OPIOID AND G-PROTEIN REGULATION OF CALCIUM CURRENTS

这项拟议研究的长期目标是了解细胞 阿片类药物抑制电压依赖性钙离子的分子机制 哺乳动物神经元上的(Ca++)通道，并确定这种调节是如何 与阿片类镇痛剂抑制神经递质的能力有关 放手。这些信息之所以被寻找，是因为它是通过这个 阿片类物质的突触前抑制作用调制 伤害性信息，发挥其镇痛作用。校长 研究的目的是描述抑制耦合的特征 阿片受体和神经末梢钙通道之间的关系 神经元胞体的类型和胞吐功能 已调制的通道，并确定其功能重要性 电压依赖性钙信号在介导阿片类药物中的变化 抑制胞吐释放。这些实验将利用全细胞 联合应用膜片钳技术记录钙电流 用膜时间分辨测量监测胞吐作用 离体肽能神经末梢和体节的电容(Cm) (仅限电流)下丘脑-神经垂体系统和 分离的成年大鼠背根神经节感觉神经元。 使用对特定类型的钙离子有选择性的药物阻滞剂 通道，以及特定的电压钳位协议，我们将 确定阿片受体的抑制性偶联模式 视上核(SON)神经元中钙离子通道的平行表达 在神经末梢的胞吐通道，调节加压素或 催产素释放。阿片类药物阻断电压之间的关系 依赖的钙离子内流和释放抑制将通过以下方式进行评估 量化特定的钙通道拮抗剂和 用Fura-2测定的[Ca++]i的受控变化对 Kappa或微阿片激动剂抑制钙电流和减弱CM 神经末梢和细胞体准备的反应。在扩展 对功能定义的DRG神经元子集的分析，我们将确定 调控在钙离子内流和下游变化中的作用 介导阿片类药物抑制的胞吐过程的组成部分 对伤害性信号传递的影响。建议数 研究，有助于从机制上理解阿片类药物 调节神经传递，应该有助于小说的设计 无耐受或滥用倾向特征的止痛药 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.