Duarte C B, Ferreira I L, Carvalho A P, Carvalho C M
Centre for Neurosciences of Coimbra, University of Coimbra, Portugal.
Pflugers Arch. 1993 May;423(3-4):314-23. doi: 10.1007/BF00374411.
The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.
γ-氨基丁酸（GABA）载体的特异性抑制剂NNC-711，即（1-[(2-二苯基亚甲基)氨基]氧基乙基）-1,2,5,6-四氢-3-吡啶羧酸盐酸盐，可阻断50 mM K⁺去极化诱导的大鼠脑突触体中[³H]GABA的非钙依赖性释放。因此，在这种抑制剂存在的情况下，有可能在完全不存在载体介导释放的条件下研究[³H]GABA的钙依赖性释放。利用Na⁺/Ca²⁺交换体的逆向转运来提高细胞内游离钙浓度（[Ca²⁺]i），以测试仅[Ca²⁺]i升高是否足以在无去极化的情况下诱导胞吐作用。我们发现，由于Na⁺/Ca²⁺交换，[Ca²⁺]i可能升高至400 nM以上，但不会诱导[³H]GABA释放，但随后的K⁺去极化会立即诱导[³H]GABA释放。因此，在通过Na⁺/Ca²⁺交换使突触体加载钙后，50 mM K⁺去极化诱导的细胞质中仅升高几纳摩尔的钙即可诱导[³H]GABA的胞吐释放，而Na⁺/Ca²⁺交换体逆向转运引起的细胞质[Ca²⁺]升高不足以诱导胞吐作用，尽管达到的[Ca²⁺]i值高于K⁺去极化诱导胞吐作用所需的值。在通过Na⁺/Ca²⁺交换使突触体最大程度加载钙后，K⁺去极化引起的电压依赖性钙内流以及随之而来的[³H]GABA释放可被50 μM维拉帕米阻断。尽管在任何研究条件下，通过Na⁺/Ca²⁺交换使突触体预加载钙都不会导致[³H]GABA释放，但Na⁺/Ca²⁺交换引起的细胞质[Ca²⁺]升高增加了后续K⁺去极化诱导的[³H]GABA胞吐释放对细胞外钙的敏感性。因此，我们的结果表明，[³H]GABA的囊泡释放对大量细胞质[Ca²⁺]相当不敏感，这与GABA胞吐作用是由通过活性区附近的钙通道进入的钙非常有效地触发的观点一致。
