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

在1994年，一種新的鴉片受體被發現，因其結構相似於傳統鴉片受體，但卻對傳統鴉片受體致效劑缺乏親和力，同時也未發現其內生性致效劑，所以一開始被稱為類鴉片孤兒受體(opioid receptor like 1, ORL1)。它的內生性致效劑在一年後被發現並命名為nociceptin或orphanin FQ，在2002年國際藥理學聯合會以其內生性致效劑之名正式將此受體命名為NOP受體(nociceptin/orphanin FQ peptide receptor)。研究顯示此N/OFQ-NOP受體系統與許多神經及精神疾病有關，如疼痛、焦慮、憂鬱、非自主運動、成癮、發作及痴呆等。 從NOP受體基因剪接變異體(splicing variants)被發現及一些受體結合和功能性研究顯示有NOP受體異質性(heterogeneity)的存在。我們之前發現Ro 64-6198這個NOP受體致效劑在中腦環導水管灰質腹側區(ventrolateral PAG)只能活化一部份的NOP受體，而在本研究中，我們發現另一個新的非胜肽NOP受體結合劑(+)-5a Compound ((3aS,6aR)-1-(cis-4-Isopropylcyclohexyl)-5'-methyl-2'–phenylhexahydro spiro[piperidine-4,1'-pyrrolo[3,4-c]pyrrole])在相同的標本也只能活化一部份NOP受體，這些可被(+)-5a Compound活化的NOP受體與Ro 64-6198所活化的NOP受體是屬於同一群，(+)-5a Compound (0.1-30 microM)能濃度相依的透過NOP受體活化35%所記錄神經細胞的G蛋白偶合向內整流鉀離子通道(GIRK channels)，其IC50為605 nM，效價(potency)及所能引發反應的最大效力(efficacy)分別為N/OFQ的1/12倍及47%，對(+)-5a Compound-insensitive的細胞，給予Ro 64-6198同樣也無法引發反應，但在這些細胞中N/OFQ則可有效的活化G蛋白偶合向內整流鉀離子通道，另外在(+)-5a Compound-sensitive的細胞，給予Ro 64-6198則反應程度並無差異，從免疫螢光染色的實驗顯示(+)-5a Compound-sensitive的細胞主要是GABAergic，此外，(+)-5a Compound-sensitive的細胞在型態上，其神經分支比(+)-5a Compound-insensitive的細胞來得複雜。 N/OFQ(1-11)是具藥理活性的N/OFQ代謝產物，從放射線標定的研究中，125I-[Tyr10]N/OFQ(1-11)及 125I-[Tyr14]N/OFQ這兩個NOP受體的放射線結合劑在鼠腦中有不同的分布，其中125I-[Tyr10]N/OFQ(1-11)的結合位置可能是125I-[Tyr14]N/OFQ所顯現出兩種不同親和力結合處中的高親和力結合處，基於這個發現，我們於是做了在環導水管灰質區對Ro 64-6198/(+)-5a Compound有反應的NOP受體就是125I-[Tyr10]N/OFQ(1-11)這個結合位置的假設，因此，我們藉由測試[Tyr10]N/OFQ(1-11)能否區別(+)-5a Compound-sensitive及-insensitive細胞來驗證這個假設。在紀錄的環導水管灰質腹側區細胞中，[Tyr10]N/OFQ(1-11)在濃度3-300 microM時能引發向內整流性鉀離子電流，其EC50為9.0 microM，比N/OFQ弱了173倍，而效力(efficacy)則與N/OFQ相似，[Tyr10]N/OFQ(1-11)顯露了與(+)-5a Compound不同的藥理特性，它不管在(+)-5a Compound-sensitive或-insensitive細胞中都有作用，這些結果顯示 [Tyr10]N/OFQ(1-11)是一個效力比N/OFQ差的NOP受體全效致效劑，而且在環導水管灰質腹側區細胞中，[Tyr10]N/OFQ(1-11)無法顯露出NOP受體功能異質性。 為了闡明NOP受體多樣性的可能，發展及鑑定新的NOP受體拮抗劑是很重要的。因此在本研究中，我們也在中腦環導水管灰質腹側區細胞中測試Compound 24 (1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4’-piperidin-1-yl]propyl]pyrrolidine-2 -carboxamide)及SB-612111 ((-)-cis-1- Methyl-7-[[4-(2,6- dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol)這兩個新發展的NOP受體拮抗劑是否具有能顯露出NOP受體功能異質性的特性，然而在我們所紀錄到可被N/OFQ所活化的全部細胞，N/OFQ的作用都會被Compound 24及SB-612111所拮抗，因此這兩種NOP受體拮抗劑並無法顯露NOP受體功能異質性。Compound 24 (0.3-10 microM)能濃度相依的抑制N/OFQ所誘導出的向內整流鉀離子電流，其IC50為2.6 microM，Compound 24在環導水管灰質腹側區神經細胞的抑制效價比在小鼠輸精管標本及表現人類NOP受體的培養細胞所得到的效價還差，而且在相同的環導水管灰質區標本，Compound 24的作用比另一個胜肽類NOP受體拮抗劑UFP-101還要慢，當Compound 24濃度高達10 microM時對NOP受體並不具有內在致效劑的活性，不過只要Compound 24的濃度高於3 microM時也會抑制mu鴉片受體致效劑DAMGO(D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin)所誘導出的向內整流鉀離子電流。而SB-612111也能濃度相依的拮抗N/OFQ所引發的向內整流鉀離子電流，從濃度反應曲線估出其IC50值為87.7 nM，而且SB-612111就算濃度高到1 microM並不會產生內在致效劑活性，也不會影響mu鴉片受體致效劑DAMGO所引發的向內整流鉀離子電流。 總結，在環導水管灰質腹側區(+)-5a Compound與Ro 64-6198一樣都只活化一部份的NOP受體，而這些對(+)-5a Compound有反應的神經細胞大多屬於GABAergic，而且對(+)-5a Compound有反應及無反應的細胞，兩者在細胞型態上並不相同，這些結果進一步支持在中腦環導水管灰質區的NOP受體有功能異質性的存在。不過[Tyr10]N/OFQ(1-11)在藥理特性上與(+)-5a Compound並不相同，而且利用[Tyr10]N/OFQ(1-11)以及Compound 24與SB-612111這兩個新的NOP受體拮抗劑也無法觀察到NOP受體功能異質性的現象。此外，在Compound 24與SB-612111的定量性研究上，證明Compound 24是一個效價普通且選擇性亦非理想的NOP受體拮抗劑，另一方面，SB-612111則是一個有效且具選擇性，同時又具非胜肽類優勢的NOP受體拮抗劑，它可說是能用來探究內生性N/OFQ生理角色最好的NOP受體拮抗劑。

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.