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General Physiology and Biophysics

Volume 34, 2015, No. 4


  The ubiquitin proteasome system as a potential therapeutic target for treatment of neurodegenerative diseases
Alena Opattova 1), Martin Cente 2), Michal Novak 3), Peter Filipcik 4)

1)Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovak Republic..
2)Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovak Republic..
3)Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovak Republic..
4)Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovak

Impairment of “protein quality control” in neurons is associated with etiopathogenesis of neurodegenerative diseases. The worn-out products of cell metabolism should be safely eliminated via the proteasome, autophago-lysosome and exocytosis. Insufficient activity of these degradation mechanisms within neurons leads to the accumulation of toxic protein oligomers, which represent a starting material for development of neurodegenerative proteinopathy. The spectrum of CNS linked proteinopathies is particularly broad and includes Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy body dementia, Pick disease, Frontotemporal dementia, Huntington disease, Amyotrophic lateral sclerosis and many others. Although the primary events in etiopathogenesis of sporadic forms of these diseases are still unknown, it is clear that aging, in connection with decreased activity of ubiquitin proteasome system, is the most significant risk factor. In this review we discuss the pathogenic role and intracellular fate of the candidate molecules associated with onset and progression of AD and PD, the protein tau and α-synuclein in context with the function of ubiquitin proteasome system. We also discuss the possibility whether or not the strategies focused to re-establishment of neuroproteostasis via accelerated clearance of damaged proteins in proteasome could be a promising therapeutic approach for treatment of major neurodegenerative diseases.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 337-352.

  Cellular and molecular mechanisms underlying the treatment of depression: Focusing on hippocampal G-protein-coupled receptors and voltage-dependent calcium channels
Michaela Pavlovicova 1), Ľubica Lacinová, Eliyahu Dremencov

1)Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.

Depression is a brain disorder characterized by severe emotional, cognitive, neuroendocrine and somatic dysfunction. Although the latest generation of antidepressant drugs has improved clinical efficacy and safety, the onset of their clinical effect is significantly delayed after treatment commencement, and a significant number of patients exhibit inadequate response to these drugs and/or depression relapse even following initially successful treatment. It is therefore essential to develop new antidepressant drugs and/or adjuncts to existing ones. Recent studies suggest that the beneficial effect of antidepressant drugs is mediated, at least in part, via stimulation of adult hippocampal neurogenesis and subsequent increase in hippocampal plasticity. Since the stimulatory effect of antidepressant drugs on hippocampal neurogenesis involves G-protein coupled receptors (GPCR) and voltage-dependent calcium channels (VDCC), greater efficacy may be available if future antidepressant drugs directly target these specific GPCR and VDCC. The potential advantages and limitations of these treatment strategies are discussed in the article.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 353-366.

  Acidotropic properties of synthetic hexahydropyridoindole antioxidants
Lucia Račková 1), Marcela Kuniaková

1)Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, 84104 Bratislava, Slovak Republic.

In acidic intracellular organelles, sequestration via a proton-trapping mechanism is observed for many amine-containing drugs. It may be related to several adverse effects of a drug, yet accumulation of amines bearing antioxidant functionality may provide efficient protection of these compartments. In the present study, a possible proton-trapping mechanism of the novel antioxidant reference stobadine (STO) and its selected derivatives was investigated also with regard to their antioxidant properties, using BV-2 microglia. Unlike its 2-ethoxycarbonyl-8-methoxy derivative EC-STO (pKa1 4.95, pKa2 –3.58), STO, bearing weakly basic piperidine moiety (pKa2 9.03), induced vacuolar response in the cells. EC-STO, compared to STO, failed to provide better protection against oxidative damage induced by tert-butyl hydroperoxide (BHP), and that in spite of its predicted improved bioavailability and antioxidant properties. However, disruption of the lysosomal proton gradient abolished the efficacy of STO in suppressing oxidants generation and injury of the cells. NT-STO, the 6-nitro derivative of stobadine, lacking antiradical efficacy, showed a lower effect in protecting the cells against BHP. In conclusion, our study suggests that weakly basic hexahydropyridoindoles may act as lysosomotropic compounds. Furthermore, their weakly basic characteristics may contribute to their improved efficacy in suppressing peroxidative processes within lysosomes, and thus possibly combating ageing-related pathologies.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 367-382.

  Biophysical characterization of inwardly rectifying potassium currents (IK1, IK, ACh, IK,Ca) using sinus rhythm or atrial fibrillation action potential waveforms
Chuyi Tang 1), Lasse Skibsbye, Lei Yuan, Bo Bentzen, Thomas Jespersen

1)Department of Cardiovascular Medicine, East Hospital, Tongji University School of Medicine, No. 150, Jimo Rd. Shanghai, China.

Although several physiological, pathophysiological and regulatory properties of IK1, IK, ACh and the small-conductance Ca2+ activated K+ current IK,Ca have been identified, quantitative biophysical details remain unclear. Both IK1 and IK, ACh are implicated in atrial fibrillation (AF), and recently also IK,Ca has been speculated to be linked with the genesis and sustainability of AF. All these three currents have been shown to be involved in the electrical remodeling in the atria of patients suffering from AF, and it is therefore important to characterize their biophysical properties and compare their relative current contribution in atrial electrophysiology in both sinus rhythm (SR) and AF. The aim of this study is to investigate the contribution of the three potassium currents when subjected to voltage protocols adapted from atrial action potentials recorded in human tissue at 1 and 3 Hz. The current recordings were performed in the HEK-293 heterologous cell system expressing either IK1, IK, ACh or IK,Ca to establish the individual contribution of each of these currents during the voltage changes of atrial action potential waveforms. IK1 primarily contributes to the atrial electrophysiology at the latter part of repolarization and during the diastolic phase, while both IK,Ca under high [Ca2+]i and IK, ACh contribute relatively most during repolarization.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 383-393.

  Lymph node mapping using quantum dot-labeled polymersomes
Rumiana Bakalova 1), Zhivko Zhelev, Biliana Nikolova, Shuhei Murayama, Desislava Lazarova, Iana Tsoneva, Ichio Aoki

1)Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.

The present study was designed to investigate whether poly-ion complex hollow vesicles (polymersomes), based on chemically-modified chitosan, are appropriate for lymph node mapping in the context of their application in the development of theranostic nanosized drug delivery systems (nano-DDS). The experiments were performed on Balb/c nude mice (colon cancer-grafted). The mice were subjected to anesthesia and quantum dot (QD705)-labeled polymersomes (d~120 nm) were injected intravenously via the tail vein. The optical imaging was carried out on Maestro EX Imaging System (excitation filter: 435–480 nm; emission filter: 700 nm). A strong fluorescent signal, corresponding to QD705 fluorescence, was detected in the lymph nodes, as well as in the tumor. A very weak fluorescent signal was found in the liver area. The half-life of QD705-labelled polymersomes was 6 ± 2 hours in the bloodstream and 11 ± 3 hours in the lymph nodes. The data suggest that polymersomes are very promising carriers for lymph node mapping using QD as a contrast agent. They are useful matrix for development of nano-formulations with theranostic capabilities.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 393-398.

  Lenalidomide treatment induced the normalization of marker protein levels in blood plasma of patients with 5q-myelodysplastic syndrome
Lucia Messingerová 1), Anna Jonášová, Miroslav Barancik, Lenka Poleková, Mário Šereš, Lenka Gibalová, Albert Breier, Zdena Sulová

1)Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.

A specific type of myelodysplastic syndrome (MDS) is associated with isolated deletion on the long arm of chromosome 5 i.e., 5q-syndrome (del(5q)). The treatment approaches for MDS del(5q) include the immunomodulating drug lenalidomide (LEN). Thirteen MDS del(5q) patients were included in this study. We found elevated activities of lactate dehydrogenase (LDH) and matrix metalloproteinase 9 (MMP-9) in the blood plasma of MDS del(5q) patients as compared with healthy controls. This was stabilized to control values after LEN treatment. Similar behavior we registered also for the thioredoxin and calnexin contents in blood plasma. Peripheral blood mononuclear cells (PBMC) from patients with MDS del(5q) prior to and after treatment with LEN did not exhibit any detectable amount of P-glycoprotein (P-gp) gene transcript. However, we detected a measurable amount of multidrug resistance associated protein 1 (MRP1) mRNA in PBMCs from three patients prior to LEN treatment and in one patient during LEN treatment but it was not present prior to treatment. These data indicated on usefulness of applied protein markers estimation for monitoring of MDS del(5q) patient treatment effectiveness by LEN. Expression of MRP1 seems to be independent on LEN treatment and reflects probably the molecular variability in the ethiopathogenesis of MDS del(5q).

General Physiology and Biophysics. Volume 34, 2015, No. 4: 399-406.

  Quercetin and hydroxytyrosol attenuates xanthine/xanthine oxidase-induced toxicity in H9c2 cardiomyocytes by regulation of oxidative stress and stress-sensitive signaling pathways
Namık Özbek 1), Elif Bali, Çimen Karasu

1)Cellular Stress Response and Signal Transduction Research Laboratory, Department of Medical Pharmacology, Faculty of Medicine, Gazi University, Beşevler 06500, Ankara, Turkey.

The increased activity of xanthine/xanthine oxidase (X/XO) has been suggested as a risk factor for heart disease and herbal polyphenols exhibits cardioprotection in vitro and in vivo. To understand the cardioprotective action mechanisms of polyphenol quercetin and hydroxytyrosol, the expression levels of stress-responsive proteins were studied in X/XO-induced toxicity model of H9c2 cardiomyocyocytes. Pretreatment with each polypenol (0.1–10 µg/ml; 24 h) enhanced viability (p < 0.01; MTT test) and inhibited reactive oxygen species (ROS) generation (p < 0.001; H2DCFDA assay) against 12 h exposure to a free radical generating system, X (0.5 mM) and XO (5 mU/ml). Western blotting experiments showed that X/XO increases the phosphorylation of downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK-2), p44/42-MAPK (Erk1/2) and cleaved caspase-3 (p < 0.001, vs. Control), however inhibits the levels of phosphorylated c-Jun and Hsp27 (p < 0.01, vs. Control). Pretreatment with quercetin or hydroxytyrosol attenuated the phosphorylation of MAPKAPK-2 and cleaved caspase-3 in X/XO-exposed cells (p < 0.01, vs. X-XO). Hydroxytyrosol enhanced the reduction of phosphorylation of a transcriptional target c-Jun and led to overphosphorylation in protective proteins, p44/42-MAPK and Hsp27 in X/XO-exposed cells (p < 0.01, vs. X-XO). Our data suggest that quercetin and hydroxytyrosol protects cardiomyocytes against X/XO-induced oxidative toxicity by diminishing intracellular ROS and the regulation of stress-sensitive protein kinase cascades and transcription factors.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 407-414.

  Pleiotropic protective roles of melatonin against aluminium-induced toxicity in rats
Mohamed Allagui 1), Rafik Hachani, Saber Saidi, Anouer Feriani, Jean Murat, Kamel Kacem, Abdelfatteh El feki

1)Laboratory of Animal Ecophysiology, Faculty of Science of Sfax, 3018 Sfax, Tunisia.

This study aimed to investigate the potential effects of melatonin on aluminium-induced toxicity in a rat model using a set of biochemical, inflammatory, oxidant, lipid profile criteria and hepatic integrity (verified by hematoxylin-eosin staining). The results indicated that AlCl3 administration during 60 days (100 mg/kg b.w.) significantly increased the activities of transaminases AST and ALT by 46% (p < 0.001) and 21% (p < 0.01), lactate dehydrogenase (LDH) by 30% (p < 0.001), the levels of bilirubin by 85% (p < 0.001), total cholesterol by 115% (p < 0.001), triglycerides by130% (p < 0.001), LDL-cholesterol by 413% (p < 0.001), oxidized LDL (oxLDL) by 51% (p< 0.01) and apolipoprotein B100 (apoB100) by 63% (p < 0.001), as compared to controls. The inflammatory markers (TNF-α, IL-2, and IL-6) were significantly increased (p < 0.001), associated to higher lipid peroxidation (TBARS) level. Also, both plasma HDL-cholesterol level and hepatic LDL receptors (p < 0.01) expression and antioxidant protein (SOD, CAT, and GPx) activities are decreased. Those physiological disturbances were, however, noted to alleviate following the co-administration of melatonin (10 mg/kg b.w.). Overall, the present study is the first to provide evidence on the anti-inflammatory, anti-oxidant, anti-lipidic and, hence, therapeutic effects of melatonin with regard to the control and prevention of aluminium-intoxication.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 415-424.

  Hypothermia mitigates neurochemical alterations in rat’s cerebral cortex during status epilepticus induced by pilocarpine
Haitham Mohammed 1)

1)Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt.

Status epilepticus (SE) is a prolonged seizure activity associated with mortality and morbidity. SE is characterized by changes in neurotransmitter systems and oxidative stress that facilitate cellular damage. These alterations represent the neurochemical mechanisms underlying the initiation and progression of seizure activity and co-existing morbidity. In the present study, amino acid levels (glutamine, glutamate, GABA, aspartate, glycine and taurine) and oxidative stress parameters malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide NO) were determined in the cerebral cortex during SE induced by pilocarpine in rats. The study has also evaluated the effects of hypothermia, as a physical non-invasive tool, on neurotransmitters and oxidative stress alterations. The results obtained revealed that there are significant increases in glutamate, GABA, glycine and taurine and NO in the cortex of pilocarpinzed rats. Hypothermia pretreatment mitigated most of the alterations induced by pilocarpine and significantly decreased GABA concentration. These findings emphasize the involvement of extrahippocampal amino acid neurotransmitters in pilocarpine-induced SE and the ameliorative role played by hypothermia.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 425-432.

  The effects of endocannabinoid receptor agonist anandamide and antagonist rimonabant on opioid analgesia and tolerance in rats
Ahmet Altun 1), Ercan Ozdemir, Kemal Yildirim, Sinan Gursoy, Nedim Durmus, Ihsan Bagcivan

1)Department of Pharmacology, Cumhuriyet University School of Medicine, Sivas, Turkey.

The role of the cannabinoid (CB) system in the tolerance to analgesic effect of opioid remains obscure. The aim of the present study was to evaluate the effects of the endocannabinoid nonselective receptor agonist anandamide (AEA) and CB1 receptor antagonist rimonabant (SR141716) on morphine analgesia and tolerance in rats. Male Wistar albino rats weighing 215–230 g were used in these experiments. To constitute morphine analgesic tolerance, a 3-day cumulative dosing regimen was used. The analgesic effects of AEA (10 mg/kg), SR141716 (10 mg/kg), and morphine (5 mg/kg) were considered at 30-min intervals by tail flick (TF) and hot plate (HP) analgesia tests. The analgesic effects of the drugs were measured as TF and HP latencies in all groups for each rat and converted to %MPE. The data were analysed by analysis of variance followed by Tukey test. The findings suggested that AEA in combination with morphine produced a significant increase in expression of analgesic tolerance to morphine. Conversely, cannabinoid receptor antagonist SR141716 attenuated morphine analgesic tolerance. In addition, administration of AEA with morphine increased morphine analgesia. In conclusion, we observed that the cannabinoid receptor agonist anandamide and CB1 receptor antagonist SR141716 plays a significant role in the opioid analgesia and tolerance.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 433-440.

  Synergism between phorbol myristate acetate and calcium ionophore in inducing proliferation of in vitro γ-irradiated murine lymphocytes
Joaquín Hernández-Godoy 1), Dolores Planelles, Baltasar Balsalobre

1)Experimental Immunology Unit, Research Centre, University Hospital La Fe, Valencia, Spain.

The objective of this study was to analyze the in vitro effects of γ-irradiation (0–5 Gy) on lymphocyte proliferation in animals sensitive to radiation as BALB/c mice. Lymphocytes were irradiated and underwent different treatments: quiescent cells were cultured with calcium ionophore A23187 (5 min or 48 h) with or without phorbol myristate acetate (PMA); lymphocytes (control cells or incubated with A23187 and PMA) were also cultured with four mitogens that are specific to the different subpopulations to determine the degree of inhibition of the response to radiation. Results obtained indicated that in quiescent cells, A23187 and PMA treatment had a mitogenic effect, which peaked with long A23187 treatment (48 h); synergism was further demonstrated between both drugs and was enhanced with higher ionizing radiation doses. However, in both irradiated and non-irradiated mitogen-stimulated cells, A23187 (48 h) and PMA had a strong inhibitory effect on cell proliferation. In conclusion these results indicate that irradiated BALB/c mice lymphocytes respond to treatment with A23187 and PMA more actively than controls. Inhibition of the post-exposure mitogen-induced proliferative response and the synergic effect between A23187 and PMA also suggest altered PKC activation mechanisms in cell membranes. Comparing with previous studies with in vivo irradiated mice, the effects of IR in vitro were less intense.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 441-447.

  Rosmarinic acid mitigates signs of systemic oxidative stress in streptozotocin-induced diabetes in rats
Ruzena Sotnikova 1), Barbara Kaprinay, Jana Navarova

1)Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic.

The aim of the work was to study the effect of rosmarinic acid (RA) on markers of oxidative stress in rats with diabetes. Diabetes was induced by streptozotocin (STZ), RA was administered orally for ten weeks. Water consumption was measured daily. Ten weeks after the first RA administration, urine was collected over 15 hours. N-acetyl-β-D-glucosaminidase (NAGA) activity, levels of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) were determined in the pancreas, kidney, and plasma. RA administration to diabetic rats ameliorated markers of oxidative stress, as well as water consumption and urination. We assume that RA may mitigate STZ-induced diabetic manifestations by protecting rat tissues against damaging effect of free radicals.

General Physiology and Biophysics. Volume 34, 2015, No. 4: 449-452.