ETB receptor activation leads to activation and phosphorylation of NHE3

In OKP cells expressing ETB endothelin receptors, activation of Na+/H+ antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+ exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETB receptors but not in cells expressing ETA receptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15-30 min. Phosphorylation was maximal with 10(-9) M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETB receptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.