title: Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase.
abstract: N-dansylhomocysteine (DnsHCys) is quenched on S-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K(M) of 2 microM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K(I) of 1 microM. The oxidized form of DnsHCys, N,N-didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilical endothelial cells and hamster lung fibroblasts were monitored as a function of extracellular BSA-NO concentration via dynamic fluorescence microscopy. The observed quenching of the DnsHCys fluorescence was an indirect measure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S-nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosarcoma cells correlated with the rate of quenching and the PDI inhibitors, 5,5'-dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phenylarsenoxide inhibited quenching. The apparent K(M) values for denitrosation of BSA-NO by csPDI ranged from 12 microM to 30 microM. Depletion of membrane N(2)O(3) with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence by approximately 70%. The K(M) for BSA-NO increased by approximately 3-fold and V(max) decreased by approximately 4-fold. These findings suggest that csPDI catalyzed NO released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O2 to produce N(2)O(3). Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Ramachandran
authors.forename: P
authors.initials: P
authors.lastname: Root
authors.forename: X M
authors.initials: XM
authors.lastname: Jiang
authors.forename: P J
authors.initials: PJ
authors.lastname: Hogg
authors.forename: B
authors.initials: B
authors.lastname: Mutus
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001152:Arsenicals / Q000494:pharmacology; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D003619:Dansyl Compounds / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005354:Fibrosarcoma / Q000473:pathology; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006710:Homocysteine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000166:cytology; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021381:Protein Transport; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014810:Vitamin E / Q000494:pharmacology
chemical_list: C426645:4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide; D000975:Antioxidants; D001152:Arsenicals; D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018832:Molecular Chaperones; C434729:N-dansylhomocysteine; D006710:Homocysteine; D014810:Vitamin E; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D004228:Dithionitrobenzoic Acid; D019704:Protein Disulfide-Isomerases; D005978:Glutathione