Rapid Kinetics of Potassium-Evoked Release of Acetylcholine from Rat Brain Synaptosomes: Analysis by Rapid Superfusion

Abstract

The rapid kinetics of spontaneous and evoked [3H]acetylcholine efflux from synaptosomes was investigated using the technique of rapid superfusion. Synaptosomes were isolated from whole rat brain and the intraterminal pool of acetylcholine was radiolabeled by preincubation with [3H]choline. Synaptosomes were retained within the superfusion system on filter disks and superfused with Krebs-bicarbonate buffer, pH 7.4, at flow rates of 0.3-0.5 ml/s. These experimental conditions provided a mixing half-life of 119 ms and efficiency of superfusion of greater than 85%. The kinetics of tritium efflux was followed on the second and subsecond time scales by collection of serial 4.8-s and 50-ms samples for a total of 67.2 and 1.0 s, respectively. Superfusion for 48 s with isoosmotic Krebs buffer containing 10, 20, 30, 50, 75, and 100 mM potassium ion stimulated concentration-dependent tritium release. All of potassium-evoked release, but only 17% of spontaneous release, was calcium-dependent. Kinetic analysis of net (total minus spontaneous) potassium-stimulated release revealed a single calcium-dependent component of release that fit a single exponential function with a half-life of 12.7 s. Analysis of the area under the tritium efflux curves observed on the millisecond time scale revealed that 0.111, 0.550, and 0.614% net tritium release was evoked by superfusion for 750 ms with isoosmotic buffer containing 20, 50, and 100 mM KCl, respectively. Consistent with the results observed on the second time scale, a small fraction of spontaneous release and all of potassium-evoked release observed on the millisecond time scale were calcium-dependent. These data indicate that the technique of rapid superfusion can be utilized for the direct investigation of spontaneous and evoked [3H]acetylcholine release, as well as the factors that regulate this release from brain synaptosomes on the second and millisecond time scales.