EFFECTS OF OPIATES ON CELLULAR MECHANISMS

阿片类药物对细胞机制的影响

• 批准号: 6801984
• 负责人: WILLIAM C DEWEY
• 金额: $ 12.15万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6801984
• 关键词: G protein; adenylate cyclase; calcium flux; drug tolerance; enzyme inhibitors; laboratory mouse; morphine; opioid receptor; phospholipase C; protein kinase A; protein kinase C; psychopharmacology; synaptosomes; western blottings

Tolerance to morphine involves numerous adaptations at multiple levels of the signal transduction pathway. Studies suggest that changes likely involve desensitization of multiple opioid receptors, decreased G-protein coupling, decreased GTPase activity, increased adenylyl cyclase activity, increased cAMP-dependent kinase activity (PKA) increased protein kinase C activities (PKC), increased Ca2+ conductance, decreased potassium conductance and induction of immediate early genes. Adaptational changes lead to elevated intercellular free calcium levels, which could in turn contribute to the decreased efficacy of morphine in inhibiting neuronal firing and neurotransmitter release. Recent studies in our laboratory have implicated protein kinases, especially PKA and PKC, in the regulation of tolerance. Morphine tolerance can be reversed by PKC and PKA inhibitors within 30 min, when central administered together with morphine. The underlying mechanism that triggers the change in morphine induced antinociception as determined in the animal's response to pain, remains elusive. The rapid onset in the effect indicates that constant Phosphorylation of proteins by kinases is essential to maintain tolerance. PKA and PKC have numerous potential target proteins, which are involved in mu opioid receptor mediated antinociception, and thus, the inhibition of PKA or PKC could interfere at numerous sites in the signal transduction cascade. The fact that two kinase controlled by different effector enzymes independently regulate tolerance suggests that a common step within the signal transduction cascade is altered by both kinases. We propose to induce morphine- tolerance and its reversal in vivo, and use membrane preparations from tolerant and reversed-tolerant animals to characterize the effects of treatment at the receptor, G-protein and effector level, by a combination of pharmacological, functional, biochemical and immunochemical methods. These studies will provide a better understanding, which steps in the signal transduction cascade are crucial for the development and maintenance of tolerance.

对吗啡的耐受性涉及信号转导途径多个水平的许多适应。研究表明，这些变化可能涉及多种阿片受体的脱敏、G蛋白偶联减少、GT3活性降低、腺苷酸环化酶活性增加、cAMP依赖性激酶活性（PKA）增加、蛋白激酶C活性（PKC）增加、Ca2+电导增加、钾电导减少和诱导立即早期基因。适应性变化导致细胞间游离钙水平升高，这反过来又可能导致吗啡抑制神经元放电和神经递质释放的功效降低。本实验室最近的研究表明，蛋白激酶，特别是PKA和PKC，参与了耐受的调节。PKC和PKA抑制剂可在30 min内逆转吗啡耐受。在动物对疼痛的反应中确定的触发吗啡诱导的抗伤害感受变化的潜在机制仍然是难以捉摸的。作用的快速起效表明激酶对蛋白质的持续磷酸化对维持耐受性至关重要。PKA和PKC具有许多潜在的靶蛋白，其参与μ阿片受体介导的抗伤害感受，因此，PKA或PKC的抑制可在信号转导级联的许多位点干扰。由不同效应酶控制的两种激酶独立调节耐受性的事实表明，信号转导级联中的共同步骤被两种激酶改变。我们建议在体内诱导吗啡耐受及其逆转，并使用耐受和逆转耐受动物的膜制剂，通过药理学、功能、生物化学和免疫化学方法的组合，在受体、G蛋白和效应器水平表征治疗的效果。这些研究将提供一个更好的理解，在信号转导级联的步骤是至关重要的耐受性的发展和维持。