Molecular Biological Study of the Signal Transduction Mechanism of the G-Protein-Coupled Receptors Involved in Pain

本研究采用分子生物学、生物化学和电生理学方法，分析了痛敏素受体和阿片受体的信号转导机制和对慢性激动剂暴露的适应机制，结果表明，将克隆的cDNA转染CHO细胞，激活痛敏素受体，诱导丝裂原活化蛋白激酶（MAPK）活化和磷脂酶A_2磷酸化，导致在钙离子载体A23187存在下花生四烯酸的释放。此外，刺激NG 108 -15细胞内源性表达的痛敏素受体可导致N型Ca^<2+>通道的抑制，激动剂刺激CHO细胞表达的μ阿片受体可诱导受体内化，随后下调，腺苷酸环化酶超敏，μ阿片受体轻微脱敏。这些细胞对慢性激动剂暴露的适应性反应可能涉及对阿片类镇痛剂的耐受性和依赖性，这在临床环境中是严重的问题。NG 108 -15细胞内源性表达的δ-阿片受体的长期激动剂暴露被证明会导致阿片诱导的N型Ca^2+通道活性抑制的脱敏。我们发现β-肾上腺素能受体激酶参与了这一脱敏机制，纳洛酮被认为是μ-、δ-和κ-阿片受体的纯拮抗剂。然而，通过分析纳洛酮对CHO细胞中异源表达的阿片受体的药理作用，我们发现纳洛酮对μ-和κ-阿片受体具有部分激动活性。

In this investigation, we analyzed signal transduction mechanism and adaptation mechanism to chronic agonist exposure of the nociceptin receptor and the opioid receptor, using molecular biological, biochemical and electrophysiological methods.Activation of the nociceptin receptor expressed by transfection of the cloned cDNA in CHO cells induced activation of mitogen-activated protein kinase (MAPK) and phosphorylation of phospholipase A_2, leading to arachidonate release in the presence of a calcium ionophore A23187. Furthermore, stimulation of the nociceptin receptor endogenously expressed in NG108-15 cells induced inhibition of the N-type Ca^<2+> channel.Agonist stimulation of the mu-opioid receptor expressed in CHO cells induced internalization of the receptor followed by down-regulation, supersensitization of adenylate cyclase and slight desensitization of the mu-opioid receptor. These cellular adaptation responses to chronic agonist exposure may be involved in tolerance and dependence to opioid analgesics, that are serious problems in clinical settings. Chronic agonist exposure of the delta-opioid receptor endogenously expressed in NG108-15 cells was demonstrated to cause desensitization of the opioid-induced inhibition of the N-type Ca^<2+> channel activity. We found that beta-adrenergic receptor kinase is involved in the desensitization mechanism.Naloxone has been so far thought as a pure antagonist of the mu-, delta- and kappa-opioid receptors. However, by analyzing pharmacological effects of naloxone on the opioid receptors expressed heterologously in CHO cells, we found that naloxone has partial agonistic activity on the mu- and kappa-opioid receptors.