ACELL Apr. 45/4

Peng, Y., O. W. Moe, T.-S. Chu, P. A. Preisig, M. Yanagisawa, and R. J. Alpern. ETB receptor activation leads to activation and phosphorylation of NHE3. Am. J. Physiol. 276 (Cell Physiol. 45): C938–C945, 1999.—In OKP cells expressing ETB endothelin receptors, activation of Na1/H1 antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na1/H1 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 receptorspecific 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 1029 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.