OPIOID AND G-PROTEIN REGULATION OF CALCIUM CURRENTS

这项研究的长期目标是了解细胞内的 以及阿片类药物抑制电压依赖性钙离子的分子机制 (Ca++）通道的哺乳动物神经元，并确定如何调节 与阿片类镇痛药抑制神经递质的能力有关 release. 这些信息正在寻找，因为它是通过这个 突触前抑制作用，阿片类药物调节的传输 伤害性信息并发挥其镇痛作用。 校长 研究的目的是表征抑制性偶联， 阿片受体和神经末梢中的Ca++通道之间的关系， 神经元胞体的类型和胞吐功能， 的功能重要性，并确定 电压依赖性Ca ~（++）信号通路的改变 抑制胞吐释放。 实验将利用全细胞 膜片钳技术记录Ca++电流， 使用膜的时间分辨测量监测胞吐作用 肽能神经末梢和胞体的电容（Cm） （仅电流）的下丘脑-神经垂体系统和 分离的成年大鼠背根神经节（DRG）感觉神经元。 使用对特定类型Ca++具有选择性的药理学阻断剂 通道，连同特定的电压钳协议，我们将 确定阿片受体的抑制偶联模式是否 视上核（SON）神经元表达的Ca ~（++）通道平行 在调节血管加压素的神经末梢中形成胞吐通道， 催产素释放 阿片类药物阻断电压- 依赖性Ca++内流和释放的抑制将通过 定量特定Ca++通道拮抗剂的作用， 使用Fura-2测量的[Ca++]i的受控变化对能力的影响 κ-或微阿片激动剂，以抑制Ca++电流并减弱Cm 神经末梢和细胞体制备的反应。 在延长 分析功能定义的DRG神经元子集，我们将确定 调节Ca++内流和下游细胞中改变的作用 阿片类抑制剂介导的胞吐过程的组分 对伤害性信号传递的影响。 拟议 研究，有助于对阿片类药物的机械理解 调节神经传递，应该有助于设计新的 没有耐受性或滥用倾向特征的镇痛剂 鸦片类药物

The long range goal of the proposed research is to understand the cellular and molecular mechanisms whereby opiates inhibit voltage-dependent calcium (Ca++) channels in mammalian neurons and to determine how this regulation relates to the ability of opiate analgesics to depress neurotransmitter release. This information is being sought, because it is through this presynaptic inhibitory action that opiates modulate the transmission of nociceptive information and exert their analgesic effects. The principal objectives of the research are to characterize the inhibitory coupling between opioid receptors and Ca++ channels in the nerve terminal and neuronal cell body with respect to the type and exocytotic function of the channels that are modulated and to determine the functional importance of alterations in voltage-dependent Ca++ signaling in mediating opioid inhibition of exocytotic release. The experiments will utilize whole-cell patch clamp techniques to record Ca++ currents in combination with monitoring of exocytosis using time-resolved measurements of membrane capacitance (Cm) in isolated peptidergic nerve endings and somata (currents only) of the hypothalamo-neurohypophysial system and in dissociated dorsal root ganglion (DRG) sensory neurons from adult rat. Using pharmacological blockers selective for specific types of Ca++ channels, together with specific voltage clamp protocols, we will determine whether the pattern of inhibitory coupling of opioid receptors to Ca++ channels expressed in supraoptic nucleus (SON) neurons parallels that for exocytotic channels in nerve endings that regulate vasopressin or oxytocin release. The relationship between opioid blockade of voltage- dependent Ca++ influx and inhibition of release will then be assessed by quantifying the effects of particular Ca++ channel antagonists and controlled alterations in [Ca++]i, measured with Fura-2, on the ability of kappa- or micro-opioid agonists to inhibit Ca++ currents and attenuate Cm responses in nerve ending and cell body preparations. In extending the analysis to functionally defined subsets of DRG neurons, we will determine the role of modulation in Ca++ influx and alterations in downstream components of the exocytotic process in mediating opioid inhibitory effects on the transmission of nociceptive signals. The proposed research, in contributing to a mechanistic understanding of opioid regulation of neurotransmission, should aid in the design of novel analgesic agents devoid of the tolerance or abuse liability characteristic of opiate drugs.