Regulation of gene expression mediated by the opioid receptor and the nociceptin receptor

本研究采用分子生物学、生物化学和电生理学方法，分析了阿片类镇痛剂和静脉麻醉剂诱导CHO细胞基因表达的变化。通过将克隆的cDNA转染CHO细胞，激活阿片受体，诱导即刻早期基因c-fos和juB的表达，这种诱导作用被百日咳毒素和PD98059抑制，提示百日咳毒素敏感的G蛋白和丝裂原激活的蛋白激酶(MAPK)参与了信号转导途径。这一机制可能与阿片类药物耐受或依赖、阿片类镇痛剂严重副作用的分子机制有关。静脉麻醉药对神经细胞基因表达的影响尚未被研究。我们发现咪达唑仑诱导即刻早期基因产物c-Fos和EGR-1的表达，而这一表达被PD98059阻断。咪达唑仑以时间和剂量依赖的方式诱导MAPK激活。咪达唑仑诱导的MAPK激活可被酪氨酸激酶和表皮生长因子受体抑制剂阻断。我们的结果提示，静脉麻醉药可能通过基因表达模式的改变而导致神经功能的长期变化。转化为表达μ-阿片受体的NG108-15细胞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。不同的μ阿片受体选择性激动剂的脱敏程度不同。利用非洲爪哇卵母细胞表达系统检测了挥发性麻醉药对钙激活钾通道亚型的影响。结果表明，氟烷、异氟烷和七氟醚不影响SK亚型，但抑制IK亚型的活性。这一发现的生理意义仍有待阐明。

In this investigation, we mainly analyzed changes in gene expression induced by opioid analgesics and intravenous anesthetics, using molecular biological, biochemical and electrophysiological methods.Activation of the opioid receptor expressed by transfection of the cloned cDNA in CHO cells induced expression of immediate early genes, c-fos and junB.This induction was inhibited by pertussis toxin and PD98059, suggesting that pertussis toxin-sensitive G-proteins and mitogen- avtivated protein kinase (MAPK) are involved in the signal transduction pathway. This mechanism might be involved in the molecular mechanism of opioid tolerance or dependence, serious side effects of opioid analgesics.Effects of intravenous anesthetics on gene expression in neuronal cells have not been investigated. We found that midazolam induces expression of immediate early gene products, c-Fos and EGR-1, that was blocked by PD98059. Midazolam induced MAPK activation in a time- and dose-dependent manner. The midazolam-induced MAPK activation was blocked by inhibitors of tyrosine kinase and epidermal growth factor receptor. Our results may suggest that intravenous anesthetics induce long-term changes in neural functions by changes in gene expression patterns.Chronic agonist exposure of the NG108-15 cells transformed to express the μ-opioid receptor was demonstrated to cause desensitization of the opioid-induced inhibition of the the N-type Ca^<2+> channel activity. The magnitude of the desensitization was different among various μ-opioid receptor-selective agonists.Effects of volatile anesthetics on Ca^<2+>-activated K^+ channel subtypes were tested by the use of Xenopus oocyte expression system. It was demonstrated that halothane, isoflurane and sevoflurane do not affect the SK subtype, but suppressed the activity of the IK subtype. The physiological implication of this finding remains to be elucidated.