Tomoyoshi Naganuma1, Mutsuko Maekawa1, Toshihiko
Murayama1,2,* and Yasuyuki Nomura1
1Department of Pharmacology, Graduate School of Pharmaceutical
Sciences, Hokkaido University, Sapporo 060-0812, Japan
2Laboratory of Chemical Pharmacology, Faculty of Pharmaceutical
Sciences, Chiba University, Chiba 263-8522, Japan
*Corresponding author (affiliationﾊ#2).ﾊﾊFAX:+81-43-290-3021
E-mail: murayama@p.chiba-u.ac.jp
Abstract: 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ﾊmM SNC. Synaptobrevin and
synapsin-1 in the cytosol fraction, and syntaxin and 43ﾊ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.
Keywords: S-Nitroso-cysteine, Exocytosis-related protein, Synaptophysin,
GTP-binding protein, PC12 cell
Copyrightｩ The Japanese Pharmacological Society 2000
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