Premium
Evidence for a Role of Protein Kinase C Substrate B‐50 (GAP‐43) in Ca 2+ ‐Induced Neuropeptide Cholecystokinin‐8 Release from Permeated Synaptosomes
Author(s) -
Hens Jacques J. H.,
Ghijsen Wim E. J. M.,
Dimjati Wati,
Wiegant Victor M.,
Oestreicher A. Beate,
Gispen Willem Hendrik,
Graan Pierre N. E.
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb02164.x
Subject(s) - protein kinase c , synaptosome , cholecystokinin , phosphorylation , chemistry , neuropeptide , gap 43 protein , streptolysin , protein kinase a , biochemistry , radioimmunoassay , medicine , biology , microbiology and biotechnology , endocrinology , receptor , in vitro , immunohistochemistry , bacterial protein , gene
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 Ca 2+ in a concentration‐dependent manner (EC 50 of ∼10 ‐5 M ). Ca 2+ ‐induced CCK‐8 release was maximal at 10 4 M Ca 2+ , amounting to ∼10% of the initial 6,000 ± 550 fmol of CCK‐8 content/mg of synaptosomal protein. Only 30% of the Ca a+ ‐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 PKC 19–36 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 Ca 2+ trigger. As CCK‐8 is stored in large densecored vesicles, we conclude that B‐50 is an essential factor in the exocytosis from this type of neuropeptide‐containing vesicle. The differential effects of the monoclonal antibodies indicate that this B‐50 property is localized in the N‐terminal region of the B‐50 molecule, which contains the PKC phosphorylation site and calmodulin‐binding domain.