Extracellular signal-regulated kinases1/2 signaling pathways are not involved in endothelin regulation of mouse inner medullary collecting duct nitric oxide production

[摘要] Aims: To determine if endothelin-1 (ET-1) stimulates the phosphorylation of ERK1/2 in the mouse inner medullary collecting duct (IMCD), and if this in turn upregulates nitric oxide (NO) production. Main methods: Confluent mouse IMCD segment-3 cells (mIMCD-3) were stimulated with 50 nM ET-1 for 24 h with and without various doses of ET receptor antagonists, BQ123 (ETA antagonist,) or BQ788 (ETB antagonist) and phosphorylation of ERK1/2 determined by immunoblots. As well, NOS isoform expression and nitrite production were assessed. Finally, increasing doses of the MEK inhibitors, PD98,059 or U0126, were incubated with mIMCD-3 cells and the ET-1 dependent nitrite production determined. Key findings: ET-1 via the ETB receptor significantly increased ERK1/2 phosphorylation, and was prevented by MEK inhibition. ET-1 also stimulates nitrite production by mIMCD-3 cells (basal: 54.5 +/- 26 pmol/mg pr/h vs ET-1: 221 +/- 28 pmol/mg pr/h; N = 4) via the ETB receptor (BQ788 + ET-1: 83.7 +/- 27 pmol/mg pr/h); however, ET-1 does not regulate NOS1 or NOS3 expression. MEK inhibition did not prevent the ET-1 stimulated nitrite production contrary to our initial hypothesis (vehicle + ET-1: 157 +/- 13 pmol/mg pr/hr vs PD98,059 + ET-1:305.7 +/- 24 pmol/mg pr/h, N = 4, P > 0.05). Significance: Although the mouse IMCD-3 cells only express the NOS1 beta splice variant, ET-1 did regulate mouse IMCD nitrite production. ET-1 stimulates ERK1/2 phosphorylation in the mouse IMCD, but ERK1/2 signaling is not involved in the ET-1 dependent increase in NO production by IMCD cells. Thus, we propose that ET-1 regulates protein-protein interactions that are necessary for NO production, that are independent of MAPK signaling cascades. Published by Elsevier Inc.

[发布日期] 2012-10-15

[效力级别]  Proceedings Paper

[关键词] Collecting duct;Nitric oxide;Nitric oxide synthase;Endothelin;ERK1/2;Phosphorylation