Inhibition of noradrenaline release from PC12 cells by the long-term treatment with cholera toxin.
Guanine nucleotide-binding (G) proteins are required for intracellular vesicular transport and endocytosis. In this study, we investigated the effects of short-term (2 h) and long-term (24 h) treatment with cholera toxin (CTX), which ADP-ribosylates proteins having arginine residues such as the alpha subunit of Gs (G(s alpha)), on exocytosis from the neurosecretory rat pheochromocytoma PC 12 cell line. Short-term treatment with CTX stimulated the accumulation of cyclic AMP, and synergistically enhanced both extracellular Ca2+-dependent [3H]noradrenaline (NA) releases (induced by high K+ and ATP) and Ca2+-independent release (induced by mastoparan, a peptide in wasp venom). Long-term treatment with CTX for 24h inhibited Ca2+-dependent and -independent stimulated [3H]NA release. The inhibitory effect of long-term CTX treatment was not derived from a cyclic AMP-dependent system, because (1) H-89, an inhibitor of protein kinase A, had no effect on the inhibition induced by CTX, (2) the long-term treatment with forskolin did not show an inhibitory effect. [32P]ADP-ribosylation of G(s alpha) and its immunoreactivity with anti-G(s alpha) antiserum in the crude membrane fraction was inhibited in the long-term CTX-treated cells, but not in the long-term forskolin-treated cells. [32P]ADP-ribosylation of G(s alpha) in the membrane fraction of short-term CTX-treated cells was approximately 90% of the level in the control cells. These findings suggest that CTX stimulates [3H]NA release via a cyclic AMP-dependent system in the short-term, and that long-term CTX treatment inhibited its release, maybe via ADP-ribosylation of CTX-sensitive proteins such as G(s alpha).
Oda
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Naganuma
T
T
Murayama
T
T
Nomura
Y
Y
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D009638:Norepinephrine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D014316:Tritium
D014316:Tritium; D000246:Adenosine Diphosphate Ribose; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium; D009638:Norepinephrine