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1: This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 2: Specific binding of [3H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with Kd values of 3.76±0.45 and 5.03±0.31?nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0?pmol?mg protein-1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. 3: Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin-A displaying a ~five-fold higher affinity than orexin-B (Ki values of 318±158 and 1516±597?nM, respectively). 4: SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5?nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2?nM, respectively) formats. 5: Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6: These studies indicate that [3H]SB-674042 is a specific, high-affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors.British Journal of Pharmacology (2004) 141, 340-346. doi:10.1038/sj.bjp.0705610 [ABSTRACT FROM AUTHOR]

Abstract Rationale: Orexins A and B have recently been discovered and shown to be derived from preproorexin, primarily expressed in the rat hypothalamus. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and, most recently, evidence for a behavioural response characterised by an increase in grooming. Objectives: Here, we have investigated the orexin-receptor subtypes involved in the grooming response to orexin-A (3 micro g, ICV) in the rat. Methods: Male rats, habituated to clear Perspex behavioural observation boxes, were pretreated with antagonists with mixed selectivity for OX[sub 1], OX[sub 2], 5-HT[sub 2B] and 5-HT[sub 2C] receptor subtypes prior to the administration of orexin-A and the intense grooming response elicited by this peptide assessed. Results: Pretreatment of rats with a mixed OX[sub 1]/5-HT[sub 2B/2C] receptor antagonist 1-(4-methylsulfanylphenyl)-3-quinolin-4-ylurea (SB-284422), revealed a significant, but incomplete, blockade of orexin-A-induced grooming. Despite the low potency of orexin-A at 5-HT[sub 2B] and 5HT[sub 2C] receptors in vitro (pKi<5), studies were undertaken to determine whether downstream 5-HT[sub 2B] or 5-HT[sub 2C] receptors mediate in the grooming-elicited by orexin-A. Whilst the selective 5-HT[sub 2B] receptor antagonist, SB-215505 (3 mg/kg, PO, 5-HT[sub 2B], pKi=8.58; OX[sub 1], pK[sub B]<5.15) failed to effect orexin-A-induced grooming, the selective 5-HT[sub 2C] receptor antagonist, SB-242084 (1 mg/kg, IP, 5-HT[sub 2C], pKi=8.95; OX[sub 1], pK[sub B]<5.1) potently antagonised the grooming response to this peptide. This suggested that the partial blockade of... [ABSTRACT FROM AUTHOR]

Abstract Rationale: Orexin-A and orexin-B are hypothalamic neuropeptides derived from a 130-amino acid precursor, prepro-orexin, and are potent agonists at both the orexin-1 (OX[sub 1]) and orexin-2 (OX[sub 2]) receptors. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and a role in the regulation of sleep-wake function. The in vivo role of orexin-B is not as clear. Objectives: To investigate the behavioural, endocrine and neurochemical effects of orexin-B in in-vivo tests. In a number of experiments, these effects were compared with those of orexin-A. Methods: Experiments were carried out in male, Sprague-Dawley rats with a guide cannula directed towards the lateral ventricle. The effects of orexin-B (ICV) upon grooming behaviour were compared with those of orexin-A. The effects of orexin-B upon the motor activity response to both novel and familiar environments were assessed in an automated activity monitor. Orexin-B was tested upon startle reactivity and body temperature. Further, plasma hormones and [DOPAC+ HVA]/[DA] and [5-HIAA]/[5-HT] ratios in six brain areas were measured 40 min post-orexin-B or orexin-A. Results: The clearest behavioural response to orexin-B was increased motor activity in both novel and familiar environments. Orexin-B-induced hyperactivity was blocked by an OX[sub 1] receptor antagonist, SB-334867-A, implicating OX[sub 1] receptors in this behavioural response. In common with orexin-A, orexin-B reduced plasma prolactin and failed to influence startle reactivity. However,... [ABSTRACT FROM AUTHOR]