Childers S R, Fleming L M, Selley D E, McNutt R W, Chang K J
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Mol Pharmacol. 1993 Oct;44(4):827-34.
BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.
BW373U86是一种强效且高度选择性的δ-阿片受体非肽类激动剂。为了确定其与G蛋白偶联第二信使系统结合的能力，本研究考察了BW373U86对腺苷酸环化酶抑制作用以及对低Km GTP酶活性刺激作用的影响。在大鼠纹状体膜中，BW373U86以GTP依赖的方式抑制基础腺苷酸环化酶活性，最大抑制水平与典型的δ激动剂[D-Ser2,Thr6]亮氨酸脑啡肽（DSLET）相似。然而，BW373U86在抑制腺苷酸环化酶方面的效力比DSLET高约100倍。对10个脑区的抑制活性分析表明，低浓度和高浓度的BW373U86均以与DSLET相似的方式抑制腺苷酸环化酶活性。BW373U86对腺苷酸环化酶的抑制作用具有δ受体选择性，因为δ受体选择性拮抗剂纳曲吲哚比纳洛酮的效力显著更高，而μ受体选择性拮抗剂D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2在阻断BW373U86的抑制作用方面无效。BW373U86还抑制了从NG108-15细胞制备的膜中的腺苷酸环化酶活性，其IC50值比DSLET低5倍。在NG108-15膜中，激动剂刺激的低Km GTP酶的浓度-效应曲线中未观察到这种效力的增加。BW373U86是[3H]二丙诺啡在NG108-15细胞膜δ受体上的竞争性抑制剂。然而，与DSLET不同，BW373U86对[3H]二丙诺啡与NG108-15细胞膜结合的置换不受钠和鸟嘌呤核苷酸的影响。这种结合缺乏GTP效应显然导致该激动剂的解离速率缓慢，因为当膜与该激动剂预孵育时，纳曲吲哚在阻断BW373U86对腺苷酸环化酶的抑制作用方面效力较低。这些结果证明了一个新发现，即完全激动剂与G蛋白偶联受体的结合不受GTP调节，并且它们还展示了受体结合缺乏调节如何影响激动剂的效力。
