Huston E, Cullen G P, Burley J R, Dolphin A C
Department of Pharmacology, Royal Free Hospital Medical School, London, U.K.
Neuroscience. 1995 Sep;68(2):465-78. doi: 10.1016/0306-4522(95)00172-f.
In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.
在本研究中，我们研究了多种钙离子通道亚型支持小脑颗粒神经元释放[3H]谷氨酸的能力，以及GABAB受体激动剂(-)-巴氯芬调节谷氨酸释放所涉及的作用机制。通过钾离子诱发的去极化刺激小脑颗粒神经元释放新合成的[3H]谷氨酸。刺激释放完全依赖于钙离子，且在存在200微摩尔镉时被消除。谷氨酸的释放不受河豚毒素或5-氨基磷酸戊酸的影响，但被二氢海因酸增强，并被6-氰基-7-硝基喹喔啉-2,3-二酮抑制。L型钙通道阻滞剂尼卡地平和N型钙通道阻滞剂ω-芋螺毒素GVIA均部分抑制刺激的谷氨酸释放；然而，P/Q型钙通道阻滞剂ω-阿加毒素IVA仅在细胞与ω-芋螺毒素GVIA预孵育后才抑制谷氨酸的释放。当在钙离子或钡离子存在下刺激时，观察到钾离子刺激的谷氨酸释放，并且在两种条件下均观察到(-)-巴氯芬对释放的类似抑制。与这些结果相反，与钾离子诱发的释放相比，离子霉素诱发的谷氨酸释放大大减少，并且不受(-)-巴氯芬的调节。单独存在ω-芋螺毒素GVIA时，(-)-巴氯芬对释放的抑制作用减弱但未消除。在阻断尼卡地平敏感通道后，(-)-巴氯芬对释放的抑制作用仍然存在，并且在预先阻断ω-芋螺毒素GVIA敏感通道后，尼卡地平的存在恢复了(-)-巴氯芬抑制谷氨酸残留释放的能力。(-)-巴氯芬对谷氨酸释放的调节不受单独存在ω-阿加毒素IVA的影响；然而，在阻断ω-芋螺毒素GVIA和ω-阿加毒素IVA敏感通道后，(-)-巴氯芬对释放的抑制作用完全消除。这些结果表明，小脑颗粒神经元突触前终末存在多种电压依赖性钙通道亚型，并支持钾离子刺激的[3H]谷氨酸释放。GABAB受体激活对释放的调节似乎依赖于该受体与多种电压敏感钙通道的相互作用，包括ω-芋螺毒素GVIA敏感和ω-阿加毒素IVA敏感通道。
