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

To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET_A and ET_B receptor cDNA. In cells overexpressing ET_B, but not ET_A receptors, ET-1 increased Na/H antiporter activity (J_Na/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET_B receptor blocker but was not inhibited by an ET_A selective receptor blocker. In ET_B-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 J_Na/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 ET_A-overexpressing cells, ET-1 increased cell [Ca²⁺] but did not increase J_Na/H. In summary, binding of ET-1 to ET_B 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.