Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.
Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.
Sulakhe
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PV
Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
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Phan
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Morris
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D012588:Scintillation Counting; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D019210:Troponin I / Q000378:metabolism*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D005576:Colforsin; D000242:Cyclic AMP; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D005609:Free Radicals; D017665:Hydroxyl Radical; D007545:Isoproterenol; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D019210:Troponin I; D000099211:Phospholamban