OPIOID EXCITATORY SIGNALLING

描述：(申请人摘要) 阿片受体激动剂刺激引起兴奋性反应 表现为细胞内游离钙浓度([Ca~(2+))i]升高 这是由于细胞内储存的钙离子释放所致。 未分化的NG108-15细胞。这项提议的总体目标是 使用电生理学、药理学和微量荧光技术 来描述介导阿片类药物诱导兴奋的途径 并确定长期接触阿片类药物对反应的影响。我们的 第一个目标是确定阿片受体与钙离子偶联的联系 动员。我们假设阿片类药物诱导的β-伽马解离 异源三聚体抑制G蛋白亚基激活磷脂酶C 从细胞内的存储中调动钙离子。这一假设提供了一种 Delta型受体如何激活效应器的合理解释 传统上被认为与抑制性G蛋白偶联。我们的第二个目标是 确定阿片受体与钙开放的联系途径 分化细胞中的通道。分化的NG108-15细胞中的阿片类物质 通过间接途径引起钙离子内流。我们假设在 分化的细胞，阿片类药物诱导的蛋白激酶激活导致 电压门控钙通道的去极化和随后的募集。 这一假说可以解释阿片类药物对神经元的某些影响。 兴奋性。我们的第三个目标是描述长期的影响 阿片类药物对激动剂诱导的钙释放的影响。我们假设 长期使用阿片类药物治疗NG108-15细胞将脱敏 阿片类药物诱导的[Ca~(2+)]i升高。这些研究预计将确定 阿片受体脱敏是否会不同程度地影响 受阿片类药物调节的各种第二信使系统。我们的第四个目标是 确定阿片类药物引起的兴奋效应的程度 在初级组织中。我们将测试大鼠中枢和外周神经元生长在 阿片类药物诱导的[Ca~(2+)]i升高的原代培养 其他信号通路，可能是磷脂酰肌醇级联 受阿片类药物调节的多个第二信使系统可能是 了解药物滥用的分子机制。特征描述 阿片类药物在NG108-15细胞中引发的兴奋性反应将提供 第二信使系统间串扰的研究基础及如何 这些关系在发育和慢性刺激过程中会发生变化。

DESCRIPTION: (Applicant's Abstract) Agonist stimulation of opioid receptors elicits an excitatory response manifest as an increase in intracellular free Ca2+ concentration ([Ca2+)i) that results from release of Ca2+ from intracellular stores in undifferentiated NG108-15 cells. The overall objective of this proposal is to employ electrophysiologic, pharmacologic and microfluorimetric techniques to characterize the pathway which mediates this opioid-induced excitation and determine how chronic exposure to opiates affects the response. Our first aim is to determine the link that couples opioid receptors to Ca2+ mobilization. We hypothesize that opioid-induced dissociation of beta gamma subunits from heterotrimeric inhibitory G proteins activates phospholipase C to mobilize Ca2+ from intracellular stores. This hypothesis provides a rational explanation for how delta receptors can activate effectors not traditionally thought to couple to inhibitory G proteins. Our second aim is to determine the pathway that links opioid receptors to the opening of Ca2+ channels in differentiated cells. In differentiated NG108-15 cells opioids evoke Ca2+ influx via an indirect pathway. We hypothesize that in differentiated cells, opioid induced activation of protein kinases leads to depolarization and subsequent recruitment of voltage-gated Ca2+ channels. This hypothesis may explain some effects of opioids on neuronal excitability. Our third aim is to characterize the effects of prolonged exposure to opioids on agonist-induced Ca2+ release. We hypothesize that chronic treatment of NG108-15 cells with opioids will desensitize opioid-induced [Ca2+]i increases. These studies are predicted to determine whether desensitization of opioid receptors affects differentially the various second messenger systems modulated by opioids. Our fourth aim is to determine the extent to which opioid-induced excitatory effects can be seen in primary tissue. We will test rat central and peripheral neurons grown in primary culture for opioid-induced increases in [Ca2+]i. Including an additional signaling pathway, possibly the phosphoinositide cascade, among the multiple second messenger systems modulated by opioids may be key to understanding the molecular mechanisms of drug abuse. Characterization of the excitatory responses elicited by opioids in NG108-15 cells will provide the basis for studies on cross talk between second messenger systems and how these relationships change during development and chronic stimulation.