Santos P F, Carvalho A L, Carvalho A P, Duarte C B
Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal.
Eur J Neurosci. 1998 Aug;10(8):2723-30. doi: 10.1046/j.1460-9568.1998.00281.x.
In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.
在本研究中，我们调查了控制从无长突细胞样神经元培养物中释放乙酰胆碱（ACh）和γ-氨基丁酸（GABA）的机制，这些培养物包含同时具有GABA能和胆碱能的细胞亚群。我们发现，培养物中81.2±2.8%的细胞用抗胆碱乙酰转移酶抗体进行免疫细胞化学染色，38.5±4.8%的细胞用抗GABA抗体染色。大多数含GABA的细胞（87.0±2.9%）是胆碱能的。乙酰胆碱和GABA的释放大多依赖Ca2+，尽管通过其转运体的逆转会发生显著的[3H]GABA释放。钾离子诱发[3H]GABA和[3H]乙酰胆碱的Ca2+依赖性释放，其EC50分别为31.0±1.0 mM和21.6±1.1 mM。1 μM河豚毒素、低浓度（30 nM）的ω-芋螺毒素GVIA（ω-CgTx GVIA）或高浓度（300 nM）的尼群地平（Nit）可显著抑制[3H]乙酰胆碱的Ca2+依赖性释放。相反，30 nM尼群地平或500 nM ω-CgTx GVIA可降低[14C]GABA的释放，但30 nM的这种毒素则无此作用。200 nM ω-银环蛇毒素IVA（ω-Aga IVA）（一种阻断P/Q型电压敏感性Ca2+通道（VSCC）的毒素）对两种递质的释放均无影响。结果表明，通过ω-CgTx GVIA敏感的N型VSCC和Nit敏感的L型VSCC的Ca2+内流诱导ACh和GABA的释放。然而，在每种神经递质释放所涉及的Ca2+通道方面观察到的显著差异表明，在同时含有GABA和乙酰胆碱的无长突细胞样神经元中，这两种神经递质可能在细胞的不同区域释放，并具有不同的VSCC群体。
