3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one] was developed as a nonpeptide agonist of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors, using bioassays at cloned receptors expressed in cell cultures. We have investigated the actions of Ro 64-6198 at native NOP receptors of the ventrolateral periaqueductal gray (PAG), a crucial site for N/OFQ-induced reversal of opioid analgesia, using the patch-clamp recording technique in rat brain slices. Ro 64-6198, like N/OFQ, activated G protein-coupled inwardly rectifying K ϩ channels (GIRK) in ventrolateral PAG neurons but displayed only 60% efficacy and 22% potency of N/OFQ. Unlike N/OFQ that activated GIRK through NOP receptors in almost all tested neurons, Ro 64-6198 affected only 62% (114/185) of the neurons recorded, among which 57% were sensitive to CompB (J-113397), a selective NOP receptor antagonist. The effect of Ro 64-6198 was not affected by naloxone (1 M), sulpiride (10 M), and [1-(2-methoxyphenyl)-4-[4-2-phthalimido)butyl]piperazine (1 M), respectively, the antagonist of opioid, dopamine D 2 , and 5-HT 1A receptors. In Ro 64-6198-unresponsive neurons, N/OFQ activated GIRK through NOP receptors. It is concluded that Ro 64-6198 is a weak agonist of NOP receptors both in terms of potency and efficacy in ventrolateral PAG neurons. Heterogeneity of NOP receptors has been proposed from binding studies and in vivo functional studies. The possibility was discussed that two subsets of NOP receptors exist in ventrolateral PAG neurons, and Ro 64-6198 activates only one subset but N/OFQ activates both of them.
G-protein coupled inwardly rectifying K+ (GIRK) channels have been reported to be targets of ethanol actions. We investigated if ethanol affects native GIRK channels in rat brain tissues at clinically relevant concentrations using brain slices containing the ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Ethanol did not affect the membrane current elicited by hyperpolarization ramps at concentrations up to 150 mM. However, at 200-300 mM, which is above the lethal level, it activated a barium-sensitive GIRK current in 30-57% of neurons. In neurons unresponsive to ethanol, baclofen, the mu-opioid or nociceptin successfully activated GIRK channels. It is suggested that GIRK channels of the ventrolateral PAG are unlikely to be targets of the analgesic action of ethanol.
A fourth opioid receptor family was cloned and named after its endogenous ligand as nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor. We have characterized several NOP receptor ligands pharmacologically at native NOP receptors of ventrolateral periaqueductal gray (vlPAG) neurons by investigating their interactions with N/OFQ in activating G protein-coupled inwardly rectifying K+ (GIRK) channels. They are listed here: (1) [Phe1Psi(CH2-NH)Gly2]N/OFQ(1-13)NH2, which was claimed to be the first selective antagonist of NOP receptors, is a partial agonist of NOP receptors in vlPAG neurons. (2) [Nphe1]N/OFQ(1-13)NH2 is a pure, selective, and competitive peptide antagonist of NOP receptors (pA2 value = 6.6). (3) CompB (J-113397) is a potent and selective nonpeptide antagonist of NOP receptors (pA2 = 8.4). (4) Naloxone benzoylhydrazone is a competitive NOP receptor antagonist but also a noncompetitive mu-opioid receptor antagonist. (5) Ro 64-6198, though being developed as a potent nonpeptide NOP receptor agonist, affected only part of vlPAG neurons and acted as a weak NOP receptor agonist. In Ro 64-6198-unresponsive neurons, N/OFQ activated GIRK channels through NOP receptors. (6) Nocistatin, a functional antagonist of N/OFQ in the spinal cord, did not affect the effect of N/OFQ in most of the recorded neurons. Our functional studies of NOP receptor ligands at native brain NOP receptors reveal that some of them act differently from those at expressed receptors of cell cultures.
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