Knapman Alisa, Abogadie Fe, McIntrye Peter, Connor Mark
1Australian School of Advanced Medicine, Macquarie University, NSW Australia.
J Biomol Screen. 2014 Feb;19(2):223-31. doi: 10.1177/1087057113501391. Epub 2013 Aug 29.
Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential-sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK (pEC₅₀, 7.3) or calcitonin (pEC₅₀, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO (pEC₅₀, 7.4; E(max), 56%), morphine (pEC₅₀, 7.0; E(max), 61%); and buprenorphine (pEC₅₀, 8.6; E(max), 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.
抑制腺苷酸环化酶（AC）活性常用于测量μ-阿片受体（MOR）的激活情况。我们试图开发一种简单、快速的全细胞AC活性检测方法，可用于研究MOR信号传导。将表达人MOR的中国仓鼠卵巢细胞（CHO-MOR细胞）接种于96孔板中，并加载膜电位敏感荧光染料。用AC激活剂福斯高林（FSK）处理CHO-MOR细胞，同时或不同时应用MOR激动剂，然后测量荧光的变化。应用FSK（pEC₅₀，7.3）或降钙素（pEC₅₀，9.4）后，CHO-MOR细胞发生超极化。亚最大有效浓度的FSK（300 nM）导致荧光降低52%±2%。同时应用阿片类药物DAMGO（pEC₅₀，7.4；E(max)，56%）、吗啡（pEC₅₀，7.0；E(max)，61%）和丁丙诺啡（pEC₅₀，8.6；E(max)，24%）可剂量依赖性抑制FSK反应，而它们自身无作用。DAMGO的作用被百日咳毒素阻断。该检测方法是实时观察CHO细胞中MOR对AC抑制作用的一种简单、可靠的方法。它是依赖于cAMP积累的终点检测方法的一种有吸引力的替代方法，可避免与MOR信号快速脱敏相关的潜在干扰。
