Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.
In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.
Shah
C M
CM
Department of Biomedical Science, University of Westminster, 115 New Cavendish Street, London, UK.
Bell
S E
SE
Locke
I C
IC
Chowdrey
H S
HS
Gordge
M P
MP
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009569:Nitric Oxide / Q000008:administration & dosage*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000378:metabolism*
D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D019704:Protein Disulfide-Isomerases; D006152:Cyclic GMP