Regulation of guinea pig heart phosphorylase kinase by cAMP, protein kinase, and calcium.
In skeletal muscle the activation of phosphorylase kinase (PK) associated with phosphorylation of the enzyme can be measured as an increase in the pH 6.8:8.2 activity ratio. Phosphorylation leads to a 20- to 30-fold increase in PK activity (PKA) at pH 6.8 and a large decrease in the Km for phosphorylase. Perfused guinea pig hearts exposed to isoproterenol (0.3 microM) showed an increase in PKA, but without an increase in the pH 6.8:8.2 activity ratio. In a 10-fold dilution of guinea pig heart cytosol exposed to cAMP + methylisobutylxanthine, PKA was stimulated twofold at pH 7.5. Addition of exogenous protein kinase stimulated PKA fourfold. Both methods of activation were reversible and were blocked by the heat-stable inhibitor of protein kinase. Guinea pig heart PK was Ca2+-dependent requiring 0.6 microM Ca2+ for half-maximal activity. Kinetic studies indicate that the Km of guinea pig heart PK for phosphorylase b at pH 6.8 is not markedly reduced after in vitro activation (35%). This could explain the observed lack of increase in the pH 6.8:8.2 activity ratio after exposure of hearts to isoproterenol. The time course for the activation of inotropic state and the glycogenolytic pathway in perfused guinea pig hearts by isoproterenol showed that these processes were maximally activated within 25 s. However, PK remained activated for 2 min, long after the other biochemical and physiological parameters had returned to control values. These data suggest that Ca2+, not phosphorylation state, is important in regulating the return of dP/dt to control levels after beta-adrenergic stimulation.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009200:Myocardial Contraction; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion; D010764:Phosphorylase Kinase / Q000378:metabolism*; D011494:Protein Kinases / Q000494:pharmacology*; D051381:Rats
D000242:Cyclic AMP; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D007545:Isoproterenol; D002118:Calcium