Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb

Abstract

Endothelin-1 (ET-1) acutely inhibits NaCl reabsorption by the thick ascending limb (THAL) by activating the ETBreceptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-1 stimulates eNOS expression via the ETBreceptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to ETBreceptors and stimulating PI3K. In primary cultures of medullary THALs treated for 24 h, eNOS expression increased by 36 ± 18% with 0.01 nM ET-1, 123 ± 30% with 0.1 nM ( P < 0.05; n = 5), and 71 ± 30% with 1 nM, whereas 10 nM had no effect. BQ-788, a selective ETBreceptor antagonist, completely blocked stimulation of eNOS expression caused by 0.1 nM ET-1 (12 ± 25 vs. 120 ± 40% for ET-1 alone; P < 0.05; n = 5). BQ-123, a selective ETAreceptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c (S6c; 0.1 μM), a selective ETBreceptor agonist, increased eNOS expression by 77 ± 30% ( P < 0.05; n = 6). Wortmannin (0.01 μM), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 μM S6c (77 ± 30 vs. −28 ± 9%; P < 0.05; n = 6). To test whether the increase in eNOS expression heightens activity, we measured NO release in response to simultaneous treatment with l-arginine, ionomycin, and clonidine using a NO-sensitive electrode. NO release by control cells was 337 ± 61 and 690 ± 126 pA in ET-1-treated cells ( P < 0.05; n = 5). Taken together, these data suggest that ET-1 stimulates THAL eNOS, activating ETBreceptors and PI3K and thereby increasing NO production.