DOMINANT-NEGATIVE C-SRC INHIBITS ANGIOTENSIN-II-INDUCED ACTIVATION OFNHE3 IN OKP CELLS

Background. Angiotensin II is a potent stimulator of the proximal tubu le apical membrane Na/H antiporter, encoded by NHE3. The nonreceptor t yrosine kinase, c-Src, plays a key role in regulation of NHE3 by acido sis in the proximal tubule, and in signaling effects of angiotensin II in vascular smooth muscle. Methods. The present studies examined the role of c-Src in mediating angiotensin II-induced NHE3 activation in c ultured OKP cells. c-Src was inhibited with herbimycin A, a tyrosine k inase inhibitor, and expression of a dominant negative c-Src, CSrc(K29 5M). Results. Herbimycin A blocked angiotensin II induced increases in Na/H antiporter activity. In two clonal cell lines expressing vector alone, angiotensin II increased Na/H antiporter activity, while in thr ee clones expressing C-SrCK295M, angiotensin II had no effect. Cyclic AMP and protein kinase A have been proposed to be key mediators in reg ulation of NHE3 by angiotensin II. 10(-4) M 8-bromo cAMP induced a 40 to 50% inhibition of Na/H antiporter activity in cells expressing C-Sr c(K295M), Similar to that seen in wild-type OKP cells. In addition, ce lls expressing C-SrCK295M responded normally to 10(-7) M dexamethasone with a 50 to 80% increase in Na/H antiporter activity. Conclusions. T hese studies demonstrate that c-Src is required for angiotensin II-ind uced increases in NHE3 activity. Thus, c-Src plays a key role in antip orter activation by acidosis and angiotensin II.