Premium
Noradrenaline Release from Streptolysin O‐Permeated Rat Cortical Synaptosomes: Effects of Calcium, Phorbol Esters, Protein Kinase Inhibitors, and Antibodies to the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP‐43)
Author(s) -
Dekker Lodewijk V.,
Graan Pierre N. E.,
Pijnappel Pim,
Oestreicher A. Beate,
Gispen Willem Hendrik
Publication year - 1991
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.1471-4159.1991.tb11404.x
Subject(s) - streptolysin , protein kinase c , protein kinase a , neuron , chemistry , calcium , synaptosome , biochemistry , antibody , biology , enzyme , in vitro , neuroscience , immunology , bacterial protein , organic chemistry , gene
We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B‐50 (GAP‐43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [ 3 H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration‐dependent manner by elevating the free Ca 2+ concentration from 10 −8 to 10 −5 M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca 2+ . Ca 2+ ‐induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na + /K + ratio in the buffer did not affect Ca 2+ ‐induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca 2+ ‐ and phorbol ester‐induced release were not additive at higher phorbol ester concentrations (>10 −7 M ). We compared the sensitivities of Ca 2+ ‐ and phorbol ester‐induced release of noradrenaline to the protein kinase inhibitors H‐7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B‐50. Ca 2+ ‐induced release was inhibited by B‐50 antibodies and polymyxin B, but not by H‐7; phorbol ester‐induced release was inhibited by polymyxin B and by H‐7, but only marginally by antibodies to B‐50. We suggest that phorbol esters and Ca 2+ stimulate noradrenaline release through different mechanisms and that the essential role of B‐50 in Ca 2+ ‐induced noradrenaline release may involve other properties of B‐50 besides protein kinase C–mediated phosphorylation.