Dominant negative c-Src inhibits angiotensin II induced activation of NHE3 in OKP cells.

Abstract

Angiotensin II is a potent stimulator of the proximal tubule apical membrane Na/H antiporter, encoded by NHE3. The nonreceptor tyrosine kinase, c-Src, plays a key role in regulation of NHE3 by acidosis in the proximal tubule, and in signaling effects of angiotensin II in vascular smooth muscle. The present studies examined the role of c-Src in mediating angiotensin II-induced NHE3 activation in cultured OKP cells. c-Src was inhibited with herbimycin A, a tyrosine kinase inhibitor, and expression of a dominant negative c-Src, c-SrcK295M. 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 three clones expressing c-SrcK295M, angiotensin II had no effect. Cyclic AMP and protein kinase A have been proposed to be key mediators in regulation 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-SrcK295M, similar to that seen in wild-type OKP cells. In addition, cells expressing c-SrcK295M responded normally to 10(-7) M dexamethasone with a 50 to 80% increase in Na/H antiporter activity. These studies demonstrate that c-Src is required for angiotensin II-induced increases in NHE3 activity. Thus, c-Src plays a key role in antiporter activation by acidosis and angiotensin II.