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Electrophysiological and molecular biological analysis on GABA_Breceptors in hypothalamic neuroendocrine cells

下丘脑神经内分泌细胞GABA_Breceptors的电生理和分子生物学分析

基本信息

批准号：
09470020
负责人：
SHIBUYA Izumi
金额：
$ 7.42万
依托单位：
University of Occupational and Environmental Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1)To elicidate the role of GABA_B receptors in the regulation of the magnocellular neurosecretory cells in the supraoptic nucleus (SON) of the hypothalamus, we measured the membrane potential, the firing frequency, and spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in rat SON slice preparations, and also the voltage-gated Ca^<2+> currents (VGC) of acutely dissociated rat SON neurons by the whole-cell patch-clamp technique. The selective GABAGABA_B agonist, baclofen suppressed the action potential discharge of SON neurons, without causing marked hyperpolarization. Baclofen educed the frequency of both the sEPSCs and sIPSCs without affecting the amplitude. The time constant of the decay phase of both the sEPSCs and sIPS Cs remained unchanged after baclofen application. The reduction of the frequency of the synaptic currents by baclofen was dose-dependent. Baclofen inhibited VGC also in a dose-dependent manner. Only the inhibition of N- and P/Q-types was significant. These results indicate that GABAGABA_B receptors are present both at the pre- and post-synaptic sites of SON neurons and mediate inhibition of EPSCs and IPSCs, and of N- and P/Q-type Ca^<2+> channels. These multiple inhibitory mechanisms mediated by pre- and postsynaptic GABAGABA_B receptors may play important roles in the regulation of SON neurons by the GABA neurons.2)To investigate the signal transduction mechanism of GABAGABA_B receptor-mediated cellular responses, poly (A)^+ RNA derived from rat brain cortex was coexpressed with CFTR CI channls or with GIRK channels. We found that upon GABAGABA_B activation, the Gbetagamma released from PTX-sensitive G-proteins activates the adenylate cyclase type II, and this process requires Gs activation by Gs-coupled receptors. We also found that GABAGABA_B receptors activated the cloned GIRKs composed GIRK1 and GIRK2 as heteromultimers.

1)为探讨GABA_B受体在调节下丘脑视上核(SON)大细胞神经分泌细胞中的作用，采用全细胞膜片钳技术，测定了大鼠SON脑片的膜电位、放电频率、自发兴奋性和抑制性突触后电流(sEPSCs和sIPSCs)以及电压门控性钙电流(VGC)。选择性GABAGABA_B激动剂巴氯芬抑制SON神经元的动作电位放电，而不引起明显的超极化。巴氯芬可使sEPSC和sIPSCs的频率降低，但不影响幅度。使用巴氯芬后，sEPSCs和sips Cs的衰变相时间常数均保持不变。巴氯芬降低突触电流的频率具有剂量依赖性。巴氯芬也以剂量依赖的方式抑制VGC。只有N型和P/Q型抑制作用显著。这些结果表明GABAGABA_B受体存在于SON神经元的突触前和突触后部位，并介导对EPSCs和IPSCs以及N和P/Q型钙通道的抑制。这些由突触前和突触后GABAGABA_B受体介导的多种抑制机制可能在GABA神经元对SON神经元的调节中起重要作用。2)为了研究GABAGABA_B受体介导的细胞反应的信号转导机制，从大鼠大脑皮质提取的Poly(A)^+RNA与CFTRCI通道或GIRK通道共表达。我们发现，在GABAGABA_B激活后，PTX敏感的G蛋白释放的Gbetagamma激活了腺苷环化酶II，这一过程需要Gs偶联受体激活Gs。我们还发现GABAGABA_B受体激活了克隆的由GIRK1和GIRK2组成的异多聚体GIRKs。