NOCICEPTIN (ORPHANIN FQ) - HIGH-AFFINITY AND HIGH-CAPACITY BINDING-SITE COUPLED TO LOW-POTENCY STIMULATION OF GUANYLYL-5'-O-(GAMMA-THIO)-TRIPHOSPHATE BINDING IN RAT-BRAIN MEMBRANES

G protein activation by the agonist-occupied nociceptin- (orphanin FQ- ) receptor in rat cerebral cortex was studied by characterizing the no ciceptin-stimulated binding of the radiolabeled guanylyl triphosphate (GTP) analog S-35-guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S ). Using H-3-Tyr(14)- and I-125-Tyr(14)-nociceptin in saturation and d isplacement receptor binding studies, a single high-affinity (K-d 21.6 -116.7 pM) and high-capacity binding site for nociceptin (orphanin FQ) in membranes and sections of rat cerebral cortex was identified. Stab le GIP analogs and NaCl lowered the affinity only moderately by 2- to 3-fold, but under these conditions nociceptin stimulated the binding o f S-35-GTP gamma S 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 K-d 45.8 to 1.57 n M of only about 6.5% of the basal binding sites for GTP gamma S, and t hat at least 10 G protein binding sites could be stimulated by one rec eptor site. The link of this nociceptin-stimulated binding of GTP to t he nociceptin receptor was further evidenced by the specificity of sti mulation, as seen with nociceptin, nociceptin(1-13), D-Ala(7)-nocicept in and nociceptin(1-9), which paralleled that of their receptor affini ties. Furthermore, the distribution in rat brain regions of the bindin g of S-35-GTP gamma S stimulated by nociceptin differed from that stim ulated by the mu opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol)]-en kephalin. Especially, no stimulation by nociceptin was observed in cau date putamen, where also the absence of ORL1 receptors had been report ed. The putative coupling of the high-affinity nociceptin receptor to the low-potency stimulation of GTP gamma S binding in rat cerebral cor tex might be explained by the switch of a low part of occupied nocicep tin binding sites to a very low-affinity state being stabilized at hig h peptide concentrations and catalytically stimulating the GTP binding .