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

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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 b-adrenoceptor agonist isoproterenol (10 µM) and with a plant diterpene forskolin (100 µM) 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 µM) 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 β-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. (Mol Cell Biochem 175: 99–107, 1997)

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  1. Department of Physiology, College of Medicine, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, S7N 5E5, Canada

    Prakash V. Sulakhe, Xuan T. Vo & Thomas E. Morris

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  1. Prakash V. Sulakhe
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  2. Xuan T. Vo
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  3. Thomas E. Morris
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Sulakhe, P.V., Vo, X.T. & Morris, T.E. Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: Modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents. Mol Cell Biochem 175, 99–107 (1997). https://doi.org/10.1023/A:1006860110618

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