title: Phospholamban and troponin I are substrates for protein kinase C in vitro but not in intact beating guinea pig hearts.
abstract: The incorporation of [32P]inorganic phosphate into membranous, myofibrillar, and cytosolic proteins was studied in Langendorff-perfused guinea pig hearts treated with phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoylglycerol (D8G), which are potent activators of protein kinase C. Control hearts were perfused with an inactive phorbol ester (4 alpha-phorbol 12,13-didecanoate), which does not cause activation of protein kinase C. To ensure the blockade of different receptor systems, the perfusions were carried out in the presence of prazosin, propranolol, and atropine. Perfusion of hearts with either PMA (4 microM) or D8G (200 microM) was associated with a negative effect on left ventricular inotropy and relaxation. Examination of the 32P incorporation into various fractions revealed that there were no increases in the degree of phosphorylation of phospholamban in sarcoplasmic reticulum, and troponin I and C protein in the myofibrils, although these proteins were found to be substrates for protein kinase C in vitro. However, in the same hearts, there were significant changes in the 32P incorporation into a 28-kDa cytosolic-protein. Examination of the activity levels of protein kinase C in hearts perfused with PMA indicated a redistribution of this activity from the cytosolic to the membrane fraction, suggesting the activation of the enzyme in vivo. These findings indicate that cardiac regulatory phosphoproteins, which may be phosphorylated by protein kinase C in vitro, are not substrates for protein kinase C in beating hearts perfused with phorbol esters or diacylglycerol analogues.
authors.affiliation: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, OH 45267-0575.
authors.forename: I
authors.initials: I
authors.lastname: Edes
authors.forename: E G
authors.initials: EG
authors.lastname: Kranias
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D006339:Heart Rate / Q000187:drug effects; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D009210:Myofibrils / Q000378:metabolism; D010477:Perfusion; D010710:Phosphates / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I; D020107:Troponin T; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D004075:Diglycerides; D015544:Inositol 1,4,5-Trisphosphate; D010710:Phosphates; D010718:Phosphatidylserines; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D014336:Troponin; D019210:Troponin I; D020107:Troponin T; D000099211:Phospholamban; C044295:1,2-dioctanoylglycerol