Role of tyrosine kinase pathways in ET(B) receptor activation of NHE3

Endothelin-1 (ET-1) binding to ET(B) receptors increases the activity of the apical membrane Na⁺/H⁺ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPET(B)6 cells, a clonal cell line of OKP cells that overexpresses ET(B) 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 ET(B) 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 p125(FAK) (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125(FAK), 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 ET(B) receptor- induced Na⁺/H⁺ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ET(B) receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.