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Expression and subcellular redistribution of PKC isoforms in chronically hypoxic rat heart
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
027. Involvement of nitric oxide and reactive oxygen species in the delayed cardioprotection induced by lipopolysaccharides Tama´s Csont a, Gabriella Fodor b, La´szlo´ Sa´rva´ry b, Pe´ter Bencsik b, Pe´ter Ferdinandy a. a Pharmahungary 2000 Ltd., Szeged, Hungary 1. b Cardiovascular Research Group, University of Szeged, Szeged, Hungary. www.pharmahungary. com, www.cardiovasc.com Bacterial lipopolysaccharides (LPS) have been shown to protect the ischemic myocardium 24 –72 h following treatment. The mechanism of LPS-induced late cardioprotection is not clear; however, the involvement of nitric oxide (NO) has been suggested. Therefore, here we further studied the effect of LPS-treatment on cardiac signalling by nitric oxide (NO) and reactive oxygen species (ROS) by systemic analysis of NO and ROS production and activities of enzymes responsible for NO and ROS signalling. Wistar rats were injected (0.5 mg/kg i.p.) with LPS (S. typhimurium) or saline. Twenty-four hours later, hearts were isolated and perfused aerobically in a ‘‘working mode’’ for 10 min. Hearts were then subjected to 30 min global ischemia followed by 20 min reperfusion. Ischemia/reperfusion decreased aortic flow from its preischemic value of 44.3 T 1.5 ml/min to 8.7 T 5.4 ml/min measured at the end of the reperfusion. LPS treatment significantly improved aortic flow (22.8 T 6.8 ml/min, P < 0.05) upon reperfusion. Basal cardiac levels of both NO and superoxide, as well as the level of serum nitrotyrosine, a marker for peroxynitrite synthesis, were significantly elevated 24 h after LPStreatment. Cardiac activities of the Ca2+-independent NO synthase and xanthine oxidoreductase were significantly increased. Activities of the Ca2+-dependent NO synthase, NADPH oxidase, and superoxide dismutase were not changed in the heart due to LPS treatment. Our results indicate that LPS, surprisingly, leads to increased formation of myocardial NO, superoxide, and their cytotoxic metabolite peroxynitrite. The mechanisms on how increased peroxynitrite may contribute to late preconditioning induced by LPS remain to be elucidated.
forms y and ( in normoxic and chronically hypoxic hearts. Adult male Wistar rats were divided into two groups that were either exposed for 5 weeks to intermittent hypobaric hypoxia of 7000 m (8 h/day, 5 days/week) or kept under normoxic conditions. Both chronically hypoxic and normoxic rats were divided into three subgroups: chelerythrine-treated (5 mg/kg), rottlerin-treated (0.3 mg/kg) and untreated controls. Hearts were collected 15 min after the intravenous administration of the inhibitors. The immunoanalysis of PKC isoforms was performed in homogenate and in particulate and cytosolic fractions (100 103 g) from the left ventricles, followed by Western blotting and chemiluminescent ECL technique. Subcellular localization of PKC isoforms was visualized in frozen apex sections (5 Am) by immunofluorescent staining followed by digital imaging microscopy. Chronic hypoxia decreased the expression of PKC( in homogenate (by 28%) and PKC inhibitors had no effect on this isoform. On the contrary, chronic hypoxia increased the expression of PKCy predominantly in the particulate fraction (by 180%). As revealed by immunofluorescent staining, chronic hypoxia induced PKCy redistribution to the sarcolemma, mitochondria and nuclear/peri-nuclear area. PKCy-selective inhibitor rottlerin partially reversed hypoxia-induced changes of this isoform. These results are in line with the inhibitory effect of rottlerin on the increased ischemic tolerance of chronically hypoxic hearts [1] and support the view that PKCy plays a role in this long-term cardioprotective mechanism.
Acknowledgment Supported by GA CR 305/04/0465 and GA UK 110/2005/ C/PrF.
Reference [1] Neckar et al. Am J Physiol Heart Circ Physiol 2005;288:1566 – 72.
doi:10.1016/j.yjmcc.2006.03.042 doi:10.1016/j.yjmcc.2006.03.043
028. Expression and subcellular redistribution of PKC isoforms in chronically hypoxic rat heart M. Hlavackova a, J. Neckar b,c, L. Nevelikova a, O. Novakova a,c, F. Kolar b,c, R.J.P. Musters d, F. Novak a. a Fac Sci, Charles University, The Netherlands. b Inst Physiol, Acad Sci, The Netherlands. c Ctr Cardiovasc Res, Prague, Czech Republic, The Netherlands. d Lab Physiol, ICaR, Free Univ Amsterdam, The Netherlands
029. Activity of MnSOD in chronically hypoxic rat heart: Effect of N-acetylcysteine P. Balkova a, O. Novakova a,c, F. Novak a, J. Neckar b,c, M. Millerova b,c, F. Kolar b,c. a Fac Sci, Charles University, Prague, Czech Republic. b Inst Physiol, Acad Sci, Prague, Czech Republic. c Ctr Cardiovasc Res, Prague, Czech Republic
Protein kinase C (PKC) is involved in the cardioprotective mechanism of chronic hypoxia (1). The aim of this study was to analyze acute effects of PKC inhibitors on the expression and subcellular localization of PKC iso-
Manganese superoxid dismutase (MnSOD) is an important antioxidant enzyme that provides primary defense against mitochondrially derived reactive oxygen species. MnSOD localizes to mitochondria and its release into the
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
027. Involvement of nitric oxide and reactive oxygen species in the delayed cardioprotection induced by lipopolysaccharides Tama´s Csont a, Gabriella Fodor b, La´szlo´ Sa´rva´ry b, Pe´ter Bencsik b, Pe´ter Ferdinandy a. a Pharmahungary 2000 Ltd., Szeged, Hungary 1. b Cardiovascular Research Group, University of Szeged, Szeged, Hungary. www.pharmahungary. com, www.cardiovasc.com Bacterial lipopolysaccharides (LPS) have been shown to protect the ischemic myocardium 24 –72 h following treatment. The mechanism of LPS-induced late cardioprotection is not clear; however, the involvement of nitric oxide (NO) has been suggested. Therefore, here we further studied the effect of LPS-treatment on cardiac signalling by nitric oxide (NO) and reactive oxygen species (ROS) by systemic analysis of NO and ROS production and activities of enzymes responsible for NO and ROS signalling. Wistar rats were injected (0.5 mg/kg i.p.) with LPS (S. typhimurium) or saline. Twenty-four hours later, hearts were isolated and perfused aerobically in a ‘‘working mode’’ for 10 min. Hearts were then subjected to 30 min global ischemia followed by 20 min reperfusion. Ischemia/reperfusion decreased aortic flow from its preischemic value of 44.3 T 1.5 ml/min to 8.7 T 5.4 ml/min measured at the end of the reperfusion. LPS treatment significantly improved aortic flow (22.8 T 6.8 ml/min, P < 0.05) upon reperfusion. Basal cardiac levels of both NO and superoxide, as well as the level of serum nitrotyrosine, a marker for peroxynitrite synthesis, were significantly elevated 24 h after LPStreatment. Cardiac activities of the Ca2+-independent NO synthase and xanthine oxidoreductase were significantly increased. Activities of the Ca2+-dependent NO synthase, NADPH oxidase, and superoxide dismutase were not changed in the heart due to LPS treatment. Our results indicate that LPS, surprisingly, leads to increased formation of myocardial NO, superoxide, and their cytotoxic metabolite peroxynitrite. The mechanisms on how increased peroxynitrite may contribute to late preconditioning induced by LPS remain to be elucidated.
forms y and ( in normoxic and chronically hypoxic hearts. Adult male Wistar rats were divided into two groups that were either exposed for 5 weeks to intermittent hypobaric hypoxia of 7000 m (8 h/day, 5 days/week) or kept under normoxic conditions. Both chronically hypoxic and normoxic rats were divided into three subgroups: chelerythrine-treated (5 mg/kg), rottlerin-treated (0.3 mg/kg) and untreated controls. Hearts were collected 15 min after the intravenous administration of the inhibitors. The immunoanalysis of PKC isoforms was performed in homogenate and in particulate and cytosolic fractions (100 103 g) from the left ventricles, followed by Western blotting and chemiluminescent ECL technique. Subcellular localization of PKC isoforms was visualized in frozen apex sections (5 Am) by immunofluorescent staining followed by digital imaging microscopy. Chronic hypoxia decreased the expression of PKC( in homogenate (by 28%) and PKC inhibitors had no effect on this isoform. On the contrary, chronic hypoxia increased the expression of PKCy predominantly in the particulate fraction (by 180%). As revealed by immunofluorescent staining, chronic hypoxia induced PKCy redistribution to the sarcolemma, mitochondria and nuclear/peri-nuclear area. PKCy-selective inhibitor rottlerin partially reversed hypoxia-induced changes of this isoform. These results are in line with the inhibitory effect of rottlerin on the increased ischemic tolerance of chronically hypoxic hearts [1] and support the view that PKCy plays a role in this long-term cardioprotective mechanism.
Acknowledgment Supported by GA CR 305/04/0465 and GA UK 110/2005/ C/PrF.
Reference [1] Neckar et al. Am J Physiol Heart Circ Physiol 2005;288:1566 – 72.
doi:10.1016/j.yjmcc.2006.03.042 doi:10.1016/j.yjmcc.2006.03.043
028. Expression and subcellular redistribution of PKC isoforms in chronically hypoxic rat heart M. Hlavackova a, J. Neckar b,c, L. Nevelikova a, O. Novakova a,c, F. Kolar b,c, R.J.P. Musters d, F. Novak a. a Fac Sci, Charles University, The Netherlands. b Inst Physiol, Acad Sci, The Netherlands. c Ctr Cardiovasc Res, Prague, Czech Republic, The Netherlands. d Lab Physiol, ICaR, Free Univ Amsterdam, The Netherlands
029. Activity of MnSOD in chronically hypoxic rat heart: Effect of N-acetylcysteine P. Balkova a, O. Novakova a,c, F. Novak a, J. Neckar b,c, M. Millerova b,c, F. Kolar b,c. a Fac Sci, Charles University, Prague, Czech Republic. b Inst Physiol, Acad Sci, Prague, Czech Republic. c Ctr Cardiovasc Res, Prague, Czech Republic
Protein kinase C (PKC) is involved in the cardioprotective mechanism of chronic hypoxia (1). The aim of this study was to analyze acute effects of PKC inhibitors on the expression and subcellular localization of PKC iso-
Manganese superoxid dismutase (MnSOD) is an important antioxidant enzyme that provides primary defense against mitochondrially derived reactive oxygen species. MnSOD localizes to mitochondria and its release into the