Smart D, Smith G, Lambert D G
University Department of Anaesthesia, Leicester Royal Infirmary, England.
J Neurochem. 1994 Mar;62(3):1009-14. doi: 10.1046/j.1471-4159.1994.62031009.x.
The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both mu- and delta-opioid receptors), we demonstrated that fentanyl, a mu-preferring opioid, caused a dose-dependent (EC50 = 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1-2 min. This response was of similar magnitude (25.4 +/- 0.8 pmol/mg of protein for 0.1 microM fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mM carbachol (18.3 +/- 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole- (a delta antagonist), reversible. Further studies using [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Pen2,5]enkephalin confirmed that the response was specific for the mu receptor. Incubation with Ni2+ (2.5 mM) or in Ca(2+)-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 microM fentanyl, 26.9 +/- 1.5 pmol/mg of protein; pertussis-treated plus 0.1 microM fentanyl, 5.1 +/- 1.3 pmol/mg of protein). In summary, we have demonstrated a mu-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca(2+)-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.
尽管目前越来越多的间接证据表明某些阿片受体可能与磷脂酶C偶联，但阿片类药物作用的细胞机制仍有待充分确定。我们使用SH-SY5Y人神经母细胞瘤细胞（同时表达μ-和δ-阿片受体），证明了芬太尼（一种偏向μ受体的阿片类药物）可引起剂量依赖性（EC50 = 16 nM）的肌醇（1,4,5）三磷酸质量形成单相增加，在15秒时达到峰值，并在1 - 2分钟内恢复到基础水平。这种反应的幅度（0.1μM芬太尼时为25.4±0.8 pmol/mg蛋白质）与用1 mM卡巴胆碱刺激后平台期（5分钟）的反应幅度（18.3±1.4 pmol/mg蛋白质）相似，并且可被纳洛酮逆转，但不能被纳曲吲哚（一种δ拮抗剂）逆转。使用[D-Ala², MePhe⁴, Gly(ol)⁵]脑啡肽和[D-Pen²,⁵]脑啡肽的进一步研究证实，该反应对μ受体具有特异性。与Ni²⁺（2.5 mM）一起孵育或在无Ca²⁺缓冲液中可消除该反应，百日咳毒素预处理（100 ng/ml，24小时）也可消除该反应（对照加0.1μM芬太尼，26.9±1.5 pmol/mg蛋白质；百日咳毒素处理加0.1μM芬太尼，5.1±1.3 pmol/mg蛋白质）。总之，我们证明了通过百日咳毒素敏感的G蛋白介导的μ-阿片受体对磷脂酶C的激活，该激活是Ca²⁺依赖性的。阿片类药物对磷脂酶C的这种刺激作用以及潜在的肌醇（1,4,5）三磷酸介导的细胞内Ca²⁺升高可能在阿片类药物作用的细胞机制中起作用。
