Studies on the role of B-50 (GAP-43) in the mechanism of Ca(2+)-induced noradrenaline release: lack of involvement of protein kinase C after the Ca2+ trigger.
The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca(2+)-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (> 60%) ATP-dependent manner as a result of an elevation of the free Ca2+ concentration from 10(-8) to 10(-5) M Ca2+. The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca(2+)-induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca(2+)-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca(2+)-induced NA release. PKC pseudosubstrate PKC19-36, which inhibited B-50 phosphorylation (IC50 value, 10(-5) M), failed to inhibit Ca(2+)-induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca(2+)-induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca(2+)-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca(2+)- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca(2+)-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA.
Hens
J J
JJ
Division of Molecular Neurobiology, Rudolf Magnus Institute, Utrecht, The Netherlands.
De Wit
M
M
Dekker
L V
LV
Boomsma
F
F
Oestreicher
A B
AB
Margolis
F
F
Gispen
W H
WH
De Graan
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000378:metabolism*; D019922:GAP-43 Protein; D007074:Immunoglobulin G; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000502:physiology*; D009638:Norepinephrine / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013301:Streptolysins / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001426:Bacterial Proteins; D019922:GAP-43 Protein; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D013301:Streptolysins; C025964:streptolysin O; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine