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

• Published in: Neurochemistry international Vol. 36; no. 7; pp. 581 - 593
• Main Authors: Walaas, S.Ivar, Sefland, Iren
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.06.2000; Elsevier
• Subjects: Actins - physiology; Animals; Biological and medical sciences; Calcium - pharmacology; Calmodulin - physiology; Central nervous system; Central neurotransmission. Neuromudulation. Pathways and receptors; Cerebral Cortex - metabolism; Cytoskeleton - physiology; Enzymes - physiology; Female; Fundamental and applied biological sciences. Psychology; Intracellular Signaling Peptides and Proteins; Male; MARCKS protein; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Norepinephrine - metabolism; Permeability; Proteins - physiology; Rats; Rats, Wistar; Synaptosomes - metabolism; Tritium; Vertebrates: nervous system and sense organs; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; BSA, bovine serum albumin; PKC, protein kinase C; NA, noradrenaline; CaM, calmodulin; CaM KII, Ca 2+-calmodulin-dependent protein kinase II; LDCV, large dense core vesicle; MARCKS, myristoylated alanine-rich C-kinase substrate; 13-phorbol myristate acetate; SLO, streptolysin-O; CaN, calcineurin; PMA, 12; DMSO, dimethyl sulfoxide; SDCV, small dense core vesicle; LDH, lactic acid dehydrogenase; Cerebral cortex; Calcium; Rat; Rodentia; Central nervous system; Catecholamine; Vertebrata; Synaptosome; Mammalia; Actin; Cytoskeleton; Norepinephrine; Calmodulin; Release; Brain (vertebrata)
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/S0197-0186(99)00159-X

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.