title: Extracellular signal-regulated kinases 1/2 signaling pathways are not involved in endothelin regulation of mouse inner medullary collecting duct nitric oxide production.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Confluent mouse IMCD segment-3 cells (mIMCD-3) were stimulated with 50 nM ET-1 for24 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.
  
  
  RESULTS
  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/hvs 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Experimental Medicine, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, United States.
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Hyndman
authors.forename: Alexander H
authors.initials: AH
authors.lastname: MacDonell
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Pollock
mesh_terms: D000818:Animals; D019332:Endothelin-1 / Q000502:physiology*; D015151:Immunoblotting; D007679:Kidney Medulla / Q000201:enzymology* / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000201:enzymology* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010456:Peptides, Cyclic / Q000008:administration & dosage; D010766:Phosphorylation; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D019332:Endothelin-1; D010456:Peptides, Cyclic; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496315:Nos3 protein, mouse; C072247:cyclo(Trp-Asp-Pro-Val-Leu)