Modulation of calcium-evoked [H-3]noradrenaline release from permeabilizedcerebrocortical 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 pe rmeabilized with streptolysin-O in the absence or presence of Ca2+. Plasma membrane permeabilization allowed efflux of cytosol and left a compartmenta lized pool of [H-3]noradrenaline intact, approximately 10% of which was rel eased 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 ca lcineurin, failed to change Ca2+-induced noradrenaline release. Evoked rele ase from permeabilized synaptosomes deficient in the vesicle-associated pho sphoprotein synapsin I was also unchanged. In contrast, addition of a synth etic 'active domain' peptide from the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein increased, while addition of calmodulin decrease d Ca2+-induced release from the permeabilized synaptosomes, the latter effe ct 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 noradrener gic vesicles. These results indicate that the MARCKS protein may modulate r elease from permeabilized noradrenergic synaptosomes, possibly by modulatin g calmodulin levels and/or the actin cytoskeleton. (C) 2000 Elsevier Scienc e Ltd. All rights reserved.