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...

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These data provide further insight into the distribution of GPR14 mRNA within the CNS and show for the first time that hU-II causes marked behavioural and endocrine effects.

1 The influence of the vascular endothelium on agonist-induced contractions and relaxations has been measured using intact segments of rat aorta. Contiguous rubbed segments were used as controls. 2 Angiotensin II, histamine, noradrenaline, U46619 and UK14304 contracted both rubbed and intact tissues. The threshold spasmogenic concentrations of these agonists were lower in rubbed tissues than in intact preparations. 3 The sensitivity and responsiveness of tissues to angiotensin II, histamine, noradrenaline and UK14304 were greater in rubbed than in intact tissues. 4 Acetylcholine and histamine relaxed the established spasms ofintact tissues but not those of rubbed preparations, These relaxant effects of acetylcholine were abolished by pre-incubation with haemoglobin. 5 In the presence of prazosin, noradrenaline or UK 14304 relaxed established contractions in intact tissues. These effects were antagonized by idazoxan or by pre-incubation with haemoglobin. 6 In intact preparations, idazoxan had no effect on the spasmogenic sensitivity and responsiveness to UK14304. 7 Pre-incubation with haemoglobin augmented the spasmogenic actions of noradrenaline, U46619 or UK 14304 in intact tissues, but had no effect on these responses in rubbed preparations. 8 Tissue concentrations of cyclic GMP were greater in intact than in rubbed tissues. A concentration of acetylcholine (10 tiM) evoking just maximal mechanical inhibition produced a significant increase in cyclic GMP concentration in intact preparations. However, no detectable changes in cyclic GMP concentration were produced by UK 14304 (10 !M) or by acetylcholine (30 nM), concentrations which were equi-effective in inhibiting mechanical activity. 9 In the presence of threshold spasmogenic concentrations of noradrenaline, the contractile effects of angiotensin II were augmented and became comparable to those observed in rubbed preparations. In the presence of greater concentrations of noradrenaline, angiotensin II always produced an additional contraction. 10 It is concluded that the presence of the vascular endothelium limits the spasmogenic action of a variety ofagonists. Although spasmogens like noradrenaline and UK 14304 can stimulate the release of endothelium-derived relaxing factor (EDRF) via M2-adrenoceptors, the inhibitory effects of EDRF largely result from the spontaneous release of this substance.

1 The mechanisms involved in the mechano-inhibitory effects of acetylcholine (ACh) have been compared with those of sodium nitroprusside (SNP) and cromakalim on the rat isolated thoracic aorta.2 Relaxations produced by ACh were endothelium-dependent, whereas those produced by SNP or cromakalim were endothelium-independent. 3 ACh, cromakalim and SNP relaxed established contractions produced by noradrenaline (NA) and KCl (20 mM) and these relaxations were well-maintained. 4 SNP was a relatively effective inhibitor of contractions produced by KCl (80mM). ACh was relatively ineffective and cromakalim was without effect against such contractions. 5 Membrane potential and cyclic GMP concentrations were higher in tissues with an intact endothelium whereas rubbed tissues had a higher 86Rb efflux rate coefficient. 6 ACh and cromakalim produced a transient and long-lasting hyperpolarization, respectively. These changes were accompanied by increases in the 86Rb efflux rate coefficient with a time course comparable to that of the electrical changes. 7 Tissue cyclic GMP concentrations were significantly increased in the presence of ACh or SNP, whereas cromakalim had no effect. 8 Transmission electron microscopy showed the presence of endothelial cells on intact tissues. On rubbed preparations, such cells were absent and some damage to the underlying smooth muscle cells was detected. 9 It is concluded that at least two inhibitory substances are released from the endothelial cells by ACh. One of these increases tissue cyclic GMP concentrations and produces an electrically-silent relaxation. The other produces a transient hyperpolarization associated with the opening of 86Rb-permeable K-channels. This event may serve to initiate relaxation processes and to close any open voltage-dependent Ca-channels.