Orexin A and orexin B are hypothalamic peptides that act on their targets via two G protein-coupled receptors (OX1 and OX2 receptors). In the central nervous system, the cell bodies producing orexins are localized in a narrow region within the lateral hypothalamus and project mainly to regions involved in feeding, sleep, and autonomic functions. Via putative pre- and postsynaptic effects, orexins increase synaptic activity in these regions. In isolated neurons and cells expressing recombinant receptors orexins cause Ca2+ elevation, which is mainly dependent on influx. The activity of orexinergic cells appears to be controlled by feeding- and sleep-related signals via a variety of neurotransmitters/hormones from the brain and other tissues. Orexins and orexin receptors are also found outside the central nervous system, particularly in organs involved in feeding and energy metabolism, e.g., gastrointestinal tract, pancreas, and adrenal gland. In the present review we focus on the physiological properties of the cells that secrete or respond to orexins.

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca 2ϩ responses (influx and release) in human OX 1 and OX 2 receptorexpressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca 2ϩ , suggesting similar activation of Ca 2ϩ influx as we have previously shown for orexin-A and OX 1 receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX 1 and OX 2 receptors, but the response mediated by the OX 2 receptor was more resistant to truncation than the response mediated by the OX 1 receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A 14 -33 . Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX 2 receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca 2ϩ dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX 2 receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10-to 100-fold lower affinity and potency for the OX 1 receptor.Recently, two novel hypothalamic peptides were isolated and subsequently named orexin-A and orexin-B (Sakurai et al., 1998) or hypocretin-1 and hypocretin-2 (de Lecea et al., 1998). Despite some initial confusion, orexin-A should now be considered identical to hypocretin-1 and orexin-B to hypocretin-2. Orexins act as agonists on two G-protein-coupled receptors called OX 1 and OX 2 receptors. Increased wakefulness and reduced sleep is a well demonstrated response to central administration of orexin, and disruption of central orexinergic signaling leads to the sleep disorder narcolepsy in animal models and probably also in man (reviewed in Beuckmann and Yanagisawa, 2002;Kukkonen et al., 2002;Sutcliffe and de Lecea, 2002). The other physiological roles for orexins may be regulation of energy homeostasis and stress response, probably both via central and peripheral mechanisms (reviewed in Willie et al., 2001;Beuckmann and Yanagisawa, 2002;Kirchgessner, 2002;Kukkonen et al., 2002;Smart and Jerman, 2002).The two orexin peptides, orexin-A and -B, are both products of the same precursor peptide, preproorexin, cleavage of which results in equimolar amounts of orexin-A and orexin-B. Orexin-A is composed of 33 amino acids and it contains two disulfide bridges, whereas orexin-B is a linear peptide of 28 residues (Sakurai et al., 1998). Although a product of a different part of the precursor peptide, orexin-B sho...

Activation of OX1 orexin receptors heterologously expressed in Chinese hamster ovary (CHO) cells led to a rapid, strong, and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly, Ca2+ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca2+ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca2+ influx pathway alone or the Ca2+ release was ineffective. If the receptor-operated pathway was blocked, an exogenously activated store-operated pathway could take its place and restore the coupling of OX1 receptors to ERK. Further experiments suggested that Ca2+ influx, as such, may not be required for ERK phosphorylation, but that Ca2+, elevated via influx, acts as a switch enabling OX1 receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation, and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca2+ influx allows them to couple to other signal pathways; in the absence of coupling to Ca2+ influx, orexin receptors can act as signal integrators by taking advantage of other Ca2+ influx pathways.

In this study, the mechanism of OX 1 orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC 50 Ϸ 1 nM) inhibition and a strong (4-fold), low potency (EC 50 ‫؍‬ Ϸ300 nM) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G i/o proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC 50 ‫؍‬ Ϸ500 nM). cAMP elevation was not caused by Ca 2؉ elevation or by G␤␥. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKC␦, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G s activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC 50 of orexin-A ‫؍‬ Ϸ10 nM and EC 50 of orexin-B ‫؍‬ Ϸ100 nM), and the response was fully dependent on PKC␦. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-G␣ s antibodies. We conclude that OX 1 receptors stimulate adenylyl cyclase via a low potency G s coupling and a high potency phospholipase C 3 PKC coupling. The former or some exogenous G s activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX 1 receptors couple to G s almost as efficiently as the orexin-A-activated receptors, in contrast to Ca 2؉ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.The neuropeptides/hormones orexin-A and -B and the corresponding G-protein-coupled receptors OX 1 and OX 2 receptor were discovered in 1998 (1, 2). Orexin-A (33 amino acids) and orexin-B (28 amino acids) share the property of being able to activate both orexin receptors. Orexins are signal substances both in the central nervous system and in the periphery. In the central nervous system, all of the orexinergic neurons have their origin in the lateral hypothalamus from where they project widely to regulate especially wakefulness and paradoxical sleep, appetite and food intake, and endocrine and autonomic processes. At most of the projection sites both OX 1 and OX 2 receptors are expressed. The orexins most often act in an excitatory manner both via putative pre-, post-, and extrasynaptic mechanisms. In the periphery, orexins and orexin receptors have been have been found in the gastrointestinal tract and in the endocrine organs. The prominent periferal effects seen so far include regulation of gastrointestinal motility and hormone production and release, especially in the adrenal gland (reviewed in Ref. 3).Based on measurements of binding affinity and the ability to elevate intracellular Ca 2ϩ and liberate inositol phosphates in heterologous expression systems, the OX 1 receptor shows a 10-fold preference for orexin-A over orexin-B in contrast to the OX 2 recep...

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