Role of tyrosine kinase pathways in ETB receptor activation of NHE3

Abstract

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.