Endothelin_A (ET_A) and ET_B receptor-mediated regulation of nitric oxide synthase 1 (NOS1) and NOS3 isoforms in the renal inner medulla

Aim: Our laboratory and others have shown that endothelin (ET)-1 directly stimulates nitric oxide (NO) production in inner medullary collecting duct (IMCD) cells. The goal of this study was to determine which NO synthase (NOS) isoforms in IMCD are sensitive to ET-1, and the role of ET_A and ET_B receptor activation in vivo and in vitro. Methods: NOS enzymatic activity and NOS isoform protein expression were examined in cultured IMCD-3 cells and isolated renal inner medulla. ET_B receptor-deficient homozygous rats (sl/sl) have elevated levels of circulating ET-1 and lack a functional ET_B signalling pathway in kidneys, and furthermore provides a unique model to study ET_A receptor signalling in the renal inner medulla in vivo. Results: Incubation of IMCD-3 cells with exogenous ET-1 (50 nm) resulted in ET_A-dependent increased NOS1 protein expression in IMCD-3 cells with no effect on NOS2 or NOS3 expression. ET_B receptor antagonism has no effect on NOS expression in IMCD-3 cells. Consistent with in vitro results, cytosolic NOS1 protein expression was significantly greater in the renal inner medulla of sl/sl rats compared with heterozygous (sl/+) controls, with no alteration in NOS3 expression. In contrast to protein expression data, NOS1- and NOS3-specific enzymatic activities decreased in the cytosolic fraction from the renal inner medulla of sl/sl compared with sl/+. Conclusion: These results provide evidence that both ET_A and ET _B receptors regulate NOS isoform activity in the renal inner medulla and specifically support the hypothesis that ET_A receptor activation increases NOS1 expression.