Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release

Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 microM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.     

Orexins in rat dorsal motor nucleus of the vagus potently stimulate gastric motor function

Orexins regulate food intake, arousal, and the sleep-wake cycle. They are synthesized by neurons in the lateral hypothalamus and project to autonomic areas in the hindbrain. Orexin A applied to the dorsal surface of the medulla stimulates gastric acid secretion via a vagally mediated pathway. We tested the hypothesis that orexins in the dorsal motor nucleus (DMN) of the vagus regulate gastric motor function. Multibarelled micropipette assemblies were used to administer vehicle, L-glutamate, orexins A (1 and 10 pmol) and B (10 pmol), and a dye marker into this site in anesthetized rats. When the pipette was positioned in the DMN rostral to the obex (where excitation of neurons by L-glutamate evoked an increase in contractility), orexins A and B increased intragastric pressure and antral motility. In contrast, 10 pmol orexin A microinjected into the DMN caudal to the obex (where L-glutamate evokes gastric relaxation through a vagal inhibitory pathway) did not significantly alter gastric motor function. In separate immunocytochemical studies, orexin receptor 1 was highly expressed in neurons in the DMN. Specifically, it was present in retrogradely labeled preganglionic neurons in the DMN that innervate the stomach. These data are consistent with the idea that orexin A stimulates vagal excitatory motor neurons. These are the first data to suggest that orexins in the DMN have potent and long-lasting effects to increase gastric contractility.     

Orexins and adipoinsular axis function in the rat

Orexin A and B are recently identified as peptides that are derived from the same precursor and their expression is highly specifically localized in neurons located in the lateral hypothalamic area, a region implicated in the feeding behaviour. These peptides appear to be a part of a complex circuit that integrates the aspects of energy metabolism, cardiovascular function, hormone homeostasis and sleep/wake behaviours. The functional linking of orexins with leptin and insulin suggests the possibility of its involvement in the regulation of the adipoinsular axis, and the present investigation was designed to examine the potential role of orexins in this axis regulation. In all the tested doses (8, 16 and 40 nmol/kg body weight (b.w.)), subcutaneous (s.c.) injections of orexin A caused the significant increase in insulin and leptin blood levels. These elevations were observed 60 and 120 min after peptide administration. On the other hand, after the orexin B administration, elevated insulin and leptin blood concentrations were found only at 60 min of the experiment, and in that time point, the increases were comparable to that evoked by orexin A. In comparison with the control animals, the administration of orexins for 7 days resulted in a significant gain in body weight. Prolonged administration of either orexin A or orexin B significantly elevated insulin and leptin blood concentrations. Under these conditions, the orexin A effect on the leptin secretion was more marked than on the insulin secretion, and this difference is reflected by the lowered insulin/leptin molar ratio. These results suggest that orexins play an important role in the adipoinsular axis function and may be a significant regulator of both insulin and leptin secretion. In this regard, we suggest the updated functional model of Kieffer and Habener [Am. J. Physiol.: Endocrinol. Metab. 27 (2000) E1] that proposed the adipoinsular axis. Our model is extended by the probable humoral links between orexins and leptin and orexins and insulin and points on the dependence of the effects evoked by orexins, leptin and insulin on the blood glucose levels.     

Contribution to sympathetic vasomotor tone of tonic glutamatergic inputs to neurons in the RVLM

The role of excitatory amino acid (EAA) receptors in the rostral ventrolateral medulla (RVLM) in maintaining resting sympathetic vasomotor tone remains unclear. It has been proposed that EAA receptors in the RVLM mediate excitatory inputs both to presympathetic neurons and to interneurons in the caudal ventrolateral medulla (CVLM), which then provide a counterbalancing inhibition of RVLM presympathetic neurons. In this study, we tested this hypothesis by determining the effect of blockade of EAA receptors in the RVLM on mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), after inhibition of CVLM neurons. In anesthetized rats, bilateral injections of muscimol in the CVLM increased MAP, HR, and RSNA. Subsequent bilateral injections of kynurenic acid (Kyn, 2.7 nmol) in the RVLM caused a modest reduction of approximately 20 mmHg in the MAP but had no effect, when compared with the effect of vehicle injection alone, on HR or RSNA. By approximately 50 min after the injections of Kyn or vehicle in the RVLM, the MAP had stabilized at a level close to its original baseline level, but the HR and RSNA stabilized at levels above baseline. The results indicate that removal of tonic EAA drive to RVLM neurons has little effect on the tonic activity of RVLM presympathetic neurons, even when inputs from the CVLM are blocked. Thus the tonic activity of RVLM presympathetic neurons under these conditions is dependent on excitatory synaptic inputs mediated by non-EAA receptors and/or the autoactivity of these neurons.     

Orexins activate histaminergic neurons via the orexin 2 receptor.

Orexins (orexin A and B) are recently identified neuropeptides implicated in the regulation of vigilance states and energy homeostasis. We have shown here the physiological significance of histaminergic neurons in the orexin-induced arousal responses. Immunohistochemical and electron microscopic techniques revealed direct synaptic interaction between orexin-immunoreactive nerve terminals and histidine decarboxylase-immunoreactive neurons in the TMN. Electrophysiological study revealed that orexins dose-dependently activate histaminergic neurons, which were freshly isolated from rats TMN region. To further evaluate, we examined the effect of pyrilamine, an H(1) receptor antagonist, on orexin-induced arousal response in rats. Simultaneously recordings of electroencephalograph and electromyograph showed that intracerebroventricular infusion of orexin A significantly increased the awake state in the light phase. Central application of pyrilamine significantly inhibited this response. These results strongly suggest that activation of histaminergic neurons by orexins might be important for modulation of the arousal.     

Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro

Dysfunction of the orexin/hypocretin neurotransmitter system leads to the sleep disorder narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness and the occurrence of cataplexy--a sudden loss of muscle tone triggered by emotionally arousing events. Both symptoms can be treated with drugs that act on dopaminergic systems. Here we have investigated the effect of orexins on the firing of dopaminergic and GABAergic neurons of the substantia nigra (SN) in brain slices. Surprisingly, dopaminergic neurons in pars compacta were unaffected by orexins. In contrast, bath application of orexin A (100 nM) or orexin B (5-300 nM) greatly increased the firing rate of GABAergic neurons in pars reticulata. The orexin B-mediated excitation was unaffected by blocking synaptic transmission (using low-Ca2+/high-Mg2+ solution). However, the effect of orexin B was reduced significantly by thapsigargin (1 microM) and inhibitors of protein kinase A. The presence of orexinergic fibres in the SN pars reticulata was demonstrated by immunohistochemical methods with the fibre density increasing in the rostrocaudal direction. The orexin excitation of SN reticulata cells may help to maintain their high firing rate during waking. Furthermore, the absence of orexin effects in narcolepsy may predispose affected individuals to attacks of cataplexy.     

Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats

Neuropeptide Y (NPY) produced in the arcuate nucleus (ARC) of the hypothalamus stimulates feeding both directly by activating NPY receptors and indirectly through release of the orexigenic peptides, galanin and beta-endorphin (beta-END), in the paraventricular nucleus (PVN) and surrounding neural sites. Orexin A and orexin B, produced outside the ARC in the lateral hypothalamic area (LH), have recently been shown to stimulate feeding. In the present studies we tested the hypothesis that NPYergic signaling may mediate feeding stimulated by orexins. In adult male rats injected intracerebroventricularly (i.c.v.) with orexin A (3, 10, 15 nmol) or orexin B (3, 10, 30 nmol) feeding was stimulated in a dose-dependent manner; maximal feeding was seen after 15 nmol orexin A and 30 nmol orexin B. To determine whether NPY may mediate this orexin stimulated feeding, we used 1229U91, a selective NPY Y1 receptor antagonist (NPY-A). Whereas NPY-A on its own was ineffective, it suppressed NPY-induced feeding. Furthermore, NPY-A completely blocked the feeding evoked by either orexin A (15 nmol) or orexin B (30 nmol). These results show that orexin A and B stimulate feeding and further suggest that these excitatory effects may be mediated by NPYergic signaling through Y1 receptors. These findings are in accord with the view that the orexin-NPY pathway may comprise a functional link upstream from NPY within the hypothalamic appetite regulating network.     

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.     

Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.     

Localization and effects of orexin on fasting motility in the rat duodenum

The orexins [orexin A (OXA) and orexin B (OXB)] are novel neuropeptides that increase food intake in rodents. The aim of this study was to determine the distribution of orexin and orexin receptors (OX1R and OX2R) in the rat duodenum and examine the effects of intravenous orexin on fasting gut motility. OXA-like immunoreactivity was found in varicose nerve fibers in myenteric and submucosal ganglia, the circular muscle, the mucosa, submucosal and myenteric neurons, and numerous endocrine cells of the mucosa. OXA neurons displayed choline acetyltransferase immunoreactivity, and a subset contained vasoactive intestinal peptide. OXA-containing endocrine cells were identified as enterochromaffin (EC) cells based on the presence of 5-hydroxytryptamine immunoreactivity. OX1R was expressed by neural elements of the gut, and EC cells expressed OX2R. OXA at 100 and 500 pmol x kg(-1) x min(-1) significantly increased the myoelectric motor complex (MMC) cycle length compared with saline. Similarly, OXB increased the MMC cycle length at 100 pmol x kg(-1) x min(-1), but there was no further effect at 500 pmol x kg(-1) x min(-1). We postulate that orexins may affect the MMC through actions on enteric neurotransmission after being released from EC cells and/or enteric neurons.     

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.     

Orexins cause epileptic activity

Orexins have been implicated in the regulation of sleep-wake cycle, energy homeostasis, drinking behavior, analgesia, attention, learning and memory but their effects on epileptic activity are controversial. We investigated whether intracortical injections of orexin A (100pmol) and B (100pmol) cause epileptic activity in rats. We observed epileptic seizure findings on these two groups rats. Orexin A and B also significantly increased total EEG power spectrum. Our findings indicate that orexins cause epileptic activity.     

延髓腹外侧区微量注射L－NNA和SNP对大鼠血压、心率和肾交感神经活动的影响

在麻醉大鼠观察了向延髓腹外侧区微量注射ＮＯ合成酶抑制剂Ｎ－硝基左旋精氨酸（ＬＮＮＡ）和硝普钢（ＳＮＰ）对血压、心率和肾交感神经活动的影响,旨在探讨中枢左旋精氨酸－ＮＯ通路在动脉血压调节中的作用及其机制。实验结果如下：（１）向延髓腹外侧头端区（ＲＶＬＭ）注射Ｌ－ＮＮＡ后,平均动脉压（ＭＡＰ）升高,肾交感神经活动（ＲＳＮＡ）增强;心率（ＨＲ）减慢,但无统计学意义。ＭＡＰ和ＲＳＮＡ的变化持续３０ｍｉｎ以上;此效应可被预先静注左旋精氨酸所逆转。（２）向ＲＶＬＭ微量注射ＳＮＰ,ＭＡＰ降低,ＲＳＮＡ减弱;但ＨＲ的变化无统计学意义。（３）向延髓腹外侧尾端区（ＣＶＬＭ）注射Ｌ－ＮＮＡ,ＭＡＰ降低,ＨＲ减慢,ＲＳＮＡ减弱。（４）向ＣＶＬＭ微量注射ＳＮＰ,ＭＡＰ升高,ＲＳＮＡ增强,而心率无明显变化。以上结果表明,中枢左旋精氨酸－ＮＯ通路对延髓腹外侧部的神经元活动有调变作用。     

Peptides that regulate food intake: orexin gene expression is increased during states of hypertriglyceridemia

Previous reports implicate the orexins in eating and body weight regulation. This study investigated possible functional relationships between hypothalamic orexins and circulating hormones or metabolites. In situ hybridization and quantitative PCR were used to examine orexin expression in the perifornical hypothalamus (PF) of rats and mice on diets varying in fat content and with differential propensity toward obesity. The results showed that orexin gene expression was stimulated by a high-fat diet in close association with elevated triglyceride levels, suggesting a functional relationship between these measures. Results obtained in obesity-prone rats and mice revealed a similar increase in orexin in close relation to triglycerides. A direct test of this orexin-triglyceride link was performed with Intralipid, which increased PF orexin expression along with circulating triglycerides. Whereas PF galanin is similarly stimulated by dietary fat, double-labeling immunofluorescence studies showed that orexin and galanin neurons are anatomically distinct. This evidence suggests that the orexins, like galanin, are "fat-responsive" peptides that respond to circulating lipids.     

Inhibition of food intake by central injection of anti-orexin antibody in fasted rats

It has been shown that intracerebroventricular injection of synthetic orexins stimulated food intake in rats. This pharmacological evidence suggests that orexins may have a role for the central regulation of feeding. In the present study, we investigated the hypothesis of whether endogenous orexins indeed play a vital role in feeding behavior. An anti-orexin polyclonal antibody was used throughout the study. First, we examined the specificity of the antibody to orexin by Western blot analysis and immunohistochemistry. Next, the effects of central injection of the orexin antibody on food intake in 24-h-fasted rats were evaluated. Western blot analysis revealed that the orexin antibody detected synthetic orexin-A. Immunohistochemical study showed that orexin-positive neurons were identified only in the lateral hypothalamic area, in agreement with previous reports. Neither control antibody nor the orexin antibody preabsorbed with excess amount of orexin-A detected neurons, indicating that the orexin antibody is specific. Intracisternal but not intraperitoneal injection of the orexin antibody dose-dependently suppressed feeding. All these results suggest that immunoneutralization of endogenous orexins in the brain reduced food intake. In other words, we suggest that endogenous brain orexin may have a physiologically relevant action on feeding behavior.     

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.     

Structure and functions of the brain orexin-containing neurons

The results of investigations of the new discovered brain orexin neurons, their chemical structure, localization and functions are reviewed. The following data are described: the specifics of orexins mRNA, orexins A and B and their receptors; connections between orexin neurons and neurons from different structures of the brain and spinal cord and the participation of the orexin neuron system in the functional regulation.     

Nitric oxide modulates the cardiovascular effects elicited by acetylcholine in the NTS of awake rats

Microinjection of acetylcholine chloride (ACh) in the nucleus of the solitary tract (NTS) of awake rats caused a transient and dose-dependent hypotension and bradycardia. Because it is known that cardiovascular reflexes are affected by nitric oxide (NO) produced in the NTS, we investigated whether these ACh-induced responses depend on NO in the NTS. Responses to ACh (500 pmol in 100 nl) were strongly reduced by ipsilateral microinjection of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol in 100 nl) in the NTS: mean arterial pressure (MAP) fell by 50 +/- 5 mmHg before L-NAME to 9 +/- 4 mmHg, 10 min after L-NAME, and HR fell by 100 +/- 26 bpm before L-NAME to 20 +/- 10 bpm, 10 min after L-NAME (both P < 0.05). Microinjection of the selective inhibitor of neuronal nitric oxide synthase (nNOS), 1-(2-trifluoromethylphenyl) imidazole (TRIM; 13.3 nmol in 100 nl), in the NTS also reduced responses to ACh: MAP fell from 42 +/- 3 mmHg before TRIM to 27 +/- 6 mmHg, 10 min after TRIM (P < 0.05). TRIM also tended to reduce ACh-induced bradycardia, but this effect was not statistically significant. ACh-induced hypotension and bradycardia returned to control levels 30-45 min after NOS inhibition. Control injections with D-NAME and saline did not affect resting values or the response to ACh. In conclusion, injection of ACh into the NTS of conscious rats induces hypotension and bradycardia, and these effects may be mediated at least partly by NO produced in NTS neurons.     

Orexins increase penicillin-induced epileptic activity

Orexins have been implicated with physiological function including sleep-wake cycle, energy homeostasis, drinking behavior, analgesia, attention, learning and memory but their effects on excitability are controversial. We investigated the effects of intracortical injections of orexin A (100 pmol) and B (100 pmol) on the electrophysiological manifestation of epileptic seizures induced by cortical penicillin application in adult male rats. In comparison to saline, orexin A and B enhanced significantly the spike number, spike amplitude and spectral power values induced by cortical penicillin. Our findings indicates that orexins enhances the hyperexcitable and hypersyncronic cortical epileptic activity induced by focal application of penicillin-G.     

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.