title: Effect of nitric oxide and peroxynitrite on type I collagen synthesis in normal and scleroderma dermal fibroblasts.
abstract: Nitric oxide ((.-)NO) is an important physiological signaling molecule and potent vasodilator. Recently, we have shown abnormal (.-)NO metabolism in the plasma of patients with systemic sclerosis (SSc), a disease that features excessive collagen overproduction as well as vascular dysfunction. The current study investigates the effects of (.-)NO and peroxynitrite (ONOO(-)) on secretion of type I collagen by SSc dermal fibroblasts, compared with those from normal dermal fibroblasts (CON) and a dermal fibroblast cell line (AG). Dermal fibroblasts were incubated with (.-)NO donors (SNP, DETA-NONOate) with or without the antioxidant ascorbic acid, or ONOO(-) for 24-72 h. In CON and AG fibroblasts, type I collagen was dose dependently decreased by SNP or DETA-NONOate. However, (.-)NO had no effect in SSc fibroblasts. Furthermore, the inhibition of collagen synthesis by (.-)NO was reversed by ascorbic acid and was not affected by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanyl cyclase, or 8-bromoguanosine cyclic 3',5'-monophosphate, a cGMP agonist. SNP also showed a significant up-regulation of matrix metalloproteinase-1 (MMP-1) protein and activity levels, an essential collagenase involved in collagen degradation, in the AG fibroblasts. Additionally, (.-)NO-treated fibroblasts had lower prolyl hydroxylase activity, an enzyme important in the post-translational processing of collagen, while there was no effect on total protein levels. There were no significant effects on type I collagen levels when dermal fibroblasts were treated with ONOO(-). Taken together, ()NO inhibits collagen secretion in normal dermal fibroblasts but regulation is lost in SSc fibroblasts, while ONOO(-) itself is ineffective. (.-)NO inhibition of collagen was by cGMP-independent regulatory mechanisms and in part may be due to up-regulation of MMP-1 and/or inhibition of prolyl hydroxylase. These differences may contribute to the observed pathology of SSc.
authors.affiliation: Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London NW3 2PF, UK.
authors.forename: Audrey
authors.initials: A
authors.lastname: Dooley
authors.forename: Beirong
authors.initials: B
authors.lastname: Gao
authors.forename: Xu
authors.initials: X
authors.lastname: Shi-Wen
authors.forename: David J
authors.initials: DJ
authors.lastname: Abraham
authors.forename: Carol M
authors.initials: CM
authors.lastname: Black
authors.forename: Michael
authors.initials: M
authors.lastname: Jacobs
authors.forename: K Richard
authors.initials: KR
authors.lastname: Bruckdorfer
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D024042:Collagen Type I / Q000096:biosynthesis* / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011190:Potassium Cyanide / Q000494:pharmacology; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology; D012867:Skin / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology
chemical_list: D024042:Collagen Type I; D030421:Peroxynitrous Acid; D009599:Nitroprusside; D009569:Nitric Oxide; D011190:Potassium Cyanide; D001205:Ascorbic Acid