Regulation of Ras-GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester - Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol la-myristate 13-acetate (PMA), endothelin-l (ET-1), and phenylephrine (PE). All three increased Ras GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin, 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras GTP loading by PMA, ET-1, or PE, The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and G(i)-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.