Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

N-dansylhomocysteine (DnsHCys) is quenched on 5-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluo rogenic substrate for the denitrosation activity of protein disulfide isome rase (PDI) with an apparent K-M of 2 muM. S-nitroso-BSA (BSA-NO) competitiv ely inhibited this reaction with an apparent K-I of 1 muM. The oxidized for m of DnsHCys, N,N ' -didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilic al endothelial cells and hamster lung fibroblasts were monitored as a funct ion of extracellular BSA-NO concentration via dynamic fluorescence microsco py. The observed quenching of the DnsHCys fluorescence was an indirect meas ure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S- nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosa rcoma cells correlated with the rate of quenching and the PDI inhibitors, 5 ,5 ' -dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phen ylarsenoxide inhibited quenching. The apparent K-M values for denitrosation of BSA-NO by csPDI ranged from 12 muM to 30 muM. Depletion of membrane N2O 3 with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quen ching rates of DnsHCys fluorescence by approximate to 70%. The K-M for BSA- NO increased by approximate to3-fold and V-max. decreased by approximate to 4-fold. These findings suggest that csPDI catalyzed No released from extrac ellular S-nitrosothiols accumulates in the membrane where it reacts with O- 2 to produce N2O3. Intracellular thiols may then be nitrosated by N2O3 at t he membrane-cytosol interface.