Adrenergic control of the cyclic AMP-dependent protein kinase and pyruvate kinase in isolated hepatocytes. Application of a synthetic peptide substrate for measuring protein kinase activity.

The cyclic AMP-dependent protein kinase (isoenzymes I and II) was measured using a synthetic peptide analog of the porcine hepatic phosphorylation site sequence (Leu-Arg-Arg-Ala-Ser-&eu-Gly). Epinephrine caused a timeand dose-dependent activation of the hepatic cyclic AMP-dependent protein kinase and inactivation of pyruvate kinase. Maximal activation of the protein kinase occurred within 1 min with an increase in the protein kinase activity ratio from 0.06 f 0.01 to 0.18 f 0.02. Both the total cyclic AMP-dependent protein kinase activity and the component stimulated by epinephrine were inhibited more than 90% by the heat-stable inhibitor protein of the protein kinase. ‘l’he apparent epinephrine-stimulated protein kinase activity ratio was stabilized by the presence of salt in the extraction buffer, suggesting that this hormone preferentially activates the cyclic AMP-dependent protein kinase isozyme II in isolated hepatocytes. Application of selected aand p-agonists and antagonists failed to reveal a strong correlation between epinephrine-mediated changes in protein kinase activity ratio, pyruvate kinase activity, and gluconeogenesis from lactate. Activation of the protein kinase by 10 pM epinephrine was partially blocked by the /3-antagonist, propranolol, (10 pM) and the a-antagonists, phenoxybenzamine (10 j&M) and phentolamine (10 PM). The inactivation of pyruvate kinase and stimulation of gluconeogenesis were preferentially blocked by the a-antagonists. Compared with epinephrine, the p-agonist, isoproterenol (0.1 mu), had a weaker effect on the activation of the protein kinase, inhibition of pyruvate kinase activity, and stimulation of gluconeogenesis. Nevertheless, the isoproterenol effects were each specifically blocked by propranolol. The effects produced by the a-agonist, phenylephrine, were similarly affected by (rand b-antagonists as were those of epinephrine; the inactivation of pyruvate kinase and stimulation of gluconeogenesis were largely blocked by phenoxybenzamine. Other a-agonists, oxymetazoline, naphazoline, and tetrahydrozoline, were without effect. Overall, our data suggest that epinephrine-stimulated hepatocyte gluconeogenesis may be mediated by activation of the cyclic AMP-dependent protein kinase.