title: Insulin-stimulated endothelial nitric oxide release is calcium independent and mediated via protein kinase B.
abstract: Insulin exerts a vasodilator effect by stimulating endothelial nitric oxide (NO) production. Studies in cultured cells suggest that insulin might activate endothelial nitric oxide synthase (eNOS) by an atypical, calcium-independent mechanism. This study investigates the mechanism of insulin-stimulated endothelial NO production in intact aortic wall. Real time fluorescence imaging with 4,5-diaminofluorescin diacetate (DAF-2 DA) or 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and FURA 2-AM was used to simultaneously visualise NO and intracellular calcium concentrations at multiple locations in the endothelium and vascular smooth muscle of isolated rat and mouse aorta after exposure to insulin. Inhibitors of intracellular insulin signalling were used to determine the pathway for insulin-stimulated NO production. Unlike acetylcholine, which stimulated endothelial NO production with a typical increase in free intracellular calcium, insulin (10(-8) to 10(-6)M) stimulated endothelial NO production without elevating intracellular calcium levels. Insulin-stimulated NO production was concentration dependent and detected within 30s of application. Peak increases in NO occurred between 60 and 120 s and declined slowly thereafter. Separate measurements of NO production by fluorescence of 2,3-diaminonaphthalene (DAN) noted that selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) inhibited insulin-stimulated NO production, whereas these inhibitors alone did not alter NO production or acetylcholine-stimulated NO production. Insulin-stimulated NO production by endothelium is an acute calcium-independent effect mediated via the PI3K-PKB signalling pathway.
authors.affiliation: Department of Pharmacology, The School of Pharmacy, University of London, WC1N 1AX, UK. nicholas.hartell@ulsop.ac.uk
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Hartell
authors.forename: Helen E
authors.initials: HE
authors.lastname: Archer
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Bailey
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction
chemical_list: D002118:Calcium; D007328:Insulin; D009569:Nitric Oxide; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt