Hirst R A, Hirota K, Grandy D K, Lambert D G
University Department of Anaesthesia, Leicester Royal Infirmary, UK.
Neurosci Lett. 1997 Aug 29;232(2):119-22. doi: 10.1016/s0304-3940(97)00594-6.
This study describes the coupling of the recombinant rat kappa-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4-7) and late (p14-17) passage after transfection. As passage increased the receptor numbers (Bmax) declined from 231 +/- 24 (early) to 31 +/- 2 fmol/mg protein (late) but the equilibrium dissociation constant (Kd) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (Ki[H]) and low (Ki[L]) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% Ki(L):50% Ki[H]), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (+/-)bremazocine, ICI-204,448 and (+/-)trans-U-50488 methanesulfonate (1 microM), compounds with activity at kappa-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not kappa-agonist specific. Collectively, these data suggest that in CHO cells, kappa-opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.
本研究描述了在中国仓鼠卵巢（CHO）细胞中表达的重组大鼠κ-阿片受体与腺苷酸环化酶的偶联以及受体密度的影响。[3H]二丙诺啡（[3H]DPN）的结合在转染后早期（p4 - 7）和晚期（p14 - 17）传代细胞制备的膜中呈剂量依赖性且可饱和。随着传代增加，受体数量（Bmax）从231±24（早期）降至31±2 fmol/mg蛋白（晚期），但平衡解离常数（Kd）未改变。螺哌啶剂量依赖性地将[3H]DPN从早期和晚期细胞制备的膜中置换出来，揭示了高亲和力（Ki[H]）和低亲和力（Ki[L]）结合位点。这些位点的比例没有显著差异（约50% Ki[L]：50% Ki[H]），虽然螺哌啶在晚期细胞中通常效力较低，但差异很小且未达到统计学显著性。相比之下，螺哌啶在全细胞中对福司可林刺激的cAMP形成产生剂量依赖性抑制，早期细胞的pIC50为8.62，晚期细胞为8.00。此外，最大抑制率从47%显著降至22%。埃托啡、（±）布瑞马佐辛、ICI - 204,448和（±）反式-U - 50488甲磺酸盐（1 microM），这些在κ-受体上有活性的化合物，在早期细胞（分别为42.2%、45.8%、50.2%和50.5%）中对cAMP形成的抑制作用比晚期细胞（分别为12.9%、11.8%、13.5%和7.8%）更大，表明cAMP形成的表达依赖性抑制并非κ-激动剂特异性。总体而言，这些数据表明在CHO细胞中，κ-阿片受体与腺苷酸环化酶的偶联取决于受体表达水平。
