Evidence for a relationship between B-50 (GAP-43) and [3H]noradrenaline release in rat brain synaptosomes.
Phosphorylation of the neuron-specific substrate of protein kinase C (PKC), B-50 (GAP-43), was studied parallel with noradrenaline release in rat brain synaptosomes. Both could be evoked by treating the synaptosomes with high K+ or veratridine. Phorbol 12,13-dibutyrate enhanced depolarization-induced B-50 phosphorylation and noradrenaline release. To investigate the involvement of PKC-mediated B-50 phosphorylation in noradrenaline release, we applied a variety of kinase inhibitors. Prior to measuring the effects of these inhibitors in intact synaptosomes, we determined their effectivity and specificity in a membrane phosphorylation assay. H-7 most specifically inhibited PKC-dependent phosphorylation, whereas calmidazolium inhibited calmodulin-dependent phosphorylation. Polymyxin B affected both protein kinase systems. Only polymyxin B effectively inhibited noradrenaline release in the intact synaptosomes. We conclude that PKC as well as calmodulin-dependent processes are important for the release event. Data are discussed in view of the presumed function of B-50 as a calmodulin-binding protein.
Dekker
L V
LV
Division of Molecular Neurobiology, Institute of Molecular Biology and Medical Biotechnology, Utrecht, The Netherlands.
De Graan
P N
PN
Spierenburg
H
H
De Wit
M
M
Versteeg
D H
DH
Gispen
W H
WH
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D019922:GAP-43 Protein; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010879:Piperazines / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
D002147:Calmodulin; D019922:GAP-43 Protein; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010761:Phosphorus Radioisotopes; D010879:Piperazines; D015240:Phorbol 12,13-Dibutyrate; D014701:Veratridine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D011188:Potassium; D009638:Norepinephrine