Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

Effects of galanin on the secretion and proliferative activity of the immature and regenerating adrenal glands of rats

The effects of galanin and the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15) on the immature and regenerating rat adrenal glands have been investigated in vivo. Adult female rats with adrenal regeneration and their offpring (20-day-old) were given three subcutaneous injections (28, 16, and 4 h before being killed) of 2 nmol/100 g galanin and/or galanin-A, and 0.1 mg/100 g vincristin 3 h before being killed. Plasma corticosterone concentration was measured by radioimmunoassay, and the mitotic index ( per thousand of metaphase-arrested cells) was evaluated. In immature rats, galanin increased plasma corticosterone concentration, without affecting mitotic index; the secretagogue effect was reversed by galanin-A, which alone was ineffective. In rats with regenerating adrenal, galanin-A increased both blood level of corticosterone and mitotic index; galanin was ineffective, but blocked the effects of galanin-A. These findings allowed us to draw the following conclusions: 1) galanin exerts a moderate glucocorticoid secretagogue action on immature rat adrenals, but endogenous galanin does not play a major physiological role in the functional control of the gland; and 2) endogenous galanin exerts a maximal tonic inhibitory control on both glucocorticoid secretion and proliferative activity of regenerating rat adrenals, whose physiological relevance remains to be investigated.     

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

Requirement of intact disulfide bonds in orexin-A-induced stimulation of gastric acid secretion that is mediated by OX1 receptor activation

Orexin-A is a neuropeptide consisting of 33 amino acids with two intrachain disulfide bonds, namely Cys6-Cys12 and Cys7-Cys14, and is a potent stimulator of food consumption and gastric acid secretion. In contrast, orexin-B, a peptide containing 28 amino acids without disulfide bond, which has no stimulatory action of gastric acid. The objective of the present study was to characterize the receptor-mediated mechanism of orexin-A-induced stimulation of gastric acid secretion using orexin-A-related peptides with modification of disulfide bonds. Intracisternal injection of orexin-A, but not orexin-B or orexin-A (15-33), that does not contain both disulfide bonds stimulated gastric acid secretion in pylorus-ligated conscious rats. The ability of the stimulation of gastric acid output was less in three alanine-substituted orexin-A, [Ala(6,12)]orexin-A, [Ala(7,14)]orexin-A, and [Ala(6,7,12,14)]orexin-A, than orexin-A. Orexins-induced calcium increase was measured in CHO-K1 cells expressing OX1R or OX2R. Orexin-A induced a transient increase in [Ca(2+)]i in CHO-K1/OX1R cells in a dose-dependent manner. EC50 values for OX1R of orexin-A, orexin-B, or orexin-A (15-33) was 0.068, 0.69 or 4.1 nM, respectively, suggesting that peptides containing no disulfide bonds have lower potency for the receptor. Agonistic activity for OX1R of the three orexin-A analogues with modification of one or both disulfide bonds was significantly reduced as compared with that of orexin-A. EC50 values for OX2R of orexin-A and orexin-B was almost equal but potency for the receptor of orexin-A (15-33) and three alanine substituted orexin-A was less than that of orexin-A. A significant inverse relationship between gastric acid output and EC50 values for OX1R, but not OX2R, was observed. These results suggested that the orexin-A-induced acid stimulation requires OX1R activation and that disulfide bonds in orexin-A may have a key role in the receptor activation.     

Orexin-A does not stimulate food intake in old rats

Aging is associated with a progressive decrease in appetite and food intake. Both A and B orexins, expressed in specific neurons of the lateral hypothalamic area, have been implicated in the regulation of sleep and feeding. In this study, the stimulatory effect of intracerebroventricular administration of the orexins on food intake was compared between young (4-mo-old) and old (25- to 27-mo-old) male Wistar rats. A stainless steel cannula was implanted stereotactically into the left lateral ventricle. After a 7-day recovery period, different doses (0-30 nmol) of orexins were injected into the left lateral ventricle without anesthesia. Food and water consumptions were measured at 1, 2, and 4 h after injection. The protein levels of orexin receptors, a specific receptor for orexin-A (OX1R) and a receptor for both orexin-A and -B (OX2R), in the hypothalamus were determined by Western blot analysis and compared between young and old rats. Intracerebroventricular administration of orexin-A stimulated food intake in a dose-dependent manner in young rats. However, no effects were observed at any dose in old rats. The protein level of OX1R in the hypothalamus was significantly lower in old rats than in young rats, although the protein level of OX2R was comparable between groups. Results of the present study indicate that the function of the orexin system is diminished in old rats. The decrease in the OX1R protein level in the hypothalamus could be responsible for orexin-A's lack of stimulation of food intake in old rats.     

Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants

In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2 alpha R (443 aa) and mOX2 beta R (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2 alpha R, and the mOX2 beta R revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP(3)) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTP gamma S revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP(3) production at all concentrations in the mOX2 beta R variant. Orexin-A, however, showed no difference in IP(3) production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2 alpha R mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2 beta R gene expression, compared with both mOX1R and mOX2 alpha R. This potentially implicates a fundamental physiological role for these splice variants.     

Orexins (hypocretins) and adrenal function.

The recently discovered neuropeptides orexin A and B regulate feeding behavior, neuroendocrine and autonomic functions, and sleep-wakefulness by central mechanisms. The expression of orexins and orexin receptors in various peripheral organs and the presence of orexin A in blood indicate the existence of a peripheral orexin system. In rat and human adrenal glands, both OX (1) and OX (2) receptor subtypes have been described with a predominant expression of OX (2) receptors in the adrenal cortex. In male rats, adrenocortical OX (2) receptors are much higher expressed than in female rats. Various experimental data demonstrate a stimulatory effect of orexins on the secretion of adrenocortical steroids, mainly on glucocorticoids. Some results also suggest the regulation of catecholamine synthesis and release by orexins. Whether the gender-dependent expression of adrenocortical OX (2) receptors has functional correlates awaits future clarification. As plasma orexin appears to rise during hunger and hypoglycemia, orexins may link adrenal functions with energy homeostasis.     

Effect of pentagastrin on steroid secretion and proliferative activity of regenerating rat adrenal cortex

The effects of three subcutaneous injections of 3 nmol/100 g body weight of the cholecystokinin type 2 (CCK2) receptor agonist pentagastrin on adrenocorticotrophic hormone (ACTH) and corticosterone secretion and proliferative activity of regenerating rat adrenal cortex were investigated. Pentagastrin did not alter either ACTH and corticosterone plasma concentrations or the adrenal mitotic index at day 5 of regeneration. In contrast, it increased (by about 50%) the adrenal mitotic index at day 8 of regeneration, and the effect was blocked by the simultaneous administration of equimolar doses of the CCK2-receptor antagonist PD-135,158. It is suggested that the activation of CCK2 receptors exerts a growth promoting action on the regenerating rat adrenal cortex.     

Effects of orexins A and B on expression of orexin receptors and progesterone release in luteal and granulosa ovarian cells

Orexin-A and orexin-B are neuropeptides controlling sleep-wakefulness, feeding and neuroendocrine functions via their G protein-coupled receptors, orexin-1R and orexin-2R. They are synthesized in the lateral hypothalamus and project throughout the brain. Orexins and orexin receptors have also been described outside the brain. Previously we demonstrated the presence of both receptors in the ovary, their increased expression during proestrous afternoon and the dependence on the gonadotropins. Here we studied the effects of orexins on the mRNA expression of both receptors, by quantitative real-time PCR, on luteal cells from superovulated rat ovaries and granulosa cells from diethylstilbestrol-treated rat ovaries. Effects on progesterone secretion were also measured. In luteal cells, 1nM of either orexin-A or orexin-B decreased progesterone secretion. Orexin-A treatment increased expression of both orexin-1R and orexin-2R mRNA. The effect on orexin-1R mRNA expression was abolished by an orexin-1R selective receptor antagonist SB-334867 and the effect on orexin-2R mRNA expression was abolished by a selective orexin-2R antagonist JNJ-10397049. Orexin-B did not modify orexin-1R mRNA expression, but increased orexin-2R mRNA expression. The effect of orexin-B on orexin-2R was abolished by a selective orexin-2R antagonist. Neither the expression of orexin receptors nor progesterone secretions by granulosa cells were affected by orexins. FSH, as positive control, increased both steroid hormones secretion, but did not induce the expression of OX receptors in granulosa cells isolated from late preantral/early antral follicles. Finally in ovaries obtained immediately after sacrifice, the expression of orexin-1R and orexin-2R was higher in superovulated rat ovaries compared to control or diethylstilbestrol treated rat ovaries. A selective presence and function of both orexinergic receptors in luteal and granulosa cells is described, suggesting that the orexinergic system may have a functional role in the ovary.     

Orexins acting at native OX(1) receptor in colon cancer and neuroblastoma cells or at recombinant OX(1) receptor suppress cell growth by inducing apoptosis

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.     

The physiological role of orexins

Orexins/hypocretins are recently discovered neuropeptides synthetized mainly by neurons located in the posterolateral hypothalamus. Hypocretin-1 and -2 are the same peptides as orexin-A and orexin-B. Orexin A is a 33 amino acid peptide with N-terminal pyroglutamyl residue and two intrachain disulphide bonds. Orexin B is a linear peptide of 28 amino acids. These two peptides are potent agonists at both the orexin-1 (OxR1) and orexin-2 (OxR2) receptors. Orexin-A is selective ligand for OxR1 and OX2 binds both orexins. The structure of orexins and their receptors is highly conservative in mammals. Orexin A sequence is identical in several mammalian species (human, mouse, rat, bovine and porcine). Intracerebroventricular administered orexin-A stimulates food intake and energy expenditure. Orexins are also involved in the regulation of neurohormones and pituitary hormones secretion as well as in the control of cardiovascular and sleep-wake function. Orexins also play a role in the pathogenesis of narcolepsy. Mutation in the gene coding preproorexin or OxR2 receptor gene results in narcolepsy in mice and canine. In patients with narcolepsy orexin neurotransmission was altered and orexin level in cerebrospinal fluid was undetectable.     

Structure, tissue distribution, and pharmacological characterization of Xenopus orexins

We isolated the Xenopus gene encoding prepro-orexin to predict the structures of orexins in submammalian chordates. Putative mature Xenopus orexin-A and -B are highly similar to each mammalian counterpart. Especially, the C-terminal 10 residues were highly conserved among these species and isopeptides. Immunohistochemical examination of Xenopus brain revealed that orexin-containing neurons were highly specifically localized in the ventral hypothalamic nucleus. A rich network of immunoreactive fibers was found in various regions of the Xenopus brain. The distribution was similar to that of mammalian orexins. Xenopus orexin-A and -B specifically bind and activate human orexin receptors expressed in Chinese hamster ovary cells. Of interest, Xenopus orexin-B had several-fold higher affinity to human OX2R compared with human orexins. These results suggest that Xenopus orexin-B might be a useful pharmacological tool as an OX2R selective high-affinity agonist.     

Food deprivation differentially modulates orexin receptor expression and signaling in rat hypothalamus and adrenal cortex

Although starvation-induced biochemical and metabolic changes are perceived by the hypothalamus, the adrenal gland plays a key role in the integration of metabolic activity and energy balance, implicating feeding as a major synchronizer of rhythms in the hypothalamic-pituitary-adrenal (HPA) axis. Given that orexins are involved in regulating food intake and activating the HPA axis, we hypothesized that food deprivation, an acute challenge to the systems that regulate energy balance, should elicit changes in orexin receptor signaling at the hypothalamic and adrenal levels. Food deprivation induced orexin type 1 (OX1R) and 2 (OX2R) receptors at mRNA and protein levels in the hypothalamus, in addition to a fivefold increase in prepro-orexin mRNA. Cleaved peptides OR-A and OR-B are also elevated at the protein level. Interestingly, adrenal OX1R and OX2R levels were significantly reduced in food-deprived animals, whereas there was no expression of prepro-orexin in the adrenal gland in either state. Food deprivation exerted a differential effect on OXR-G protein coupling. In the hypothalamus of food deprived rats compared with controls, a significant increase in coupling of orexin receptors to Gq, Gs, and Go was demonstrated, whereas coupling to Gi was relatively less. However, in the adrenal cortex of the food-deprived animal, there was decreased coupling of orexin receptors to Gs, Go, and Gq and increased coupling to Gi. Subsequent second-messenger studies (cAMP/IP3) have supported these findings. Our data indicate that food deprivation has differential effects on orexin receptor expression and their signaling characteristics at the hypothalamic and adrenocortical levels. These findings suggest orexins as potential metabolic regulators within the HPA axis both centrally and peripherally.     

The signalling profile of recombinant human orexin-2 receptor

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A

Presence of appetite-regulating peptides orexin-A and orexin-B in mucosal endocrine cells suggests a role in physiological control of the intestine. Our aim was to characterize orexin-induced stimulation of duodenal bicarbonate secretion and modulation of secretory responses and mucosal orexin receptors by overnight food deprivation. Lewis x Dark Agouti rats were anesthetized and proximal duodenum cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Orexin-A was administered intra-arterially close to the duodenum, intraluminally, or into the brain ventricles. Total RNA was extracted from mucosal specimens, reverse transcribed to cDNA and expression of orexin receptors 1 and 2 (OX1 and OX2) measured by quantitative real-time PCR. OX1 protein was measured by Western blot. Intra-arterial orexin-A (60-600 nmol.h(-1).kg(-1)) increased (P < 0.01) the duodenal secretion in fed but not in fasted animals. The OX1 receptor antagonist SB-334867, which was also found to have a partial agonist action, abolished the orexin-induced secretory response but did not affect secretion induced by the muscarinic agonist bethanechol. Atropine, in contrast, inhibited bethanechol but not orexin-induced secretion. Orexin-A infused into the brain ventricles (2-20 nmol.kg(-1).h(-1)) or added to luminal perfusate (1.0-100 nM) did not affect secretion, indicating that orexin-A acts peripherally and at basolateral receptors. Overnight fasting decreased mucosal OX1 and OX2 mRNA expression (P < 0.01) as well as OX1 protein expression (P < 0.05). We conclude that stimulation of secretion by orexin-A may involve both receptor types and is independent of cholinergic pathways. Intestinal OX receptors and secretory responses are markedly related to food intake.