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 fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K-M of 2 muM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K-I of 1 muM. 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 muM to 30 muM. Depletion of membrane N2O3 with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching 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 to4-fold. These findings suggest that csPDI catalyzed No released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O-2 to produce N2O3. Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.