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，分析了伤害素受体和阿片受体对慢性激动剂作用的信号转导机制和适应机制。在钙离子载体A23187存在的情况下，通过在CHO细胞中转导表达的伤害素受体，可诱导丝裂原活化蛋白激酶(MAPK)激活和磷脂酶A2磷酸化，导致花生四烯酸释放。此外，刺激NG108-15细胞内源性表达的伤害素受体可抑制N型钙通道。激动剂刺激表达于CHO细胞的Mu-阿片受体可诱导该受体内化，随后腺苷环化酶过度敏感，Mu-阿片受体轻度脱敏。这些对慢性激动剂暴露的细胞适应性反应可能涉及对阿片类镇痛剂的耐受和依赖，这在临床环境中是严重的问题。在NG108-15细胞中内源性表达的β-阿片受体的慢性激动剂暴露可导致阿片类药物对N型钙通道活动的抑制的脱敏。我们发现，β-肾上腺素能受体激酶参与了脱敏机制。到目前为止，纳洛酮一直被认为是u-阿片受体、β-阿片受体和kappa-阿片受体的纯拮抗剂。然而，通过分析纳洛酮对中国仓鼠卵巢细胞异源表达的阿片受体的药理作用，我们发现纳洛酮对Mu阿片受体和kappa阿片受体具有部分激动性。

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.