EFFECTS OF OPIATES ON CELLULAR MECHANISMS

对吗啡的耐受涉及信号转导途径的多个水平上的许多适应。研究表明，这些变化可能涉及多个阿片受体脱敏、G蛋白偶联减少、GTP酶活性降低、腺苷环化酶活性增加、cAMP依赖的激酶活性(PKA)增加、蛋白激酶C(PKC)活性增加、钙电导增加、钾电导降低和即刻早期基因的诱导。适应性变化导致细胞内游离钙水平升高，进而导致吗啡抑制神经元放电和神经递质释放的效果降低。我们实验室最近的研究表明，蛋白激酶，特别是PKA和PKC，参与了耐受性的调节。联合应用PKC和PKA抑制剂可在30min内逆转吗啡耐受。在动物对疼痛的反应中，触发吗啡诱导的抗伤害感觉变化的潜在机制仍然难以捉摸。这一效应的迅速出现表明，蛋白的持续磷酸化对于维持耐受性是必不可少的。PKA和PKC有许多潜在的靶蛋白，它们参与了u阿片受体介导的抗伤害性感受，因此，抑制PKA或PKC可以在信号转导级联中的许多位置进行干扰。由不同的效应酶控制的两个激酶独立地调节耐受性的事实表明，信号转导级联中的一个共同步骤被这两个激酶改变。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，结合药理、功能、生化和免疫化学方法，在受体、G蛋白和效应水平上表征治疗的效果。这些研究将提供更好的理解，即信号转导级联中的哪些步骤对耐受性的发展和维持至关重要。

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.