K Mackay1, D Mochly-Rosen. 1. Department of Molecular Pharmacology, Stanford University School of Medicine, 269 Campus Drive, CCSR 3145, Stanford, CA 94305-5332, USA.
Abstract
OBJECTIVES: Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. METHODS: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. RESULTS: Arachidonic acid resulted in translocation of delta and epsilonPKC but not alpha, betaII, eta or zetaPKC isozymes, indicating activation of only delta and epsilonPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of epsilonPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4beta-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilonPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. CONCLUSIONS: These results demonstrate that arachidonic acid selectively activates delta and epsilonPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.
OBJECTIVES:Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. METHODS: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. RESULTS:Arachidonic acid resulted in translocation of delta and epsilonPKC but not alpha, betaII, eta or zetaPKC isozymes, indicating activation of only delta and epsilonPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of epsilonPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4beta-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilonPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. CONCLUSIONS: These results demonstrate that arachidonic acid selectively activates delta and epsilonPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.
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