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 [3H]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 (greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Dekker
L V
LV
Division of Molecular Neurobiology, Rudolf Magnus Institute, Utrecht, The Netherlands.
De Graan
P N
PN
Pijnappel
P
P
Oestreicher
A B
AB
Gispen
W H
WH
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism*; D019922:GAP-43 Protein; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D010539:Permeability; D010703:Phorbol Esters / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013301:Streptolysins / Q000494:pharmacology*; D013574:Synaptosomes / Q000378:metabolism*
D000906:Antibodies; D001426:Bacterial Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D013301:Streptolysins; C025964:streptolysin O; D002118:Calcium; D009638:Norepinephrine