title: Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha.
abstract: Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: S
authors.initials: S
authors.lastname: Lamas
authors.forename: T
authors.initials: T
authors.lastname: Michel
authors.forename: B M
authors.initials: BM
authors.lastname: Brenner
authors.forename: P A
authors.initials: PA
authors.lastname: Marsden
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D002956:Citrulline / Q000096:biosynthesis; D006152:Cyclic GMP / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D012077:Renal Artery / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D006152:Cyclic GMP