Jjh. Hens et al., EVIDENCE FOR A ROLE OF PROTEIN-KINASE-C SUBSTRATE B-50 (GAP-43) IN CA-2-INDUCED NEUROPEPTIDE CHOLECYSTOKININ-8 RELEASE FROM PERMEATED SYNAPTOSOMES(), Journal of neurochemistry, 61(2), 1993, pp. 602-609
To study the involvement of the protein kinase C (PKC) substrate B-50
[also known as growth-associated protein-43 (GAP-43), neuromodulin, an
d F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purifie
d synaptosomes from rat cerebral cortex were permeated with the bacter
ial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptos
omes, determined quantitatively by radioimmunoassay, could be induced
by Ca2+ in a concentration-dependent manner (EC50 of approximately 10(
-5) M). Ca2+-induced CCK-8 release was maximal at 10(-4) M Ca2+, amoun
ting to approximately 10% of the initial 6,000 +/- 550 fmol of CCK-8 c
ontent/mg of synaptosomal protein. Only 30% of the Ca2+-induced CCK-8
release was dependent on the presence of exogenously added ATP. Two di
fferent monoclonal anti-B-50 antibodies were introduced into permeated
synaptosomes to study their effect on Ca2+-induced CCK-8 release. The
N-terminally directed antibodies (NM2), which inhibited PKC-mediated
B-50 phosphorylation, inhibited Ca2+-induced CCK-8 release in a dose-d
ependent manner, whereas the C-terminally directed antibodies (NM6) af
fected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibit
ors PKC19-36 and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7),
which inhibited B-50 phosphorylation in permeated synaptosomes, had n
o effect on Ca2+-induced CCK-8 release. Our data strongly indicate tha
t 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 dens
e-cored vesicles, we conclude that B-50 is an essential factor in the
exocytosis from this type of neuropeptide-containing vesicle. The diff
erential effects of the monoclonal antibodies indicate that this B-50
property is localized in the N-terminal region of the B-50 molecule, w
hich contains the PKC phosphorylation site and calmodulin-binding doma
in.