Evidence for a role of protein kinase C substrate B-50 (GAP-43) in Ca(2+)-induced neuropeptide cholecystokinin-8 release from permeated synaptosomes.
To study the involvement of the protein kinase C (PKC) substrate B-50 [also known as growth-associated protein-43 (GAP-43), neuromodulin, and F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca2+ in a concentration-dependent manner (EC50 of approximately 10(-5) M). Ca(2+)-induced CCK-8 release was maximal at 10(-4) M Ca2+, amounting to approximately 10% of the initial 6,000 +/- 550 fmol of CCK-8 content/mg of synaptosomal protein. Only 30% of the Ca(2+)-induced CCK-8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti-B-50 antibodies were introduced into permeated synaptosomes to study their effect on Ca(2+)-induced CCK-8 release. The N-terminally directed antibodies (NM2), which inhibited PKC-mediated B-50 phosphorylation, inhibited Ca(2+)-induced CCK-8 release in a dose-dependent manner, whereas the C-terminally directed antibodies (NM6) affected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibitors PKC19-36 and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), which inhibited B-50 phosphorylation in permeated synaptosomes, had no effect on Ca(2+)-induced CCK-8 release. Our data strongly indicate that B-50 is involved in the mechanism of presynaptic CCK-8 release, at a step downstream of the Ca2+ trigger. As CCK-8 is stored in large dense-cored vesicles, we conclude that B-50 is an essential factor in the exocytosis from this type of neuropeptide-containing vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)
Hens
J J
JJ
Division of Molecular Neurobiology, Rudolf Magnus Institute, Utrecht, The Netherlands.
Ghijsen
W E
WE
Dimjati
W
W
Wiegant
V M
VM
Oestreicher
A B
AB
Gispen
W H
WH
De Graan
P N
PN
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000648:ultrastructure; D019922:GAP-43 Protein; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012844:Sincalide / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D012844:Sincalide; D011188:Potassium; D002118:Calcium