Effect of alpha adrenergic agents and phorbol esters on phosphorylation of sarcolemmal proteins in beating guinea pig hearts.

STUDY OBJECTIVE

The aim was to determine whether activation of protein kinase C by alpha adrenergic agonists or phorbol esters would be associated with increased phosphorylation of the 15 kDa sarcolemmal protein in guinea pig hearts.

DESIGN

Intact, beating guinea pig hearts were perfused with modified Krebs-Henseleit buffer containing [32P]Pi and freeze clamped in a control condition or at the peak of the inotropic response to noradrenaline. Membrane vesicles enriched in sarcolemma were isolated and then subjected to SDS polyacrylamide gel electrophoresis and autoradiography. Phosphorylated proteins were identified and 32P incorporation was quantitated. In some cases, hearts were perfused with phorbol 12-myristate, 13-acetate, or dioctanoyl-glycerol, which are known to be potent activators of protein kinase C. EXPERIMENTAL PREPARATIONS: Whole hearts from 55 anaesthetised guinea pigs weighing 500-600 g were used.

MEASUREMENTS AND MAIN RESULTS

Perfusion of guinea pig hearts with noradrenaline resulted in increases in contractility and tissue inositol 1,4,5-triphosphate levels, but there were no increases in the phosphorylation of the 15 kDa sarcolemmal protein observed. Furthermore, perfusion with phorbol 12-myristate, 13-acetate, or dioctanoylglycerol failed to stimulate the phosphorylation of the 15 kDa sarcolemmal protein.

CONCLUSIONS

These data indicate that the 15 kDa sarcolemmal protein, which may be phosphorylated by protein kinase C in vitro, is not a substrate for the same enzyme in beating guinea pig hearts.