A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.
1 This study describes the in vitro characterization of two potent and selective 5-HT 6 receptor antagonists at the rat and human recombinant 5-HT 6 receptor. 2 In binding assays with [ 3 H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pK i values +s.e.mean at the rat 5-HT 6 receptor of 7.35+0.04 and 7.83+0.01, respectively and pK i values at the human 5-HT 6 receptor of 7.26+0.06 and 7.91+0.02, respectively. 3 Both compounds were found to be over 100 fold selective for the 5-HT 6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. 4 In functional studies, neither compound had any signi®cant e ect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT 6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT 6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean +s.e.mean pA 2 values of 6.75+0.07 and 7.10+0.09, respectively. 5 In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT 6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. 6 Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identi®cation of 5-HT 6 receptors in natural tissues and the study of their physiological function.
1 The present study examined the eects of the selective 5-HT 6 receptor antagonist 4-amino-N-(2, 6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) on locomotor activity and unconditioned behaviour in male Sprague Dawley rats (230 ± 300 g). , i.p.) was signi®cantly greater than that observed in saline-treated rats. The yawning behaviour, however, was not dose-dependent nor was the number of yawns in any of the drug treated groups signi®cantly greater than in those treated with saline. 6 These data suggest that systemic injection of the 5-HT 6 antagonist, Ro 04-6790, produces a stretching behaviour that appears to be mediated by an increase in cholinergic neurotransmission in the CNS and which could be a useful functional correlate for 5-HT 6 receptor blockade. There is no evidence for dopamine D 2 -like receptor involvement in this behaviour.
Ro 63-0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [3H]Ro 63-0563, the first selective 5-HT6 receptor radioligand. Specific binding of [3H]Ro 63-0563 (nonspecific binding defined in the presence of 10 microM methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nM and 4.96 nM, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [3H]Ro 63-0563 (methiothepin > D-lysergic acid diethylamide > clozapine approximately Ro 63-0563 > lisuride > ergotamine approximately Ro 04-6790 > 5-HT > amitriptyline approximately metergoline approximately mianserin approximately ritanserin > methysergide > mesulergine) was similar to that obtained by using either [3H]D-lysergic acide diethylamide or [3H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [3H]Ro 63-0563 labeled a single binding site with Kd and Bmax values of 11. 7 nM and 175 fmol/mg protein, respectively. In porcine striatal membranes, [3H]Ro 63-0563 also labeled a single binding site with Kd and Bmax values of 8.0 nM and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6 receptor, which suggested the presence of 5-HT6 receptors in porcine striatum.
1 4-Amino-N-(2,4 bis-methylamino-pyrimidin-4-yl) benzene sulphonamide (Ro 04-6790) is a potent, selective and competitive antagonist for the 5-HT 6 receptor which can be detected in the cerebro-spinal uid (CSF) of rats following intraperitoneal administration. Since 5-HT 6 receptor mRNA and 5-HT 6 receptor-like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the e ect of 5-HT 6 receptor antagonism on haloperidol-and SCH 23390-induced catalepsy in mice and on the turning behaviour of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle. 2 Ro 04-6790 (3, 10 and 30 mg kg 71 i.p.) did not induce catalepsy and had no e ect on catalepsy induced by either haloperidol or SCH 23390. 3 Ro 04-6790 (3, 10 and 30 mg kg 71 i.p.) did not itself induce rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle nor did it a ect the rotational behaviour induced by either L-Dopa or amphetamine. 4 5-HT 6 receptor antagonism inhibited the rotational behaviour of 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine. 5 The data support earlier conclusions from experiments with antisense oligonucleotides that the 5-HT 6 receptor is involved in the control of acetylcholine neurotransmission in the rat brain.
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