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 12-myristate 13-acetate (PMA), endothelin-1 (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 Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.
National Heart and Lung Institute Division (Cardiac Medicine), Imperial College School of Medicine, London SW3 6LY, United Kingdom.
A
A
Chiloeches
H F
HF
Paterson
R
R
Marais
A
A
Clerk
C J
CJ
Marshall
P H
PH
Sugden
D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D019332:Endothelin-1 / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000819:agonists*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D037342:Pertussis Toxin; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism*
D019332:Endothelin-1; D010566:Virulence Factors, Bordetella; D010656:Phenylephrine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate