80.

 
  Extremely low frequency magnetic fields induce oxidative stress in rat brain
Pavan Manikonda 1), Pilankatta Rajendra, D Devendranath, B Gunasekaran, Channakeshava, Shivakumara Aradhya, Rao Sashidhar, Chivukula Subramanyam

1)Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, AP, India. sashi_rao@yahoo.com.


The present investigation was conducted to understand the influence of long-term exposure of rats to extremely low frequency magnetic fields (ELF-MF), focusing on oxidative stress (OS) on different regions of rat’s brain. Male Wistar rats (21-day-old) were exposed to ELF-MF (50 Hz; 50 and 100 µT) for 90 days continuously; hippocampal, cerebellar and cortical regions from rats were analyzed for (i) reactive oxygen species (ROS), (ii) metabolites indicative of OS and (iii) antioxidant enzymes. In comparison to control group rats, the rats that were continuously exposed to ELF-MF caused OS and altered glutathione (GSH/GSSG) levels in dose-dependent manner in all the regions of the brain. Accumulation of ROS, lipid peroxidation end products and activity of superoxide dismutase in different regions was in the descending order of cerebellum < hippocampus < cortex. Decrement in GSH/GSSG levels and increment in glutathione peroxidase activity were in the descending order of hippocampus < cerebellum < cortex. The continuous exposure to ELF-MF caused OS in all the examined regions of brain more significantly at 100 µT than at 50 µT. Varied influences observed in different regions of the brain, as documented in this study, may contribute to altered metabolic patterns in its related regions of the central nervous system, leading to aberrant neuronal functions.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 81-90.

 
  Mitochondrial oxidative energy metabolism in guanethidine-induced sympathectomized ducklings
Younes Filali-Zegzouti 1), Jean-Louis Rouanet, Toufiq Fechtali, Damien Roussel

1)Université Moulay Ismail de Meknès, EPPE-FST, Errachidia, Morocco. filali.younes@gmail.com.


Here we investigate the possible involvement of the sympathetic nervous system in the respiratory properties of intermyofibrillar and subsarcolemmal mitochondrial populations from heart and gastrocnemius muscles. Mitochondrial oxidative phosphorylation was assessed polarographically by using succinate (plus rotenone), and ascorbate plus N,N,N’,N’-tetramethyl-p-phenyl-enediamine (plus antimycin) as respiratory substrates. We report that chronic chemical sympathectomy with guanethidine (150 mg/kg, daily for 3 weeks) induced a marked decrease in whole body metabolic and heart rates, in plasma metabolites (fatty acids and glucose) and norepinephrine levels. Guanethidine treatment decreased mainly the oxidative phosphorylation capacity of subsarcolemmal mitochondria in heart, irrespective of the substrate used. In contrast, both mitochondrial populations were affected by the treatment in skeletal muscle. This suggests that sympathetic nervous system activity can alter the energetic status of muscle cells, and to some extent play a thermogenic role in birds.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 91-97.

 
  β3-Adrenoceptor-mediated responses in diabetic rat heart
Gizem Kayki-Mutlu 1), Ebru Arioglu-Inan, Isil Ozakca, Arif Ozcelikay, Vecdi Altan

1)Department of Pharmacology, Faculty of Pharmacy, Ankara University, 06100, Tandogan, Ankara, Turkey. gkayki@ankara.edu.tr.


β3-adrenoceptors mediate negative inotropic effect in contrast to classical β1- and β2-adrenoceptors. Cardiac β3-adrenoceptors are upregulated in experimental diabetes. Thus, cardiodepressant effect mediated by β3-adrenoceptors has been proposed to contribute to the impaired cardiac function in this pathology. In our study, we investigated the influence of streptozotocin-diabetes on cardiac contractility to β3-adrenoceptors stimulation by using Langendorff-perfused rat hearts. BRL 37344, a selective β3-adrenoceptor agonist, induced dose-dependent decreases in left ventricular developed pressure (LVDP) in hearts from control rats. BRL 37344 also dose-dependently decreased +dP/dt and –dP/dt values. Effects of BRL 37344 were abolished by SR 59230, but not altered by nadolol pre-treatment. On the other hand, these effects of BRL 37344 were all significantly increased in hearts from diabetic rats. We also observed that diabetes significantly increased the mRNA levels encoding cardiac β3-adrenoceptors. In addition, Giα2 mRNA expressions were found to be increased in the cardiac tissue of diabetic rats as well. The effect of BRL 37344 on cardiac contractility was normalized upon treatment of diabetic rats with insulin. These data demonstrate an increased effect of β3-adrenoceptor stimulation on hemodynamic function of the heart in accordance with an increased mRNA levels encoding cardiac β3-adrenoceptors in 8-week diabetic rats.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 99-109.

 
  Gender differences in ghrelin response to chronic immobilization stress in rats: possible role of estrogen
Eman Elbassuoni 1)

1)Physiology Department, Minia University School of Medicine, Minia, Egypt. emanelbassuoni@yahoo.com.


Ghrelin is a peptidergic hormone known to be one of the main hormones involved in the regulation of energy balance. Here we evaluated ghrelin response to stress in rats after ovariectomy and during estradiol benzoate (EB) therapy and compared results of males and females, to know whether ghrelin is involved in disordered eating behaviors in response to stress, and for understanding differences between males and females in food intake and weight gain especially during stress. 96 adult rats were classified into; male, female, ovariectomized (Ovx), Ovx with EB. Half animals of each group were exposed to immobilization stress 20 min/day for 21 days. We found that chronic stress significantly augments serum ghrelin levels in both males and females, which is correlated with an increase in food intake and body weight. Females displayed significant higher ghrelin than males especially in response to stress, ovariectomy suppresses serum ghrelin in both unstressed and stressed females which is rescued by replacement with EB. EB replacement augments ghrelin response to stress in Ovx female, and reduces food intake and body weight. In conclusion, there is a clear sex difference in ghrelin secretion in response to stress caused by EB, since it amplifies ghrelin response to stress in females.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 111-120.

 
  Electrophysiological variability in the SH-SY5Y cellular line
Silvia Santillo 1), Aniello Schiano Moriello, Vito Di Maio

1)Institute of Cybernetics “E. Caianiello” of the CNR, c/o Comprensorio “Olivetti”, Via Campi Flegrei 34, 80078, Pozzuoli (NA), Italy. s.santillo@cib.na.cnr.it.


The basic electrophysiological properties of SH-SY5Y tumour cells have been studied by whole cell patch-clamp in voltage clamp configuration. The results shown the existence of a large variability of the response among individual cells in the same experimental conditions. Experiments conducted by using different ionic concentrations of the recording pipette filling solution, yielded a significant variability of peak current amplitude for all the filling solutions used. In addition, variability among groups was detected. The inter-group variability was not dependent on the different ionic components among the groups. Our results confirm earlier findings that this cell line is not constituted of an uniform population of cells. Moreover, from the present results we can conclude that these cells have diverse regulatory patterns of membrane conductance, probably due to both the expression as well as a non precise regulation of the membrane density of the different channels.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 121-129.

 
  Effect of Pb2+ ions on photosynthetic apparatus
Frantisek Sersen 1), Katarina Kralova, Matus Pesko, Marek Cigan

1)Faculty of Natural Sciences, Institute of Chemistry, Comenius University in Bratislava, Bratislava, Slovak Republic. sersen@fns.uniba.sk.


Using model lead compounds Pb(NO3)2 and Pb(CH3CHOO)2, the mechanism and the site of action of Pb2+ ions in the photosynthetic apparatus of spinach chloroplasts were studied. Both compounds inhibited photosynthetic electron transport (PET) through photosystem 1 (PS1) and photosystem 2 (PS2), while Pb(NO3)2 was found to be more effective PET inhibitor. Using EPR spectroscopy the following sites of Pb2+ action in the photosynthetic apparatus were determined: the water-splitting complex and the Z•/D• intermediates on the donor side of PS2 and probably also the ferredoxin on the acceptor side of PS1, because cyclic electron flow in chloroplasts was impaired by treatment with Pb2+ ions. Study of chlorophyll fluorescence in suspension of spinach chloroplasts in the presence of Pb2+ ions confirmed their site of action in PS2. Using fluorescence spectroscopy also formation of complexes between Pb2+ and amino acid residues in photosynthetic proteins was confirmed and constants of complex formation among Pb2+ and aromatic amino acids were calculated for both studied lead compounds.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 131-136.

 
  Triptolide induces apoptosis through the SERCA 3 upregulation in PC12 cells
Olga Krizanova 1), Jana Markova, Karel Pacak, Ludovit Skultety, Andrea Soltysova, Sona Hudecova

1)Institute of Molecular Physiology and Genetics, Center of Excellence for Studying Metabolic Aspects of Development, Diagnostics and Treatment of the Oncologic Diseases, Slovak Academy of Sciences, Bratislava, Slovak Republic. sona.hudecova@savba.sk.


Diterpenoid triepoxide – Triptolide (TTL) – increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.

General Physiology and Biophysics. Volume 33, 2014, No. 1: 137-144.