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

Abstract

In order to examine intracellular modulation of CNS catecholamine release, cerebrocortical synaptosomes were prelabeled with [ 3H]noradrenaline and permeabilized with streptolysin-O in the absence or presence of Ca 2+. Plasma membrane permeabilization allowed efflux of cytosol and left a compartmentalized pool of [ 3H]noradrenaline intact, approximately 10% of which was released by addition of 10 −5 M Ca 2+. Addition of activators or inhibitors of protein kinase C, as well as inhibitors of Ca 2+-calmodulin kinase II or calcineurin, failed to change Ca 2+-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 Ca 2+-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, Ca 2+-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.