Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells.

AIM: The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr(10)]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of (125)I-[Tyr(14)]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr(10)]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr(10)]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP-MOP receptors. We also examined if [Tyr(10)]N/OFQ(1-11) distinguished heteromeric NOP-MOP receptors from homomeric NOP receptors. MAIN METHODS: The NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors. KEY FINDINGS: In vlPAG neurons, [Tyr(10)]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC(50): 8.98µM) but equi-efficacious as N/OFQ. [Tyr(10)]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr(10)]N/OFQ(1-11) displayed similar concentration-response curves in decreasing cAMP accumulation. SIGNIFICANCE: [Tyr(10)]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP-MOP and homomeric NOP receptors.

Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)-induced disinhibition.

Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1'-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGLα, but not DAGLß, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.

Quantitative study of the antagonistic effect of (-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) on nociceptin/orphanin FQ-mediated potassium channel activation in rat periaqueductal gray slices.

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, a non-opioid branch of the opioid receptor family, shows structural similarities to traditional opioid receptors but binds opioid with very poor affinity. This receptor has been implicated in many physiological functions including pain regulation. This study quantitatively investigated the effect of (-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111), a novel non-peptide ligand of NOP receptor, on the native NOP receptors in the midbrain ventrolateral periaqueductal gray (vlPAG), a crucial region for pain regulation. SB-612111 concentration-dependently antagonized N/OFQ-induced G-protein coupled inwardly rectifying K(+) (GIRK) current in vlPAG neurons. The IC(50) value of SB-612111 estimated from dose-response curves is 87.7±1.2nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, a mu-opioid receptor agonist, when tested at concentrations of up to 1µM. It is concluded that SB-612111 is a pure, potent and selective antagonist of NOP receptors that mediate GIRK channel activation in the vlPAG neurons.

Functional heterogeneity of nociceptin/orphanin FQ receptors revealed by (+)-5a Compound and Ro 64-6198 in rat periaqueductal grey slices.

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a non-opioid branch of the opioid receptor family 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 grey (vlPAG). In this study, we found that a new NOP receptor ligand, (+)-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 in vlPAG neurons. (+)-5a Compound (0.1-30 µm) concentration-dependently activated G-protein-coupled inwardly-rectifying potassium (GIRK) channels mediated 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 activated 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. It is suggested that (+)-5a Compound activates a subset of NOP receptors, similar to the Ro 64-6198-sensitive subset, in the vlPAG neurons which are mostly GABAergic. These results further support the presence of functional heterogeneity of NOP receptors in the midbrain PAG.

Orexins/hypocretins: pain regulation and cellular actions.

Orexin A and B (also named hypocretin 1 and 2) are 33 and 28 amino acid-containing neuropeptides, respectively, derived from prepro-orexin (prepro-hypocretin) which is localized in the the lateral and perifonical areas of the hypothalamus. Two G-protein coupled receptor subtypes, OX1 and OX2, were identified. Orexin-containing fibers and OX receptors are widely distributed in the central nervous system. Orexins have been implicated in the arousal, rewarding, energy homeostasis, autonomic central control and antinociceptive systems. Subtype-selective peptide agonists and antagonists and non-peptide antagonists, but not non-peptide agonists, have been developed. This review summarizes the studies investigating the antinociceptive effects of orexins in various animal models of pain, including trigeminovascular pain, and their cellular mechanisms. Orexins are antinociceptive at both spinal and supraspinal levels. The antinociceptive effect of orexin A is comparable to opioids but orexin B is less or not effective. This effect is opioid-independent and mainly mediated through OX1 receptors. Some animal studies suggest that endogenous orexins may be released during postoperative and inflammatory, but not acute, pain states, or during some stress conditions, which may contribute to stress-induced analgesia. Purinergic P(2X) and glycine receptors are proposed to be involved in orexin-induced spinal antinociception. The supraspinal sites of actions might involve the posterior hypothalamus, which contributes to the trigeminovascular nociception, and the ventrolateral periaqueductal gray, which mediates descending pain inhibition. Endocannobinoids and nociceptin/orphanin FQ were found to interplay with orexins in nocicpetive processing. Further studies are required to elucidate the receptor subtype-specific mechanism(s) and clinical implications of orexin-induced antinociception.

1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) antagonizes NOP receptor-mediated potassium channel activation in rat periaqueductal gray slices.

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, the fourth member of opioid receptor family, shares 60-70% homology with traditional opioid receptors but displays little affinity for opioids. This receptor was implicated in many neurological functions and its functional heterogeneity has been proposed. Therefore, it is imperative to develop and characterize new ligands for NOP receptors. 1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) is a new non-peptide ligand of NOP receptor having antagonistic actions in cloned and peripheral NOP receptors. In this study, we quantitatively characterized its effect on the native NOP receptors in the midbrain slices containing ventrolateral periaqueductal gray (vlPAG), a region with dense NOP receptors and involved in pain regulation. In vlPAG neurons, N/OFQ induced G-protein-coupled inwardly rectifying potassium (GIRK) current through NOP receptors. Compound 24, at 0.3-10 microM, attenuated N/OFQ-induced GIRK current concentration-dependently. The antagonistic potency of Compound 24 in vlPAG neurons (IC(50): 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 [Nphe(1), Arg(14), Lys(15)]N/OFQ-NH(2) (UFP-101), a peptide antagonist, in the same preparation. Compound 24 had no intrinsic agonistic activity at NOP receptors at the concentration up to 10 microM. However, at concentrations higher than 3 microM, it also attenuated the GIRK current induced by [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, a mu-opioid receptor agonist. It is concluded that Compound 24 acts as a pure antagonist at the native NOP receptors in the vlPAG with moderate potency and selectivity.

Quantitative study of [(pF)Phe4,Arg14,Lys15]nociceptin/orphanin FQ-NH2 (UFP-102) at NOP receptors in rat periaqueductal gray slices.

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a novel member of the opioid receptor family with little affinity for traditional opioids. This receptor and its endogenous ligand, N/OFQ, are widely distributed in the brain and are implicated in many physiological functions including pain regulation. [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102) is a newly developed peptide agonist of NOP receptors. In this study, we quantitatively investigated the effect of UFP-102 at native NOP receptors of the ventrolateral periaqueductal gray (PAG), a crucial midbrain area involved in pain regulation and enriched with NOP receptors, using blind patch-clamp whole-cell recording technique in rat brain slices. UFP-102, like N/OFQ, induced an outward current in ventrolateral PAG neurons and increased the membrane current elicited by a hyperpolarization ramp from -60 to -140 mV. The current induced by UFP-102 was characterized with inward rectification and had a reversal potential near the equilibrium potential of K(+) ions, indicating that UFP-102 activates G-protein coupled inwardly rectifying K(+) channels. The effect of UFP-102 was concentration-dependent with the maximal effect similar to that of N/OFQ. The EC(50) value was 11+/-2 nM, which is 5 fold lower than that of N/OFQ. The effect of UFP-102 was not affected by naloxone while competitively antagonized by UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2)), a potent NOP receptor antagonist, with a pA(2) value of 6.7. These results suggest that UFP-102 is a full agonist at the postsynaptic NOP receptors of the midbrain of rats and is 5 fold more potent than N/OFQ.

[Nphe1,Arg14,Lys15]N/OFQ-NH2 is a competitive antagonist of NOP receptors in the periaqueductal gray.

Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptors are implicated in many physiological functions including pain regulation. This study quantitatively investigated the interaction of a novel NOP receptor antagonist, UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ-NH2), with N/OFQ in the ventrolateral periaqueductal gray, a crucial midbrain area for pain regulation. N/OFQ concentration-dependently activated G-protein coupled inwardly rectifying K+ (GIRK) channels in ventrolateral neurons of periaqueductal gray slices. UFP-101 antagonized N/OFQ-induced GIRK channel activation in a concentration-dependent manner and produced a parallel shift of the concentration-response curve of N/OFQ. The pA2 value estimated from Schild plot is 6.92+/-0.06. At concentrations up to 1 microM, UFP-101 had no effect on membrane current per se and did not affect the GIRK current activated by [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin, a mu-opioid receptor agonist. It is concluded that UFP-101 is a potent and competitive peptide antagonist of NOP receptors that mediate GIRK channel activation in ventrolateral periaqueductal gray neurons.

Does gabapentin act as an agonist at native GABA(B) receptors?

Gabapentin, a novel anticonvulsant and analgesic, is a gamma-aminobutyric acid (GABA) analogue but was shown initially to have little affinity at GABA(A) or GABA(B) receptors. It was recently reported to be a selective agonist at GABA(B) receptors containing GABA(B1a)-GABA(B2) heterodimers, although several subsequent studies disproved that conclusion. In the present study, we examined whether gabapentin is an agonist at native GABA(B) receptors using a rat model of postoperative pain in vivo and periaqueductal gray (PAG) slices in vitro; PAG contains GABA(B) receptors, and their activation results in antinociception. An intrathecal injection of gabapentin or baclofen, a GABA(B) receptor agonist, induced antiallodynia in this postoperative pain model. Intrathecal injection of GABA(B) receptor antagonists CGP 35348 and CGP 55845 antagonized baclofen- but not gabapentin-induced antiallodynia. In ventrolateral PAG neurons, baclofen activated G-protein-coupled inwardly rectifying K(+) (GIRK) channels in a manner blocked by CGP 35348 or CGP 55845. However, gabapentin displayed no effect on the membrane current. In neurons unaffected by gabapentin, baclofen activated GIRK channels through GABA(B) receptors. It is concluded that gabapentin is not an agonist at GABA(B) receptors that are functional in baclofen-induced antiallodynia in the postoperative pain model in vivo and in GIRK channel activation in ventrolateral PAG neurons in vitro.