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

Life Sciences
Publication Date
DOI: 10.1016/j.lfs.2012.01.014
  • Collecting Duct
  • Nitric Oxide
  • Nitric Oxide Synthase
  • Endothelin
  • Erk1/2
  • Phosphorylation
  • Biology


Abstract 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 50nM ET-1 for 24h 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±26pmol/mgpr/h vs ET-1: 221±28pmol/mg pr/h; N=4) via the ETB receptor (BQ788+ET-1: 83.7±27pmol/mgpr/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±13pmol/mgpr/hr vs PD98,059+ET-1: 305.7±24pmol/mgpr/h, N=4, P>0.05). Significance Although the mouse IMCD-3 cells only express the NOS1β 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.

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