Traynor John R, Clark Mary J, Remmers Ann E
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
J Pharmacol Exp Ther. 2002 Jan;300(1):157-61. doi: 10.1124/jpet.300.1.157.
Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.
作用于其受体的阿片类激动剂通过启动各种类型的Gi/Go蛋白激活来改变细胞内事件。这可以通过稳定的GTP类似物[（35）S]鸟苷-5'-O-（3-硫代）三磷酸（[（35）S]GTPγS）的结合来测量。在本研究中，激动剂效力由阿片类药物刺激[（35）S]GTPγS结合的程度来定义。这允许对完全激动剂和部分激动剂进行定义；完全激动剂比部分激动剂对[（35）S]GTPγS结合的刺激更大。使用表达μ-或δ-阿片受体的C6胶质瘤细胞膜测试了激动剂刺激的[（35）S]GTPγS结合速率取决于激动剂效力的假设。在最大浓度下，激动剂刺激的[（35）S]GTPγS结合速率遵循μ-激动剂刺激[（35）S]GTPγS结合的效力，即[D-Ala（2），N-Me-Phe（4），Gly（5）-ol]-脑啡肽>吗啡>哌替啶>布托啡诺>纳布啡。在μ-或δ-完全激动剂的亚最大浓度下，[（35）S]GTPγS结合速率也降低，使得[（35）S]GTPγS结合速率与结合的[（35）S]GTPγS的程度相关，无论这种结合是由完全激动剂还是部分激动剂刺激的。激动剂也刺激[（35）S]GTPγS解离，表明这种稳定核苷酸的结合是可逆的。比较δ-激动剂[D-Ser（2），Leu（5）]-脑啡肽-Thr和（±）-4-（（α-R*）-α-（（2S*，5R*）-4-烯丙基-2,5-二甲基-1-哌嗪基）-3-羟基苄基）-N，N-二乙苯甲酰胺（一种具有缓慢解离动力学的化合物）表明，测量的G蛋白激活速率不受激动剂在受体之间切换的影响。结果与完全或部分激动剂诱导的受体活性状态相同，但激活受体的数量决定G蛋白激活速率的观点一致。
