EndothelinB receptor activates NHE-3 by a Ca2+ -dependent pathway in OKP cells

Abstract

To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ETA and ETB receptor cDNA. In cells overexpressing ETB, but not ETA receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ETB receptor blocker but was not inhibited by an ETA selective receptor blocker. In ETB-overexpressing cells, 10-8 M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in JNa/H were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ETA-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ETB receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Cacalmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation.