以藥理學方法在大鼠中腦環導水管灰質腦切片研究Nociceptin/Orphanin FQ受體之功能異質性

Abstract

In 1994, a novel opioid receptor family, opioid receptor like 1 (ORL1) orphan receptors, was identified to be structurally similar to classical opioid receptors, but insensitive to traditional opioids. Its endogenous ligand was later identified to be a heptadecapeptide and termed nociceptin or orphanin FQ. This receptor was consensusly renamed after its endogenous ligand as nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor in IUPHAR2002. The N/OFQ-NOP receptor system was implicated in several neurological and psychological disorders, such as pain, anxiety, depression, involuntary movement, addiction, seizure and dementia. Heterogeneity of NOP receptors has been proposed based on the findings of splicing variants and from binding and functional studies. We have previously reported that Ro 64-6198, a NOP receptor agonist, activated a subset, but not all, of N/OFQ-sensitive NOP receptors in midbrain ventrolateral periaqueductal gray (vlPAG). In this study, we found that a new non-peptide ligand of NOP receptors, (+)-5a Compound ((3aS,6aR)-1-(cis-4-Isopropylcyclohexyl)-5'-methyl-2'-phenylhexahydrospiro[piperidine -4,1'-pyrrolo[3,4-c]pyrrole]), also activated a subset of NOP receptors as the same subset affected by Ro 64-6198 in vlPAG neurons. (+)-5a Compound (0.1-30 microM) concentration-dependently activated G-protein-coupled inwardly rectifying potassium (GIRK) channels through the NOP receptors in about 35% of the recorded vlPAG neurons. (+)-5a Compound (EC50: 605 nM) was less potent (1/12) and efficacious (47%) than N/OFQ. In (+)-5a Compound-insensitive neurons, Ro 64-6198 was also ineffective, and vice versa, but N/OFQ was effective in activating GIRK channels through NOP receptors. In (+)-5a Compound-sensitive neurons, (+)-5a Compound precluded the effect of Ro 64-6198. Immunofluorecent and morphometric studies showed that most of the (+)-5a Compound-sensitive neurons were multipolar with intensive dendritic arborization and immunoreactive to glutamic acid decarboxylase-67. N/OFQ(1-11) is a pharmacologically active metabolite of N/OFQ. The distribution of its radioligand, 125I-[Tyr10]N/OFQ(1-11), resembled the high affinity, but not all, binding sites of 125I-[Tyr14]N/OFQ in rodent brains. Based on this finding we hypothesize that the Ro 64-6198/(+)-5a Compound-sensitive NOP receptor in the PAG is the binding site of 125I-[Tyr10]N/OFQ(1-11). Here, we validated this hypothesis by examining if [Tyr10]N/OFQ(1-11) can differentiate (+)-5a Compound-sensitive and -insensitive vlPAG neurons. [Tyr10]N/OFQ(1-11), like N/OFQ, induced GIRK current through NOP receptors in the vlPAG neurons. It was 173 times less potent (EC50: 9.0 microM) but equi-efficacious, as compared with N/OFQ. [Tyr10]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound. It was effective in both (+)-5a Compound-sensitive and -insensitive neurons. These results suggest that [Tyr10]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. The functional heterogeneity of NOP receptors, therefore, can not be revealed by [Tyr10]N/OFQ(1-11) in vlPAG neurons. To clarify the possible diversity of NOP receptors, it is important to develop and characterize novel NOP receptor antagonists. In this study, we also examined if two newly developed NOP receptor antagonists, 1-Benzyl-N-[3-[spiroisobenzofuran -1(3H),4’-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) and (-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111), can reveal the heterogeneity of NOP receptors in vlPAG slices. Both Compound 24 and SB-612111 antagonized the effect of N/OFQ in all the recorded neurons. Therefore, the heterogeneity of NOP receptors can not be revealed by these two antagonists. However, we further quantitatively characterized their interactions with N/OFQ in vlPAG slices. Compound 24, at 0.3-10 microM, attenuated N/OFQ-induced GIRK current concentration-dependently. The antagonistic potency of Compound 24 in vlPAG neurons (IC50: 2.6 + 0.6 microM) was, however, lower than that obtained in mouse vas deferens preparations or expressed human NOP receptors. The action kinetic of Compound 24 was slower than UFP-101, a peptide antagonist, in the same preparations. Compound 24 had no intrinsic agonistic activity at NOP receptors at concentrations up to 10 microM. However, at concentrations higher than 3 microM, it also attenuated the GIRK current induced by DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin), a mu-opioid receptor agonist. As for SB-612111, it also concentration-dependently antagonized N/OFQ-induced GIRK current in vlPAG neurons. The IC50 value of SB-612111 estimated from concentration-response curve is 87.7+1.2 nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by DAMGO when tested at concentrations of up to 1 microM. In conclusion, (+)-5a Compound activates a subset of NOP receptors, similar to the Ro 64-6198-sensitive subset, in vlPAG neurons which are mostly GABAergic. Moreover, (+)-5a Compound-sensitive or -insensitive neurons are morphologically distinct. These results further support the presence of functional heterogeneity of NOP receptors in the PAG. However, the pharmacological profiles of [Tyr10]N/OFQ(1-11) are unlike that of (+)-5a Compound and the functional heterogeneity of NOP receptors can not be revealed by either [Tyr10]N/OFQ(1-11) or new NOP receptor antagonists, Compound 24 and SB-612111. Quantitative studies of Compound 24 and SB-612111 in the vlPAG showed that Compound 24 acts as a competitive NOP receptor antagonist but its potency and selectivity are moderate. On the other hand, SB-612111 is a pure, potent and selective antagonist of NOP receptors with the merits of non-peptide nature, high potency, and selectivity. SB-612111 is the best NOP receptor antagonist available for exploring the functional roles of endogenous N/OFQ.