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General Physiology and Biophysics

Volume 31, 2012, No. 3


  Analysis of blood clot degradation fragment sizes in relation to plasma flow velocity
Franci Bajd 1), Jernej Vidmar, Aleš Blinc, Igor Serša

1)Jožef Stefan Institute, Ljubljana 1000, Slovenia.

Thrombolytic therapy aims to dissolve blood clots and restore vessel patency. Our hypothesis is that the therapy depends not only on chemical reactions of the fibrinolytic system, but also on mechanical forces exerted by streaming blood on the clot surface. The aim of the study was to analyze the role of mechanical forces in promoting thrombolysis and their relation to the maximum size of removed clot fragments. Non-occlusive whole-blood clots were exposed to flow of plasma containing the thrombolytic agent rt-PA. Plasma, perfusing through the clot, was collected and the sizes of clot degradation fragments in the plasma were analyzed by optical microscopy. Theoretical models for the maximum clot fragment size as a function of blood flow velocity were developed based on the relation between surface or volume clot binding forces and the opposing forces of the streaming plasma in the laminar or turbulent flow regime. The best agreement between experimental results and models was obtained for the volume binding forces and the laminar flow model, in which the maximum clot fragment size was linearly dependent on the plasma flow velocity. Such result could not be obtained if thrombolysis would be purely a biochemical process. Therefore, the result confirms our hypothesis that thrombolysis is also strongly influenced by the mechanical forces of streaming plasma.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 237-245.

  Effect of lesions of A5 or A7 noradrenergic cell group or surgical transection of brainstem catecholamine pathways on plasma catecholamine levels in rats injected subcutaneously by formalin
Boris Mravec 1), Ibolya Bodnar, Gabriela Uhereczky, Richard Kvetnansky, Miklos Palkovits

1)Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

The pain-induced activation of the sympatho-adrenal system is modulated by several brain areas, including brainstem catecholamine cell groups. In the present study, we evaluated the effect of bilateral lesions of the A5 or A7 cell groups or bilateral transections of brainstem catecholaminergic pathways on plasma catecholamine levels in Sprague-Dawley rats injected subcutaneously by formalin or saline. Plasma levels of both epinephrine and norepinephrine were slightly elevated after formalin injections within 15–30 min in rats with or without lesions of the A7 catecholamine cell group. However, saline but not formalin elicited a significant increase in plasma epinephrine level in both sham-operated and A5-lesioned groups. It is more likely, that formalin blocks the effect of the handling and the painful injection procedure. In rats with bilateral partial transections of the lower brainstem, formalin was more effective than saline in the elevation of plasma epinephrine and norepinephrine levels at several time-points through the investigation period. Our data indicate the involvement of A5 and A7 norepinephrine neurons and brainstem catecholaminergic pathways in the regulation of the activity of the sympatho-adrenal system during acute painful situations. Their modulatory effect, however, seems to be a very rapid one, short and moderate.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 247-254.

  Role of fenofibrate in restoring angiogenesis in diabetic and control hind limb ischemic rats
Ensieh Salehi 1), Majid Khazaei, Bahman Rashidi

1)Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran.

In this study, we examined the effect of fenofibrate, an agonist of peroxisome proliferator-activated receptor α (PPARα), on angiogenesis and serum nitric oxide (NO), vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) in diabetic and control hind limb ischemic rats. Male Wistar rats underwent left hind limb ischemia. The experimental groups consisted of rats: control, diabetic, control receiving fenofibrate (100 mg/kg/day, by gavage) and diabetic receiving fenofibrate. After 21 days, blood samples were taken and capillary density and capillary/fiber ratio of the ischemic hind limb muscle was evaluated by immunohistochemistry. Activation of PPARα by fenofibrate restored neovascularization in diabetic and control hind limb ischemic rats (p < 0.05). Fenofibrate administration significantly increased serum nitrite concentration, the main metabolite of NO, without significant changes in serum VEGF and VEGFR-2 concentrations. It seems that fenofibrate enhances angiogenesis in hind limb ischemia possibly through increasing of NO bioavailability and can be considered for treatment of diabetic peripheral vascular diseases in future human studies.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 255-260.

  Prolonged oxytocin treatment in rats affects intracellular signaling and induces myocardial protection against infarction
Maria Ondrejcakova 1), Miroslav Barancik, Monika Bartekova, Tana Ravingerova, Daniela Jezova

1)Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

Oxytocin is a hormone, which is released into the circulation in response to acute or chronic stress stimuli. One of the important targets of oxytocin is cardiovascular system. Present studies were aimed at testing the hypothesis that prolonged treatment with oxytocin (simulation of stress-induced rise in circulating oxytocin) activates intracellular signaling pathways playing a role in ischemia/reperfusion injury. Furthermore, we tested protective effects of oxytocin treatment in vivo against cardiac injury induced by ischemia/reperfusion of isolated hearts. Male Wistar rats were treated with oxytocin or vehicle continuously via osmotic minipumps for 2 weeks. The hearts were used for biochemical measurements or isolated for Langendorff perfusion. Treatment with oxytocin resulted in a significant increase in specific phosphorylation (activation) of p38-MAPK and Akt kinase, an increase in phosphorylated Hsp27 and an elevation in atrial natriuretic peptide (ANP) levels in left ventricular heart tissue. There were no significant changes in the activation of MMP-2 and ERK in the left heart ventricle of oxytocin-treated rats. Postischemic recovery of functional parameters LVDP, RPP, +dP/dtmax and -dP/dtmax was better in the hearts of oxytocin-treated rats compared to that in the controls. Oxytocin treatment significantly reduced infarct size to 15.1 + 3.2% as compared to 32.4 + 3.5% in vehicle-treated rats (p < 0.01). This is the first evidence for cardioprotective effects of oxytocin administered in vivo simulating chronic stress-induced elevation in plasma oxytocin. The present results show that positive effects of oxytocin that may ameliorate negative consequences of stress on the heart are, at least in part, mediated through p38-MAPK and Akt kinase pathways.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 261-270.

  The influence of ETA and ETB receptor blockers on LPS-induced oxidative stress and NF-κB signaling pathway in heart
Aleksandra Piechota-Polanczyk 1), Paulina Kleniewska, Anna Gorąca

1)Department of Cardiovascular Physiology, Medical University of Lodz, Lodz, Poland.

The aim of this study was to assess whether an endothelin-A receptor (ETA-R) blocker, BQ123, or an endothelin-B (ETB-R) receptor blocker, BQ788, influences nuclear factor kappa beta (NF-κB) pathway, free radical generation, tumor necrosis factor-alpha (TNF-α) concentration, and glutathione redox system in hearts obtained from lipopolysaccharide (LPS)-induced endotoxic rats. The study was performed on rats divided into groups: 1) saline, 2) saline + LPS (15 mg/kg), 3) BQ123 (1 mg/kg b.w.) + LPS, 4) BQ123 (0.5 mg/kg b.w.) + LPS, 5) BQ788 (3 mg/kg b.w.) + LPS. The ETA-R and ETB-R antagonists were injected i.v. 30 min before LPS administration. In rats, BQ123 caused a significant decrease in TBARS (p < 0.05) but not in H2O2 concentration. It also decreased tissue protein level and improved tissue redox status (p < 0.01). Only a dose of 1 mg/kg decreased TNF-α concentration (p < 0.05). BQ788 lowered TBARS, H2O2 and protein concentration (p < 0.05; p < 0.02; p < 0.001, respectively), however, it did not affect TNF-α concentration. Neither ETA-R nor ETB-R blockers influenced LPS-induced increase in p65 subunit level and activation of NF-κB pathway. Our results demonstrated that ETA-R blockage is more effective in inhibiting free radical generation and improving heart antioxidant properties than ETB-R blockage under oxidative stress. NF-κB pathway is not incorporated in ETA-R and ETB-R influence on ROS production.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 271-278.

  Reactivity of the mesenteric bed arteries of normotensive rats exposed to chronic social stress
Angelika Puzserova 1), Jozef Torok, Ruzena Sotnikova, Anna Zemancikova, Iveta Bernatova

1)Institute of Normal and Pathological Physiology, Centre of Excellence for Examination of Regulatory Role of Nitric Oxide in Civilization Diseases, Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic.

The aim of this study was to investigate the effects of chronic social stress on endothelium-dependent relaxation in the superior mesenteric artery (SMA) and its first branches (1MA) as well as on neurogenic contractions of SMA in adult, male Wistar-Kyoto (WKY) rats. Mesenteric arteries were isolated from control (living space: 480 cm2/rat) or stressed rats exposed to 8-week-lasting crowding stress (living space: 200 cm2/rat). Blood pressure (BP) and heart rate, determined by tail-cuff plethysmography, were not affected by crowding. Stress increased neurogenic contractions of SMA elicited by electrical stimulation of perivascular nerves and significantly elevated vasoconstriction induced by exogenous noradrenaline in SMA, without modulation of its endothelial function. In 1MA, nitric oxide (NO)-dependent component of endothelium-dependent relaxation to acetylcholine was investigated. In 1MA, stress failed to affect noradrenaline- and phenylephrine-induced vasoconstriction, total acetylcholine-induced relaxation as well as its NO-dependent and NO-independent components. Moreover, endothelium-independent sodium nitroprusside-induced relaxations of 1MA from the stressed rats did not differ from those of controls. In conclusion, chronic stress produced by crowding failed to induce an increase of BP, presumably because endothelial function of SMA and vascular function of small mesenteric arteries, which are rather important in BP regulation, remained preserved.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 279-290.

  Song-related dopamine receptor regulation in Area X of zebra finch male
Eva Bosíková 1), Ľubor Košťál, Martina Cviková, Boris Bilčík, Ľubica Niederová-Kubíková

1)Laboratory of Neurobiology and Physiology of Behaviour, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic.

Song learning and production have many parallels with speech and the mechanisms of their control have been studied extensively. There is an increasing amount of evidence that the dopaminergic system is involved in song learning and maintenance. Dopamine receptors show distinct expression in most of the song nuclei and the highest levels in Area X of the striatum. Here we have investigated whether the mRNA expressions for D1A, D1B, and D2 receptors in Area X are associated with quantitative and/or qualitative characteristics of zebra finch song. We found that quantitative parameters of song such as the amount of songs sang, motif duration, and numbers of distinct syllables and/or notes per motif did not correlate with expression of D1A, D1B nor D2 receptors in Area X or surrounding striatum. However, the mean accuracy of the song correlated negatively with D1A receptor expression levels and the sequential match correlated positively with D2 receptor expression levels in Area X relative to the surrounding striatum. These data suggest that dopamine receptor densities in Area X are associated with song variability.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 291-298.

  The effects of REV5901 on intracellular calcium signalling in freshly isolated bovine articular chondrocytes
Ala Qusous 1), Eleanor Parker, Niwa Ali, Sajid Mohmand, Mark Kerrigan

1)School of Life Sciences, University of Westminster, 115 New Cavendish Street, London, United Kingdom.

REV5901 is an inhibitor of regulatory volume decrease (RVD) a mechanotransduction pathway regulating cell volume in response to hypotonicity, with protective properties upon chondrocyte trauma impact in situ. As the mechanism of action of REV5901 is unknown and changes in intracellular calcium ([Ca2+]i) have been linked to REV5901-loading, we investigated the effects of REV5901 on a known calcium signalling pathway. Upon REV5901 loading, there was significant increase in [Ca2+]i reaching 37.97 ± 5.67%, above basal levels which was reduced to 27.86 ± 3.15% in the presence of 2 mmol/l EGTA. In the presence of U73122 or neomycin there was a decrease in calcium with inhibition factors (I.F.) of 0.39 ± 0.09 and 0.37 ± 0.08, respectively, whereas rottlerin abolished the REV5901-induced [Ca2+]i rise. The role of calcium channels in contributing to the REV5901-induced calcium rise was investigated whereby the calcium rise was inhibited in the absence of extracellular sodium and by the addition of Gd3+ and Ruthenium red. These data show a phospholipase Cβ3-dependent release of calcium from intracellular stores as well as a sodium calcium exchanger-mediated influx in response to REV5901 loading, suggesting a potential role for calcium signalling in mediating the action of REV5901 in chondrocytes.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 299-307.

  Promoter-context as a determinant of glucocorticoid receptor-responsiveness to activation of p38 and JNK mitogen-activated protein (MAP) kinases
Zoltan Szatmary 1), Anton Kebis

1)Slovak Medical University, Limbová 12, 833 03 Bratislava, Slovak Republic.

MAP kinases JNK and p38 play an important role in many immune and inflammatory processes, whereas glucocorticoids exert immunosuppressive and anti-inflammatory activities. We found previously that activation of p38 or JNK inhibits glucocorticoid receptor (GR)-mediated transcriptional activation of a mouse mammary tumor virus (MMTV) promoter-driven luciferase construct in HeLa cells. It appears that this effect is DNA regulatory element-specific, since p38 or JNK activation stimulates GR-dependent transcription from TAT3-ADH promoter-luciferase construct in the same cells. The apparent promoter-specificity of this action suggests that not all glucocorticoid-activated genes are negatively regulated by p38 or JNK. Using different MMTV/TAT3 chimeric reporters we demonstrate that the presence of other accessory binding sites of the MMTV construct contributes to the inhibitory effect of activated p38 or JNK on the MMTV-driven transcriptional activity; and diminishes, but does not reverse the stimulation observed using the TAT GREs from the TAT3-ADH promoter-luciferase construct. On the other hand, comparison of the effects of GRE sequences, either in isolation or in the context of the MMTV LTR accessory binding sites, demonstrates that the responsiveness of the GR depends on the GRE sequence; indicating that in addition to transcription factors bound nearby, interaction with the DNA itself modulates GR activity.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 309-322.

  Ursolic acid and rosiglitazone combination alleviates metabolic syndrome in high fat diet fed C57BL/6J mice
Arjunan Sundaresan 1), Ranganathan Harini, Kodukkur Pugalendi

1)Faculty of Science, Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608 002, Tamilnadu, India.

The aim of this study was to examine the combined effect of ursolic acid (UA) and rosiglitazone (RSG) on metabolic syndrome in C57BL/6J mice. Upon feeding high fat diet (HFD) C57BL/6J mice developed obesity, insulin resistance, dyslipidemia and hypertension. The male mice were randomly divided into six groups, and fed normal diet, normal diet plus UA and RSG, HFD alone, HFD plus UA, HFD plus RSG, and HFD plus UA and RSG, respectively. HFD fed mice showed increase in body weight, elevated plasma glucose and insulin. Activities of gluconeogenic enzymes such as glucose 6-phosphatase, fructose 1,6-bisphosphatase increased while the activity of glycolytic enzyme, glucokinase, decreased in the liver along with glycogen content. Total cholesterol, triglyceride, low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol and free fatty acid levels significantly increased in the plasma, whereas high-density lipoprotein cholesterol significantly decreased in high fat diet fed mice. In addition, both systolic and diastolic blood pressure was increased significantly. Combined treatment with UA and RSG improved the above parameters towards normality and pronounced more responses than UA or RSG lone treatment. The inclusion of UA in treatment with RSG may reduce the body weight gain, one of adverse side effect associated with the RSG-therapy.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 323-333.

  Evaluation of Opuntia ficus indica f. inermis fruit juice hepatoprotective effect upon ethanol toxicity in rats
Hichem Alimi 1), Najla Hfaeidh, Sakhria Mbarki, Zouhour Bouoni, Mohsen Sakly, Khémais Ben Rouma

1)Research unit of Macromolecular Biochemistry and Genetic, Faculty of Sciences of Gafsa, 2112 Gafsa, Tunisia.

The aim of our present study is to investigate the effect of Opuntia ficus indica f. inermis prickly pear juice (PPJ) against ethanol-induced liver injury in rats. Chronic ethanol administration (3 g/kg b.w.) during 90 days to Wistar rats, significantly (p < 0.01) increased the liver lipid and protein oxidation, reduced the glutathione content and the activities of liver antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and conversely elevated the liver injury biochemical markers like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase, cholesterol, triglycerides and caused a severe histopathologic injuries. Conversely pre-treatment of ethanol-fed rats with PPJ (20 and 40 ml/kg b.w., orally), interestingly reduced liver lipid and protein oxidation, histopathologic lesions and inhibited the alterations of antioxidant enzymes and the release of biochemical markers. The hepatoprotective effect of PPJ could be due to their capacity to end free radicals chain reactions or to enhance the endogenous antioxidants activities.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 335-342.

  The activity of G-ROS and the predominant role of Type II reaction in the photodynamic therapy using 9-hydroxypheophorbide-α for HeLa cell lines
Jin-Chul Ahn 1), Phil-Sang Chung

1)Medical Laser Research Center, Dankook University, Cheonan, Chungnam 330-714, Korea.

Photodynamic therapy (PDT) is a treatment modality that destroys the tumor. It activates the photosensitizer with the light of a specific wavelength, where the light is well absorbed by the photosensitizer, thus causing a fatal injury and thereby leading to a tumor necrosis. To date, a hematoporphyrin-derived photosensitizer has been widely used. It is disadvantageous, however, in that it causes a long-term photo-toxicity and has a poor selectivity for the tumor. This had led to the development of a chlorophyll-derived photosensitizer. We conducted this study to elucidate the mechanisms by which the activity of ROS is involved in the PDT using a novel type of chlorophyll-derived photosensitizer, 9-hydroxypheophorbide-α (9-HpbD-α), for the HeLa cell lines. Besides, we also attempted to determine which reaction plays a predominant role in the synthesis of ROS, either Type I reaction or Type II one, when both reactions are involved in the synthesis of ROS during the PDT using 9-HpbD-α. Our results showed not only that the activity of ROS is involved in the PDT using 9-HpbD-α in human uterine cervical cancer cell lines but also that the mechanisms of PDT are based on Type II reaction where the singlet oxygen is involved.

General Physiology and Biophysics. Volume 31, 2012, No. 3: 343-350.