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General Physiology and Biophysics

Volume 33, 2014, No. 2


  Potent cough suppression by physiologically active substance in human plasma
Norio Akaike 1), Yushi Ito, Sachie Ogawa, Megumi Maeda, Masahito Wakita, Kazuo Takahama, Tetsuro Noguchi, Shintaro Kamei, Takayoshi Hamamoto, Misako Umehashi, Hiroaki Maeda

1)Research Division for Life Science, Kumamoto Health Science University, 325 Izumi-machi, Kita-ku, Kumamoto 861-5598, Japan.

Human plasma contains wide variety of bioactive proteins that have proved essential in therapeutic discovery. However many human plasma proteins remain orphans with unknown biological functions. Evidences suggest that some plasma components target the respiratory system. In the present study we adapted heparin affinity chromatography to fractionate human plasma for functional bioassay. Fractions from pooled human plasma yielded particular plasma fractions with strong cough suppressing effects. Purification yielded a fraction that was finally identified as an activated blood coagulation factor fXIa using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). The fraction almost completely suppressed coughs induced by either chemical or mechanical stimulation applied to larynx or bifurcation of guinea-pig trachea. Cough suppressing effect of the fraction and commercially available fXIa were one million times stronger than codeine and codeine only partially suppressed the mechanically triggered coughing in animal model. Recent reviews highlighted prominent shortcomings of current available antitussives, including narcotic opioids such as codeine and their unpleasant or intolerable side effects. Therefore, safer and more effective cough suppressants would be welcome, and present findings indicate that fXIa in human plasma as a very promising, new therapeutic candidate for effective antitussive action.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 145-155.

  Long-term effects of somatostatin 14 on the pituitary-ovarian axis in rats
Nataša Nestorović 1), Milica Manojlović-Stojanoski, Svetlana Trifunović, Nataša Ristić, Branko Filipović, Branka Šošić-Jurjević, Verica Milosevic

1)Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia.

The long-term effects of somatostatin 14 (SST-14) on the pituitary-ovarian axis were examined. Female Wistar rats received 20 µg/100g b.w. doses subcutaneously twice daily for 5 consecutive days in the infantile (from 11th to 15th day) or peripubertal (from 33rd to 37th day) period of life. Females treated as infants were killed in the peripubertal (38th day) or adult period of life (80th day), and those treated during peripuberty as adults (80th day). Pituitary follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) cells, and ovaries were analyzed by stereology and morphometry. Serum FSH and LH concentrations were determined by RIA. FSH and LH cell volumes were significantly decreased in pituitaries of peripubertal females treated with SST-14 as infants, and in adult females treated during peripuberty. GH cell volume was decreased in all treated rats. In the ovaries, enlargement of the non-growing pool of follicles was detected in adult females treated during peripuberty. SST-14 applied to infant rats did not lead to changes in initial follicular recruitment, but it disturbed follicle growth and development at later stages. It can be concluded that SST-14 exerted long-term inhibitory effects on the pituitary-ovarian axis and GH cells in rats.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 157-168.

  Placental infiltration of inflammatory markers in gestational diabetic women
Ines Mrizak 1), Oussama Grissa, Benoit Henault, Mariem Fekih, Ali Bouslema, Imen Boumaiza, Monia Zaouali, Zouhair Tabka, Naim Khan

1)University of Burgundy, INSERM U866, Faculty of Life Sciences, Dijon, France.

Gestational diabetes mellitus (GDM) is pathology of glucose intolerance during pregnancy. It is influenced by maternal hyperglycemia and insulinemia through placental circulation. The study was undertaken to investigate the implication of pro-inflammatory factors in the placenta of GDM women. Thirty GDM women have delivered macrosomic babies, and 30 healthy age-matched pregnant women have delivered non macrosomic babies, were recruited in the study. The mRNAs encoding for IL-6, TLR4, TGF-β, CD68, CD14, EMR-1, CCL2, TCR-α, T-bet, GATA-3, leptin and adiponectin were quantified in placental samples by using RT-qPCR. The mRNA expression of the pro-inflammatory factors, i.e., IL-6, TLR4 and TGF-β, was increased in GDM placenta. The mRNA expression of markers of infiltration of macrophage, i.e., CD68, CD14 and EMR-1, was higher in the GDM placenta than the control placenta. The expression of mRNA of TCR-α, an indicator of T-cell infiltration, was significantly higher in the GDM placenta. Interestingly, the expression of mRNA of GATA-3, an indicator of Th2 phenotype differentiation, was unregulated in the GDM placenta. Leptin and adiponectin mRNAs were also significantly increased in the placenta of the GDM group. Our results revealed that there is an increase of inflammation in the GDM placenta which might be involved, in part, in the pathogenesis of macrosomia.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 169-176.

  Approximation of A1 adenosine receptor reserve appertaining to the direct negative inotropic effect of adenosine in hyperthyroid guinea pig left atria
Krisztian Pak 1), Csaba Papp, Zoltan Galajda, Tamas Szerafin, Balazs Varga, Bela Juhasz, David Haines, Andras Szentmiklosi, Arpad Tosaki, Rudolf Gesztelyi

1)Department of Pharmacology, Medical and Health Science Center, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98, Hungary.

Hyperthyroidism elevates cardiovascular mortality by several mechanisms, including increased risk of ischemic heart disease. Therefore, therapeutic strategies, which enhance tolerance of heart to ischemia-reperfusion injury, may be particularly useful for hyperthyroid patients. One promising cardioprotective approach is use of agents that cause (directly or indirectly) A1 adenosine receptor (A1 receptor) activation, since A1 adenosinergic pathways initiate protective mechanisms such as ischemic preconditioning. However, previously we found great A1 receptor reserve for the direct negative inotropic effect of adenosine in isolated guinea pig atria. This phenomenon suggests that weakening of atria is a possible side effect of A1 adenosinergic stimulant agents. Thus, the goal of the present investigation was to explore this receptor reserve in hyperthyroidism. Our recently developed method was used that prevents the rapid intracellular elimination of adenosine, allowing sufficient time for exogenous adenosine administered for the generation of concentration-response curves to exert its effect. Our method also allowed correction for the bias caused by the consequent endogenous adenosine accumulation. Our results demonstrate that thyroxine treatment does not substantially affect the A1 receptor reserve for the direct negative inotropic effect of adenosine. Consequently, if an agent causing A1 receptor activation is administered for any indication, the most probable adverse effect affecting the heart may be a decrease of atrial contractility in both eu- and hyperthyroid conditions.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 177-188.

  Single- and multiple-dose pharmacokinetics of arginase inhibitor Nω-hydroxy-nor-L-arginine, and its effect on plasma amino acids concentrations in Wistar rats
Zuzana Havlínová 1), Miloš Hroch, Andrea Nagy, Luděk Šišpera, Milan Holeček, Jaroslav Chládek

1)Department of Pharmacology, Faculty of Medicine, Charles University, Hradec Králové, Czech Republic.

Arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) augments synthesis of nitric oxide (NO) exerting therapeutic effects in rodent models for cardiovascular and airway diseases. This study examined single- and multiple-dose pharmacokinetics and effects of nor-NOHA on plasma amino acids in Wistar rats. Animals were administered 30 mg/kg nor-NOHA in a single bolus intravenous (i.v.) or intraperitoneal (i.p.) injection, or five once-daily i.p. injections at the same dose, or vehicle. Nor-NOHA and amino acids were assayed in blood plasma by high-performance liquid chromatography. After a bolus i.v. injection, the elimination of nor-NOHA was rapid (the mean residence time was 12.5 min). The area under the concentration-time curve and maximum concentration were higher by 17% and 31%, respectively, after the fifth as compared to the first i.p. injection. A shift in arginine utilization towards the synthesis of NO was indicated by elevated citrulline-to-ornithine and citrulline-to-arginine ratios. No changes in plasma arginine were observed. Increased glutamine concentrations might indicate an alternative detoxification pathway for ammonia due to inhibition of hepatic arginase. In conclusion, pharmacokinetic data of the present study can guide rational dosing of nor-NOHA in future studies. Limitations of the strategy of NO modulation via arginase inhibition should be further explored.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 189-198.

  Association of specific diplotypes defined by common rs1800682 and rare rs34995925 single nucleotide polymorphisms within the STAT1 transcription binding site of the FAS gene promoter with preeclampsia
Zora Lasabova 1), Imrich Zigo, Iveta Svecova, Gabor Szabo, Andrea Stanclova, Maria Skerenova, Pavol Zubor, Kristina Biskupska-Bodova, Janos Rigo, Balint Nagy, Jan Danko

1)Institute of Molecular Biology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak Republic.

The tolerance of fetal antigens by intradecidual T-cell involving the Fas-mediated apoptosis plays an important role in the physiological course of pregnancy. Objective of this study is to determine the association of diplotypes of common rs1800682G and rare rs34995925C alleles within the STAT1 transcription binding site of the FAS promoter region with preeclampsia. There were 116 preeclamptic women and 123 healthy control subjects from Hungary and Slovakia enrolled in the study. The presence of the GG or GA genotypes on rs1800682 was confirmed in 91 patients and 85 controls (OR = 1.628, 95%CI 0.907-2.92). The rare rs34995925 C allele laying 7 bp further from rs1800682 within STAT1 transcription binding site was detected in 3 preeclamptic cases and none healthy subjects. Haplotypes GT and AC were defined by common rs1800682G and rare rs34995925C alleles, respectively, and were considered as “low” FAS-producing. The combinations of GT or AC with normal FAS-producing haplotypes AT were considered as “low” FAS-producing diplotypes in dominant model. The “low” FAS –producing diplotype group of GT/GT, GT/AT, and AC/AT compared to the normal FAS-producing diplotype group of AT/AT showed OR = 1.91 (95%CI 1.04-3.48) and p = 0.03 for the association with preeclampsia.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 199-204.

  Possible role of nitric oxide in hepatic injury secondary to renal ischemia-reperfusion (I/R) injury
Abdelaziz Hussein 1), Hazem Khaled, Mohamed Seisa, Azza Baiomy, Mie Mohamed, Dina Eltantawy, Amel Mahmoud, Hussein Sheashaa, Mohamed Sobh

1)Medical Physiology Department, Mansoura Faculty of Medicine, Mansoura, Egypt.

Hepatic injury secondary to renal I/R injury has been documented in several studies. This study aimed to investigate the role of NO in hepatic injury secondary to renal I/R in rat model. Sprague-Dawley rats (n = 48) were divided into 4 equal groups; sham-operated, I/R injury group (45 min of bilateral renal ischemia), L-arginine group (I/R with 300 mg/kg L-arginine, 20 min before ischemia), L-NAME group (I/R with 50 mg/kg L-NAME, 20 min before ischemia). L-NAME (NO synthase inhibitor) caused significant elevation in serum creatinine, BUN, liver enzymes, liver histopathological damage score (p ≤ 0.05) and MDA production (p ≤ 0.001); on the other hand significantly decreased NO and GSH levels (p ≤ 0.05). L-arginine significantly decreased serum creatinine, BUN and GSH (p ≤ 0.05) and caused significant elevation in liver enzymes and NO (p ≤ 0.05), and also in MDA levels (p ≤ 0.001) in liver tissues. We conclude that endogenous NO might have protective effect against hepatic injury induced by renal I/R injury and inhibition of this endogenous NO by L-NAME or exogenous administration of NO (by L-arginine) might be harmful.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 205-213.

  Adrenomedullin and the calcitonin receptor-like receptor system mRNA expressions in the rat heart and sensory ganglia in experimentally-induced long-term diabetes
Eliska Mistrova 1), Silke Wiegand, Jitka Sviglerova, Uwe Pfeil, Jitka Kuncova, Jana Slavikova, Wolfgang Kummer, Magdalena Chottova Dvorakova

1)Department of Physiology, Faculty of Medicine in Pilsen, Charles University in Prague, Lidicka 1, Pilsen, Czech Republic.

Both adrenomedullin and calcitonin gene-related peptide (CGRP) regulate vascular tone in the heart, being cardioprotective in hypoxia. Additionally, adrenomedullin exhibits antiproliferative and antiapoptotic functions in the myocardium, while CGRP exerts positive chronotropic effect. Their actions are mediated through the specific G protein-coupled receptor, CRLR, whose ligand affinity is determined by receptor activity modifying proteins RAMP1-3. CGRP binds to the complex formed by CRLR/RAMP1, whereas CRLR/RAMP2 and CRLR/RAMP3 serve as receptors for adrenomedullin. Here, we quantified expression of this signaling system in the rat heart and supplying sensory ganglia (dorsal root ganglia T1-T4 and vagal nodose ganglia) in streptozotocin-induced diabetes. In the course of diabetes, an increase of CRLR mRNA was noticed in the right ventricle 8 weeks and of RAMP3 mRNA in the left ventricle and right atrium 26 weeks after induction of diabetes. Relative expressions of other tested genes were not significantly altered. In the nodose vagal supplying specific cardiac afferents, but not in dorsal root ganglia which provide cardiac pain fibres, a small upregulation of CGRP expression was detected. In summary, the shifts observed in diabetes may favour a trend of a pronounced adrenomedullin signaling. These observations may provide a new possible therapeutic strategy for diabetic cardiomyopathy.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 215-225.

  Crystallization, solubility and thermodynamics of the highly thermostable glucose isomerase from Streptomyces sp. strain
Mohamed Borgi 1), Moez Rhimi, Adel Kadri

1)Laboratory of Enzymes and Metabolites of Prokaryotes, CBS, B.P 1177, Sfax, Tunisia.

The crystallization behaviour of the highly thermostable glucose isomerase from the Streptomyces sp. strain isolated from Tunisian soil was investigated using ammonium sulfate as a precipitating agent. We established phase diagrams at different temperatures and protein concentrations. It was found that the solubility increased with increasing temperature and decreased with increasing salt concentration. The temperature-dependent solubility was used to characterize the thermodynamic parameters of crystallization such as enthalpy, entropy and free energy.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 227-234.

  Antimanic drug sensitizes breast cancer cell line to ionizing radiation
Maryam Rouhani 1), Bahram Goliaei, Fariba Khodagholi, Alireza Nikoofar

1)Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

Breast cancer is one of the most prevalent types of cancer among women. Lithium chloride (LiCl) is an FDA-approved drug for bipolar disorder. Breast cancer is reported to occur with higher rate in women with bipolar disorder. The effect of LiCl on the response of breast cancer cells to ionizing radiation has not been studied. We studied the effect of LiCl on the radiosensitivity of radioresistant T47D breast cancer cell line. Treatment of T47D cells with 20 mM LiCl for 24 hours decreased the radioresistance of these cells indicated by clonogenic survival assay. Comet assay demonstrated decreased DNA repair in LiCl-treated cells. LiCl treatment also decreased the meiotic recombination 11 (Mre11) mRNA level. Mre11 is an essential protein for DNA repair whose transcription is regulated by β-catenin protein. Western blot analysis indicated that the β-catenin protein level was decreased in LiCl-treated cells. LiCl increased glycogen synthase kinase-3β (GSK-3β) protein that is involved in β-catenin degradation. The results demonstrated that LiCl could radiosensitize T47D cells by decreasing DNA repair, partially through Mre11 repression. GSK-3β/β-catenin/Mre11 pathway might be the connection between LiCl treatment and the decreased DNA repair in T47D cells.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 235-242.

  The effects of acute intermittent hypoxia on cardiovascular parameters in normotensive and chronic hypobaric hypoxia-induced hypertensive rabbits
Muhittin Yaman 1), Ibrahim Guner, Hafize Uzun, Gulderen Sahin, Nermin Yelmen

1)Department of Physiology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey.

The effects of both chronic hypoxia and acute intermittent hypoxia (AIH) on cardiovascular system are unclear. We designed this study to develop a rabbit model of hypertension by exposure to chronic hypobaric hypoxia (CHH) and to investigate the effects of AIH on hypertensive rabbits. Present study was performed in 13 albino rabbits that divided into CHH and control groups. To develop hypertension, the rabbits were placed in a hypobaric chamber (390 mmHg; 22 hours/day, 30 days). Afterwards, AIH protocol was applied (8% FIO2 (Fraction of Inspired Oxygen) 1 min + 5 min normoxia, 20 cycles, 2 hours) to rabbits anesthetized with urethane and alpha-chloralose. Mean arterial pressure (MAP), heart rate (HR) and hematocrit values have been determined. Also asymmetric dimethylarginine (ADMA), endothelial nitric oxide synthase (eNOS), endothelin-1 and norepinephrine values have been analyzed in blood. We developed a model of hypertension in rabbits via exposure to severe CHH and we believe that ADMA is an important parameter in the development and permanence of CHH-induced hypertension. The main finding of this sudy was the depressor effect of AIH on blood pressure and heart rate in CHH- induced hypertension model. Finally, we believe that AIH protocol may be applicable for prevention and treatment of hypertension if properly developed.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 243-250.

  Chronic consumption of three forms of palm oil diets alters glomerular filtration rate and renal plasma flow
Favour Beshel 1), Atim Antai, Eme Osim

1)Department of Physiology, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Nigeria.

The effects of chronic consumption of three types of palm oil diets on glomerular filtration rate (GFR), renal plasma flow (RPF) and blood pressure were studied. Wistar rats were randomly assigned into four groups of ten rats each, respectively: control, fresh (FPO), photoxidized (PPO), thermoxidized (TPO) palm oil diet-fed rats. The control group was fed rat chow only, while experimental groups had different palm oil diets at 15% wt/wt for twelve weeks and tap water ad libitum. After the feeding period, GFR, RPF, systolic and diastolic blood pressures were measured. GFR and RPF of the TPO (0.07 ± 0.01 ml/min and 1.50 ± 0.24 ml/min) and PPO (0.14 ± 0.01 and 2.54 ± 0.11) groups were significantly (p < 0.001) reduced compared with control (0.77 ± 0.04 and 5.3 ± 0.30) and FPO (0.81 ± 0.02 and 4.8 ± 0.13) groups. The GFR and RPF of the TPO group was significantly (p < 0.05) higher than that of the PPO group. Systolic and diastolic blood pressures of the TPO group (140 ± 3 mmHg and 106 ± 4 mmHg) were significantly (p < 0.01) increased when compared with the control (112 ± 6.4 and 78 ± 5), FPO (118 ± 5 and 81 ± 6) and PPO (122 ± 5 and 89 ± 5) groups. These results suggest that chronic consumption of TPO and PPO caused a decrease in GFR and RPF, but increased blood pressure in rats, while FPO did not adversely affect blood pressure, GFR and RPF.

General Physiology and Biophysics. Volume 33, 2014, No. 2: 251-256.