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)

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 [H-3]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration-dependent manner by elevating the free Ca2+ concentration from 10(-8) to 10(-5) M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+.Ca2+-induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2+-induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+- and phorbol ester-induced release were not additive at higher phorbol ester concentrations (> 10(-7) M). We compared the sensitivities of Ca2+- 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. Ca2+-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 Ca2+ stimulate noradrenaline release through different mechanisms and that the essential role of B-50 in Ca2+-induced noradrenaline release may involve other properties of B-50 besides protein kinase C-mediated phosphorylation.