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 [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.
Walaas
S I
SI
Neurochemical Laboratory, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1115-Blindern, N-0317, Oslo, Norway. ivar.walaas@basalmed.uio.no
Sefland
I
I
D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000502:physiology; D002540:Cerebral Cortex / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology; D004798:Enzymes / Q000502:physiology; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009638:Norepinephrine / Q000378:metabolism*; D010539:Permeability; D011506:Proteins / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism*; D014316:Tritium
D000199:Actins; D002147:Calmodulin; D004798:Enzymes; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D014316:Tritium; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D002118:Calcium; D009638:Norepinephrine