The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.Since the discovery of the orexins (1) investigations of their functions have been guided by evidence for their hypothalamic distribution (1, 2), focusing on feeding, energy homeostasis (1, 3), and neurocrine functions (3). Our studies now show the presence of orexin A immunoreactive fibers and varicosities in extrahypothalamic areas, particularly the locus coeruleus, and demonstrate that the functions of orexin A extend beyond the hypothalamus.Orexin A and B are derived from a 130-aa precursor, prepro-orexin, which is encoded by a gene localized to human chromosome 17q21 (1). Prepro-orexin, or preprohypocretin (2), was identified in the rat hypothalamus by directional tag PCR subtractive hybridization (2) and has been shown by Northern blot analysis to be abundant in the brain and detectable at low levels in testes but not in a variety of other tissues (1, 2). Hypocretins had been identified as hypothalamic neuropeptides, but their biological role was not described (2). Nucleotide sequence alignment shows that hypocretins 1 and 2 have sequence in common with orexins A and B, respectively, but additional amino acids are present in both hypocretins. In situ hybridization maps confirm dense prepro-orexin mRNA expression in the hypothalamus (1, 2). Immunocytochemical mapping of orexin A has identified a population of mediumsized neurones within the hypothalamus, median eminence (3), and ventral thalamic nuclei of rat brain (1, 3). This distribution has been confirmed in human tissue (4).Orexin A binds with high affinity to the novel G proteincoupled receptors orexin 1 (OX 1 ) (IC 50 20 nM) and orexin 2 (OX 2 ) (IC 50 38 nM). Calcium mobilization assays in transfected HEK293 cells confirm that orexin A is a potent agonist at both OX 1 (EC 50 30 nM) and OX 2 (EC 50 34 nM) (1). Emerging evidence suggests the existence of an extensive extrahypothalamic projection of orexin-immunoreactive neurones. Peyron et al. (5), in addition to confirming the presence of immunoreactive cell somata within the hypothalamus, reported immunolabeled fibers throughout extrahypothalamic regions, including septal nuclei, substantia nigra, and raphe nucle...
The dispositions of 50 marketed central nervous system (CNS) drugs into the brain have been examined in terms of their rat in situ (P) and in vitro apparent membrane permeability (P app ) alongside lipophilicity and free fraction in rat brain tissue. The inter-relationship between these parameters highlights that both permeability and brain tissue binding influence the uptake of drugs into the CNS. Hydrophilic compounds characterized by low brain tissue binding display a strong correlation (R 2 ϭ 0.82) between P and P app , whereas the uptake of more lipophilic compounds seems to be influenced by both P app and brain free fraction. A nonlinear relationship is observed between logP oct and P over the 6 orders of magnitude range in lipophilicity studied. These findings corroborate recent reports in the literature that brain penetration is a function of both rate and extent of drug uptake into the CNS.The development of new drugs targeting the central nervous system (CNS) is the fastest growing franchise within the pharmaceutical sector, although this growth has been tempered by relatively poor success of novel candidates (Alavijeh et al., 2005). One of the significant challenges in treating CNS conditions is drug passage across the blood-brain barrier (BBB), a layer of endothelial cells connected with tight junctions that express numerous drug-metabolizing enzymes and efflux transporters (Pardridge, 1997;Tamai and Tsuji, 2000). Therefore, investigation of drug properties that are favorable for CNS delivery can greatly improve efforts in drug discovery.A number of methods are available to determine the rate of uptake of drugs from blood into brain parenchyma (Begley, 1999). In the pharmaceutical industry, CNS penetration is usually assessed in rodents following either intravenous or oral dosing to determine the brain-to-blood concentration ratio. This takes into account not only BBB penetration but also binding, metabolism, and clearance. However, there can be marked species differences in the influence of these parameters on overall BBB penetration; hence, there is significant value in removing some of this complexity and assessing brain penetration at the level of the BBB in situ. Considering that the BBB is conserved across species (Cserr and Bundgaard, 1984), this may represent a more meaningful indicator of the intrinsic ability of the compound to cross the BBB in humans. Furthermore, in situ techniques offer an ideal validation tool for assessing in vitro BBB models, and they also provide further insight into the molecular descriptors that are crucial for BBB penetration.Brain perfusion has been used in neurochemical research for more than 50 years. Early methods focused on long-term perfusion of isolated brain and required extensive surgical Article, publication date, and citation information can be found at
The pharmacology of various peptide and non-peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX 1 ) or orexin-2 (OX 2 ) receptors by measuring intracellular calcium ([Ca 2+ ] i ) using Fluo-3AM. Orexin-A and orexin-B increased [Ca 2+ ] i in CHO-OX 1 (pEC 50 =8.38+0.04 and 7.26+0.05 respectively, n=12) and CHO-OX 2 (pEC 50 =8.20+0.03 and 8.26+0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM ± 10 mM) displayed neither agonist nor antagonist properties in either cell-line. SB-334867-A (1-(2-Methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses (pK B =7.27+0.04 and 7.23+0.03 respectively, n=8), but had no eect on the UTP (3 mM)-induced calcium response in CHO-OX 1 cells. SB-334867-A (10 mM) also inhibited OX 2 mediated calcium responses (32.7+1.9% versus orexin-A). SB-334867-A was devoid of agonist properties in either cell-line. In conclusion, SB-334867-A is a non-peptide OX 1 selective receptor antagonist.
The hypothalamic peptide orexin-A and the orexin-1 receptor are localized in areas of the brain and spinal cord associated with nociceptive processing. In the present study, localization was confirmed in the spinal cord and demonstrated in the dorsal root ganglion for both orexin-A and the orexin-1 receptor. The link with nociception was extended when orexin-A was shown to be analgesic when given i.v. but not s.c. in mouse and rat models of nociception and hyperalgesia. The efficacy of orexin-A was similar to that of morphine in the 50 degrees C hotplate test and the carrageenan-induced thermal hyperalgesia test. However, involvement of the opiate system in these effects was ruled out as they were blocked by the orexin-1 receptor antagonist SB-334867 but not naloxone. Orexin-1 receptor antagonists had no effect in acute nociceptive tests but under particular inflammatory conditions were pro-hyperalgesic, suggesting a tonic inhibitory orexin drive in these circumstances. These data demonstrate that the orexinergic system has a potential role in the modulation of nociceptive transmission.
Intracerebroventricular (i.c.v.) administration of the novel hypothalamic neuropeptide orexin-A stimulates food intake in rats, and delays the onset of behavioural satiety (i.e. the natural transition from feeding to resting). Furthermore, preliminary findings with the selective orexin-1 receptor antagonist, SB-334867, suggest that orexin-A regulation of food intake is mediated via the orexin-1 receptor. At present, however, little is known about either the intrinsic effects of SB-334867 on the normal structure of feeding behaviour, or its effects upon orexin-A-induced behavioural change. In the present study, we have employed a continuous monitoring technique to characterize the effects of SB-334867 (3-30 mg/kg, i.p.) on the microstructure of rat behaviour during a 1-h test with palatable wet mash. Administered alone, SB-334867 (30 mg/kg, but not lower doses) significantly reduced food intake and most active behaviours (eating, grooming, sniffing, locomotion and rearing), while increasing resting. Although suggestive of a behaviourally nonselective (i.e. sedative) action, the structure of feeding behaviour was well-preserved at this dose level, with the reduction in behavioural output clearly attributable to an earlier onset of behavioural satiety. As previously reported, orexin-A (10 microg per rat i.c.v.) stimulated food intake, increased grooming and delayed the onset of behavioural satiety. Pretreatment with SB-334867 dose-dependently blocked these effects of orexin-A, with significant antagonism evident at dose levels (3-10 mg/kg) below those required to produce intrinsic behavioural effects under present test conditions. Together, these findings strongly support the view that orexin-A is involved in the regulation of feeding patterns and that this influence is mediated through the orexin-1 receptor.
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