DEPOLARIZATION BY K- VESICULAR VERSUS NONVESICULAR RELEASE OF GABA( AND GLUTAMATE ACTIVATES DIFFERENT NEUROTRANSMITTER RELEASE MECHANISMS IN GABAERGIC NEURONS )

Neurotransmitter release and changes in the concentration of intracell ular free calcium ([Ca++]i) were studied in cultured GABAergic cerebra l cortical neurons, from mice, upon depolarization with either an unph ysiologically high potassium concentration (55 mM) or the physiologica l excitatory neurotransmitter glutamate (100 muM). Both depolarizing s timuli exerted prompt increases in the release of preloaded [H-3]GABA as well as in [Ca++]i. However, the basic properties of transmitter re lease and the increase in [Ca++]i under a variety of conditions were d ifferent during stimulation with K+ or glutamate. Potassium-evoked rel ease of [H-3]GABA consisted of two phases, a rapid, large and transien t phase followed by a smaller, more persistent second phase. The rapid phase was inhibited (60%) by nocodazole which reduced the number of v esicles in the neurites by 80%. This rapid phase of the GABA release w as also reduced by organic (verapamil) and inorganic (Co++) Ca++ chann el blockers but was insensitive to the GABA transport inhibitor SKF 89 976A. In contrast, the second phase was less sensitive to nocodazole a nd Ca++ channel antagonists but could be inhibited by SKF 89976A. The glutamate-induced [H-3]GABA release, which was mainly mediated by N-me thyl-D-aspartate receptors, consisted of a single, sustained phase. Th is was insensitive to nocodazole, partly inhibited by verapamil and co uld be blocked by Co++ as well as SKF 89976A. The action of Co++ could be attributed to a block of N-methyl-D-aspartate-associated ion chann els. These findings strongly suggest that the majority of the K+-stimu lated GABA release is dependent upon vesicles whereas the glutamate in duced release is non-vesicular and mediated by a depolarization-depend ent reversal of the direction of high-affinity GABA transport. The bas ic differences in the mode of action of the two depolarizing stimuli w ere reflected in the properties of the increase in [Ca++]i elicited by 55 mM K+ and 100 muM glutamate, respectively. The K+-induced increase in [Ca++]i was reduced by both verapamil and Ca++-free media whereas the corresponding glutamate response was only sensitive to Ca++-free c onditions. Exposure of the cells to nocodazole or SKF 89976A had no ef fect on the ability of K+ or glutamate to increase [Ca++]i. Altogether , the results clearly demonstrate that K+-induced transmitter release from these GABAergic neurons is vesicular in nature whereas that induc ed by the neurotransmitter glutamate is not.