The kidney is important in the long-term regulation of blood pressure and sodium homeostasis. Stimulation of ETB receptors in the kidney increases sodium excretion, in part, by decreasing sodium transport in the medullary thick ascending limb of Henle and in collecting duct. However, the role of ETB receptor on Na(+)-K(+) ATPase activity in renal proximal tubule (RPT) cells is not well defined. The purpose of this study is to test the hypothesis that ETB receptor inhibits Na(+)-K(+) ATPase activity in rat RPT cells, and investigate the mechanism(s) by which such an action is produced. In RPT cells from Wistar-Kyoto rats, stimulation of ETB receptors by the ETB receptor agonist, BQ3020, decreased Na(+)-K(+) ATPase activity, determined by ATP hydrolysis (control=0.38+/-0.02, BQ3020=0.26+/-0.03, BQ788=0.40+/-0.06, BQ3020+BQ788=0.37+/-0.04, n=5, P<0.01). The ETB receptor-mediated inhibition of Na(+)-K(+) ATPase activity was dependent on an increase in intracellular calcium, because this effect was abrogated by a chelator of intracellular-free calcium (BAPTA-AM; 5 x 10(-3) M 15 min(-1)), Ca(2+) channel blocker (10(-6) M 15 min(-1) nicardipine) and PI3 kinase inhibitor (10(-7) M per wortmannin). An inositol 1,4,5-trisphosphate (IP3) receptor blocker (2-aminoethyl diphenyl borate; 10(-4) M 15 min(-1)) also blocked the inhibitory effect of the ETB receptor on Na(+)-K(+)ATPase activity (control=0.39+/-0.06, BQ3020=0.25+/-0.01, 2-APB=0.35+/-0.05, BQ3020+ 2-APB=0.35+/-0.06, n=4, P<0.01). The calcium channel agonist (BAY-K8644; 10(-6) M 15 min(-1)) inhibited Na(+)-K(+) ATPase activity, an effect that was blocked by a phosphatidylinositol-3 kinase inhibitor (10(-7) M 15 min(-1) wortmannin). In rat RPT cells, activation of the ETB receptor inhibits Na(+)-K(+) ATPase activity by facilitating extracellular Ca(2+) entry and Ca(2+) release from endoplasmic reticulum.