DUAL OPIOID MODULATION OF SPINAL CORD-GANGLION ACTIVITY

双阿片类药物对脊髓神经节活动的调节

• 批准号: 3207088
• 负责人: STANLEY M CRAIN
• 金额: $ 33.66万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3207088
• 关键词: action potentials; adenylate cyclase; analgesics; calcium channel; calcium channel blockers; cyclic AMP; dorsal root; drug addiction antagonist; drug tolerance; electrophysiology; enzyme inhibitors; forskolin; laboratory mouse; morphine; naloxone; neuropharmacology; opiate alkaloid; opioid receptor; protein kinase A; radiotracer; tissue /cell culture; voltage /patch clamp

Our major objective are: 1) to analyze mechanisms underlying the dual receptor-mediated excitatory and inhibitory modulation of the action potentials (APs) of mouse dorsal-root ganglion (DRG) neurons that we have observed in explants and dissociated cell cultures; 2) to test the hypothesis that enhancement of the adenylate cyclase (AC)/cyclic AMP system is causally related to the tolerance that occurs in DRG neuron APs and dorsal-horn network response after chronic opioid treatment of DRG-cord cultures. Whereas the duration of the AP calcium component (APD) of naive DRG neurons is generally shortened by acute exposure to high (muM) concentrations of opioids, low (nM) levels of specific mu, delta and kappa opioids elicit APD prolongation in many of the same cells. Both excitatory and inhibitory opioid effects are prevented by naloxone or diprenorphine. Intracellular and whole- cell patch-clamp recording will be used to analyze this dual opioid modulation of DRG neurons in explant and dissociated-cell cultures. Excitatory vs. inhibitory opioid receptors will be characterized by electrophysiologic tests during systematic treatments with specific opioid agonists, antagonists and receptor inactivators. These physiologic studies will be coordinated with opioid binding assays and receptor autoradiography after similar treatments, including pertussis (PTX) vs. cholera toxin (CTX). To further evaluate our evidence that opioid-induced APD prolongation is mediated by receptors positively coupled via Gs to the AC/cAMP/protein kinase A(PKA) system, the opioid responsivity of DRG neurons will be tested extracellular or intracellular perfusion with CTX vs. PTX, cAMP, PKA, inhibitors of AC and PKA, specific ion channel blockers, or altered ion concentrations. Linkages of excitatory opioid receptors with specific K+ and Ca2+ conductances in DRG cells will be analyzed. Correlative biochemical assays of AC activity of these neuros will made, especially during the physiologic expression of tolerance and during the increase in opioid excitatory responsiveness in DRG cells chronically exposed to specific mu, delta, or kappa opioids. Correlative binding assays and autoradiography will be made to determine if specific subtypes of opioid receptors are up-regulated or down-regulated in tolerant DRG neurons. These studies will provide valuable insights into cellular compensatory mechanisms that mediate tolerance to opioid analgesic effects in primary afferent synaptic networks and clues to some of the addictive and euphoric effects of opioids in the CNS.

我们的主要目标是：1）分析机制的基础上， 双重受体介导的兴奋性和抑制性调节 小鼠背根神经节动作电位 我们在外植体和分离的细胞中观察到的神经元 2）为了验证假设，即增强 腺苷酸环化酶（AC）/环AMP系统与 背根神经节神经元AP和背角发生的耐受 慢性阿片类药物治疗后DRG-脊髓的网络反应 cultures. 然而，未处理组AP钙成分（APD）的持续时间 DRG神经元通常因急性暴露于高浓度的 (muM)阿片类药物浓度，低水平（nM）的特定μ， δ和κ阿片类药物引起APD延长，在许多 同样的细胞。 阿片类药物的兴奋性和抑制性作用都是 通过纳洛酮或二丙诺啡预防。 细胞内和整个- 细胞膜片钳记录将被用来分析这一双重 阿片对背根节神经元的调节作用 cultures. 兴奋性与抑制性阿片受体将是 以系统性电生理测试为特征 使用特定的阿片激动剂、拮抗剂和 受体灭活剂。 这些生理学研究将是 与阿片样物质结合试验和受体结合试验相协调 类似治疗后的放射自显影，包括百日咳 (PTX)与霍乱毒素（CTX）相比。 为了进一步评估我们的证据 阿片类药物诱导的APD延长由受体介导， 通过Gs与AC/cAMP/蛋白激酶A（PKA）正偶联 系统，将测试DRG神经元的阿片样物质反应性 细胞外或细胞内灌注CTX与PTX，cAMP， PKA、AC和PKA抑制剂、特异性离子通道阻滞剂，或 改变离子浓度。 兴奋性阿片类药物的联系 DRG细胞中具有特异性K+和Ca 2+传导的受体 将被分析。 AC活性的相关生化测定 尤其是在生理过程中， 耐受性的表达和阿片类药物增加期间 DRG细胞的兴奋性反应性 特异性μ、δ或κ阿片样物质。 相关结合试验 并进行放射自显影，以确定是否特异性 阿片受体的亚型被上调或下调 在耐受性DRG神经元中。 这些研究将提供有价值的见解细胞 调节阿片耐受性的代偿机制 初级传入突触网络的镇痛作用及其线索 阿片类药物的一些成瘾性和欣快作用， CNS。