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 ne onatal rat cardiac fibroblasts. However, the signaling events that lea d to fibroblast cell growth in response to AII remain to be elucidated . Mitogen-activated protein (MAP) kinases are cytosolic serine/threoni ne kinases which have been shown to be activated in quiescent cells by diverse growth stimuli, thereby being linked to growth regulatory pat hways. This study was designed to determine whether MAP-kinase activat ion occurred in response to AII/receptor coupling in neonatal rat card iac fibroblasts and the role of MAP-kinase activation in the AII-induc ed proliferation of these cells. Immunoblot analysis of MAP-kinase iso forms revealed predominantly p44 with less p42 MAP-kinase in rat cardi ac fibroblasts. Both isoforms were activated upon stimulation of the c ells with AII for 5 min or platelet derived growth factor-BE for 10 mi n. Angiotensin II stimulated MAP-kinase in a dose-dependent fashion wi th 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 pro tein, P<0.05, n=4, was observed in response to AII between 120-150 min utes following receptor occupancy, The responses to AII were markedly attenuated by the AT(1) receptor antagonist EXP3174. Stimulation of th e cells with carbachol induced the first but not the second phase of M AP-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 A II was necessary to induce DNA synthesis and fibroblast proliferation. These results indicate that AII stimulates a biphasic activation of M AP-kinase by the AT(1) receptor and that this pathway may participate in the AII induced mitogenic response in cardiac fibroblasts.