Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter
Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter
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DOI：
10.1124/mol.66.1.137
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发表时间：
2004-07-01
影响因子：
3.6
通讯作者：
Galli, A
中科院分区：
文献类型：
--
作者：
Gnegy, ME;Khoshbouei, H;Galli, A
虽然苯丙胺诱导的多巴胺(DA)释放是由多巴胺转运体(DAT)介导的，是这种精神刺激剂行为行为所必需的，但这种释放的机制尚不清楚。在这项研究中，我们探讨了苯丙胺诱导的DA外流和人DAT介导的电流对细胞内钙的需求。将膜片钳技术应用于稳定转染人DAT细胞(HDAT细胞)的Na+和DA预负载的人胚胎肾293细胞。通过在全细胞移液管中加入50微米的BAPTA来螯合细胞内的钙离子，减少了电压依赖的苯丙胺诱导的HDAT电流，在正电压下的影响最大。电流法测定，1，2-二(2-氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸(BAPTA)可减少苯丙胺诱发的DA外流。此外，用50um的BAPTA乙酰氧基甲酯(AM)或thapsigargin预先孵育HDAT细胞，也可阻断苯丙胺诱导的HDAT细胞释放预加载的N-甲基-4-[H-3]苯基吡啶。BAPTA-AM还可减少纹状体突触体内DA的释放。安非他明还导致细胞内钙离子增加，这一作用可被5微米他西格金或10微米可卡因阻断。这些研究表明，苯丙胺诱导的DAT介导的电流和底物外流需要内钙离子，苯丙胺可通过与DAT相互作用来调节胞浆内钙离子水平，从而刺激多巴胺外流。
Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca2+ in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na+ and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intra-cellular Ca2+ by inclusion of 50 muM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 muM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[H-3]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca2+ that was blocked by prior treatment with 5 muM thapsigargin or 10 muM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca2+ and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca2+ levels through its interaction with DAT.