Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb.
Endothelin-1 (ET-1) acutely inhibits NaCl reabsorption by the thick ascending limb (THAL) by activating the ET(B) receptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-1 stimulates eNOS expression via the ET(B) receptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to ET(B) receptors 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 ET(B) receptor 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 ET(A) receptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c (S6c; 0.1 microM), a selective ET(B) receptor agonist, increased eNOS expression by 77 +/- 30% (P < 0.05; n = 6). Wortmannin (0.01 microM), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 microM 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 ET(B) receptors and PI3K and thereby increasing NO production.
Herrera
Marcela
M
Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI 48202-2689, USA.
Garvin
Jeffrey L
JL
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000008:administration & dosage / Q000494:pharmacology / Q000502:physiology*; D008138:Loop of Henle / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009994:Osmolar Concentration; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044023:Receptor, Endothelin B / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D019332:Endothelin-1; D044023:Receptor, Endothelin B; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019869:Phosphatidylinositol 3-Kinases