Albrecht E, Samovilova N N, Oswald S, Baeger I, Berger H
Department of Peptide Pharmacology, Research Institute of Molecular Pharmacology, Berlin, Germany.
J Pharmacol Exp Ther. 1998 Aug;286(2):896-902.
G protein activation by the agonist-occupied nociceptin- (orphanin FQ-) receptor in rat cerebral cortex was studied by characterizing the nociceptin-stimulated binding of the radiolabeled guanylyl triphosphate (GTP) analog 35S-guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS). Using 3H-Tyr14- and 125I-Tyr14-nociceptin in saturation and displacement receptor binding studies, a single high-affinity (Kd 21.6-116.7 pM) and high-capacity binding site for nociceptin (orphanin FQ) in membranes and sections of rat cerebral cortex was identified. Stable GTP analogs and NaCl lowered the affinity only moderately by 2- to 3-fold, but under these conditions nociceptin stimulated the binding of 35S-GTPgammaS to G proteins in the membranes with a potency about 100-fold lower (EC50 9.11 nM). It was estimated that this stimulation was due to a 29-fold increase in the affinity from Kd 45. 8 to 1.57 nM of only about 6.5% of the basal binding sites for GTPgammaS, and that at least 10 G protein binding sites could be stimulated by one receptor site. The link of this nociceptin-stimulated binding of GTP to the nociceptin receptor was further evidenced by the specificity of stimulation, as seen with nociceptin, nociceptin(1-13), D-Ala7-nociceptin and nociceptin(1-9), which paralleled that of their receptor affinities. Furthermore, the distribution in rat brain regions of the binding of 35S-GTPgammaS stimulated by nociceptin differed from that stimulated by the mu opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol)]-enkephalin. Especially, no stimulation by nociceptin was observed in caudate putamen, where also the absence of ORL1 receptors had been reported. The putative coupling of the high-affinity nociceptin receptor to the low-potency stimulation of GTPgammaS binding in rat cerebral cortex might be explained by the switch of a low part of occupied nociceptin binding sites to a very low-affinity state being stabilized at high peptide concentrations and catalytically stimulating the GTP binding.
通过对放射性标记的鸟苷三磷酸（GTP）类似物35S - 鸟苷 - 5'-O -（γ - 硫代） - 三磷酸（GTPγS）与伤害感受素刺激结合的特性进行研究，探讨了激动剂占据的伤害感受素 - （孤啡肽FQ -）受体在大鼠大脑皮层中对G蛋白的激活作用。在饱和和置换受体结合研究中使用3H - Tyr14 - 和125I - Tyr14 - 伤害感受素，在大鼠大脑皮层的膜和切片中鉴定出一个单一的高亲和力（Kd 21.6 - 116.7 pM）和高容量的伤害感受素（孤啡肽FQ）结合位点。稳定的GTP类似物和NaCl仅适度降低亲和力2至3倍，但在这些条件下，伤害感受素刺激35S - GTPγS与膜中G蛋白的结合，其效力约低100倍（EC50 9.11 nM）。据估计，这种刺激是由于仅约6.5%的GTPγS基础结合位点的亲和力从Kd 45.8 nM增加到1.57 nM，增加了29倍，并且一个受体位点至少可以刺激10个G蛋白结合位点。伤害感受素刺激的GTP结合与伤害感受素受体的联系通过刺激的特异性进一步得到证明，如伤害感受素、伤害感受素（1 - 13）、D - Ala7 - 伤害感受素和伤害感受素（1 - 9）所见，其与它们的受体亲和力平行。此外，伤害感受素刺激的35S - GTPγS结合在大鼠脑区的分布与μ阿片样激动剂[D - Ala2，N - Me - Phe4，Gly5 - ol)] - 脑啡肽刺激的分布不同。特别是，在尾状壳核中未观察到伤害感受素的刺激，在该区域也已报道不存在ORL1受体。在大鼠大脑皮层中，高亲和力的伤害感受素受体与GTPγS结合的低效刺激之间的假定偶联可能是由于一部分占据的伤害感受素结合位点转变为极低亲和力状态，在高肽浓度下稳定并催化刺激GTP结合来解释。
