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 wi th [P-32]-sodium phosphate to label intracellular ATP stores, Incubati ons of the [P-32]-labelled cardiomyocytes with a beta-adrenoceptor ago nist isoproterenol (10 mu M) and with a plant diterpene forskolin (100 mu M) Which directly stimulates adenylyl cyclase increased the phosph orylation of an inhibitory subunit of troponin (TN-I) and phospholamba n (PLN), Brief exposure (1 min) of labelled myocytes to the hydroxyl r adical generating system (H2O2 plus FeCl2) decreased markedly the stim ulatory action of isoproterenol and forskolin on TN-I and PLN phosphor ylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic a cid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory ef fect on the isoproterenol or forskolin stimulated TN-I and PLN phospho rylation. In contrast exposure of myocytes to low concentrations (< 50 mu M) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augme nted the stimulatory effect of isoproterenol on TN-I and PLN phosphory lation. 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 phosphoryla tion appeared related to greater increase in the isoproterenol-stimula ted cyclic AMP accumulation in the NEM-treated cardiomyocytes. The res ults 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 depresse d rate of relaxation of the myocardium exposed to oxygen free radicals .