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Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters


Address correspondence and reprint requests to Dr. M. Verhage at Rudolf Magnus Institute, Institute for Molecular Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.


Abstract: In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2+-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2+-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+. Furthermore, the relationship between the volume average [Ca]i (measured with fura-2) and the extent of release is more or less linear for the release of CCK-LI, whereas this relationship is clearly nonlinear for the release of endogenous glutamate and γ-aminobutyric acid in similar preparations. We hypothesize that these differences between the neuropeptide CCK-8 and classical transmitters are caused by three differences: (a) vesicle type; (b) Ca2+ sensitivity of the release mechanism; and (c) release site.