Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.

Abstract

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.