Inhibition of regulated catecholamine secretion from PC12 cells by the Ca2+/calmodulin kinase II inhibitor KN-62.
When stimulated by the cholinergic agonist carbachol, PC12 cells rapidly secrete a large fraction of the intracellular catecholamines by exocytotic release from the large dense-core secretory vesicles in a Ca(2+)-dependent manner. To investigate whether Ca2+/calmodulin kinase II plays a role in the regulated secretion of catecholamines, we examined the effect of the specific Ca2+/calmodulin kinase II inhibitor KN-62 on the carbachol-induced release of norepinephrine from PC12 cells. Approximately 50% of the regulated release of norepinephrine, stimulated either by carbachol or direct depolarization, was inhibited by pretreatment with KN-62, while the remaining 50% was resistant to KN-62 and therefore independent of Ca2+/calmodulin kinase II. In contrast, H7, an inhibitor of protein kinase C, had no effect on any of the stimulated release. FURA 2 imaging experiments demonstrated that KN-62 does not act by blocking the stimulation-induced increase in intracellular [Ca2+]. The most likely model consistent with these data is that all the dense-core vesicles fuse with the plasma membrane in a Ca(2+)-dependent process, but that approximately 50% of the vesicles require an additional step that is dependent on the action of Ca2+/calmodulin kinase II. This step occurs between the influx of Ca2+ and the fusion of vesicle membranes with the plasma membrane, and may be analogous to the Ca2+/calmodulin kinase II phosphorylation of synapsin which mobilizes small, clear synaptic vesicles for exocytosis at the synapse.
Schweitzer
E S
ES
Department of Anatomy, UCLA School of Medicine 90024, USA.
Sanderson
M J
MJ
Wasterlain
C G
CG
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007546:Isoquinolines / Q000494:pharmacology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D010879:Piperazines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D014268:Trifluoperazine / Q000494:pharmacology
D018492:Dopamine Antagonists; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007546:Isoquinolines; D010879:Piperazines; C049925:fura-2-am; D014268:Trifluoperazine; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium; D016257:Fura-2; D009638:Norepinephrine