SEQUENTIAL ACTIVATION OF MAP KINASE CASCADE BY ANGIOTENSIN-II IN OPOSSUM KIDNEY-CELLS

Angiotensin II (Ang II) is a potent regulator of proximal tubule funct ions, including transport, metabolism, and cell proliferation. The opo ssum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated an d activated in response to various agonists. We investigated Ang II ef fects on serine/threonine kinase cascades in OK cells. The major findi ngs of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it incr eased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(- 11) M). The time course of activation was sequential; the peak stimula tion was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistei n and herbimycin) did not inhibit Ang II-induced MAPK activity. This a ctivation of MAPK was also inhibited via AT(I) receptor antagonist, Du p753 and pertussis toxin. This evidence suggests that the activation o f serine/threonine cascades by Ang II is largely dependent, on PMA-sen sitive PKC, and is not dependent on tyrosine kinase and pertusis toxin .