Effects of S-nitroso-cysteine on proteins that regulate exocytosis in PC12 cells: inhibitory effects on translocation of synaptophysin and ADP-ribosylation of GTP-binding proteins.
S-Nitroso-cysteine (SNC) inhibits Ca2+-induced noradrenaline (NA) release from PC12 cells. Since SNC stimulated Ca2+ mobilization from intracellular Ca2+ pools and SNC-induced inhibition of NA release was not washed-out, SNC may modify exocytosis-related proteins that overcome Ca2+ mobilization. In the present study, we investigated the effects of SNC on exocytosis-related proteins in PC12 cells. Ionomycin stimulated NA release and increased the immunoreactivity of synaptophysin in the cytosol fraction. A 25-kDa synaptosome-associated protein (SNAP-25), which localizes to plasma membranes and vesicles, increased in the cytosol fraction after stimulation. The increases in these proteins by ionomycin were inhibited in PC12 cells treated with 0.6 mM SNC. Synaptobrevin and synapsin-1 in the cytosol fraction, and syntaxin and 43 kDa growth-associated protein in the membrane fraction were not affected by ionomycin or SNC. Incubation of each protein with SNC did not affect antibody immunoreactivity. [32P]ADP-ribosylation of GTP-binding proteins (Gi/Go) by pertussis toxin, but not Gs by cholera toxin, was inhibited in SNC-treated PC12 cells and by co-addition of SNC to the assay mixture. These findings suggest that 1) SNC inhibits translocation of vesicles containing synaptophysin and SNAP-25, and 2) SNC reacts with cysteine residues in Gi/Go, causing inhibition of ADP-ribosylation by pertussis toxin.
Naganuma
T
T
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Maekawa
M
M
Murayama
T
T
Nomura
Y
Y
D020727:ADP-Ribosylation Factors / Q000187:drug effects / Q000502:physiology; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000502:physiology; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000502:physiology; D009603:Nitroso Compounds / Q000494:pharmacology*; D016716:PC12 Cells; D050683:R-SNARE Proteins; D051381:Rats; D026403:S-Nitrosothiols; D016704:Synapsins / Q000187:drug effects; D016708:Synaptophysin / Q000187:drug effects* / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25; D014178:Translocation, Genetic / Q000187:drug effects* / Q000502:physiology
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009603:Nitroso Compounds; D050683:R-SNARE Proteins; D026403:S-Nitrosothiols; C494210:Snap25 protein, rat; D016704:Synapsins; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25; D009569:Nitric Oxide; D015759:Ionomycin; C021315:S-nitrosocysteine; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D003545:Cysteine