ANGIOTENSIN-II IS A POTENT STIMULATOR OF MAP-KINASE ACTIVITY IN NEONATAL RAT CARDIAC FIBROBLASTS

We have previously shown that angiotensin II (AII) is a mitogen for neonatal rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to AII remain to be elucidated. Mitogen-activated protein (MAP) kinases are cytosolic serine/threonine kinases which have been shown to be activated in quiescent cells by diverse growth stimuli, thereby being linked to growth regulatory pathways. This study was designed to determine whether MAP-kinase activation occurred in response to AII/receptor coupling in neonatal rat cardiac fibroblasts and the role of MAP-kinase activation in the AII-induced proliferation of these cells. Immunoblot analysis of MAP-kinase isoforms revealed predominantly p44 with less p42 MAP-kinase in rat cardiac fibroblasts. Both isoforms were activated upon stimulation of the cells with AII for 5 min or platelet derived growth factor-BE for 10 min. Angiotensin II stimulated MAP-kinase in a dose-dependent fashion with an EC(50) of 2.5 nM. Two minutes following stimulation with 1 mu M AII MAP-kinase activity increased from 90+/-17.9 to 477.5+/-75.9 pmol/min/mg protein, P<0.05, n=4. A smaller, sustained, secondary increase in MAP-kinase activity from 37.7+/-5.3 to 110.9+/-15.3 pmol/min/mg protein, P<0.05, n=4, was observed in response to AII between 120-150 minutes following receptor occupancy, The responses to AII were markedly attenuated by the AT(1) receptor antagonist EXP3174. Stimulation of the cells with carbachol induced the first but not the second phase of MAP-kinase activity and this compound had no effect on cellular growth. The second phase of MAP-kinase activity 2-2.5 h after AII stimulation, paralleled data demonstrating that a 2-3 h receptor occupancy with AII was necessary to induce DNA synthesis and fibroblast proliferation. These results indicate that AII stimulates a biphasic activation of MAP-kinase by the AT(1) receptor and that this pathway may participate in the AII induced mitogenic response in cardiac fibroblasts.