Modulation of calcium-evoked [3H]noradrenaline release from permeabilized cerebrocortical synaptosomes by the MARCKS protein, calmodulin and the actin cytoskeleton

In order to examine intracellular modulation of CNS catecholamine release, cerebrocortical synaptosomes were prelabeled with [H-3]noradrenaline and permeabilized with streptolysin-O in the absence or presence of Ca2+. Plasma membrane permeabilization allowed efflux of cytosol and left a compartmentalized pool of [H-3]noradrenaline intact, approximately 10% of which was released by addition of 10(-5) M Ca2+. Addition of activators or inhibitors of protein kinase C, as well as inhibitors of Ca2+-calmodulin kinase II or calcineurin, failed to change Ca2+-induced noradrenaline release. Evoked release from permeabilized synaptosomes deficient in the vesicle-associated phosphoprotein synapsin I was also unchanged. In contrast, addition of a synthetic 'active domain' peptide from the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein increased, while addition of calmodulin decreased Ca2+-induced release from the permeabilized synaptosomes, the latter effect being reversed by a peptide inhibitor of calcineurin. Moreover, addition of the actin-destabilizing agent DNase I, as well as antibodies to MARCKS, appeared to increase spontaneous, Ca2+-independent release from noradrenergic vesicles. These results indicate that the MARCKS protein may modulate release from permeabilized noradrenergic synaptosomes, possibly by modulating calmodulin levels and/or the actin cytoskeleton. (C) 2000 Elsevier Science Ltd. All rights reserved.