Localization of orexin B and orexin-2 receptor in the rat epididymis

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R^?/?) and OX1R/OX2R double knock-out (OX1R^?/?; OX2R^?/?) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.     

Orexins and receptor OX2R in the gastroenteric apparatus of two teleostean species: Dicentrarchus labrax and Carassius auratus

Orexin A and B peptides and the receptor OX2R were studied in sea bass and goldfish gastroenteric tract by immunoblotting combined with densitometric analysis using NIH Image J software and immunohistochemical techniques. These teleost species present a different gut organization and diverse feeding habits. Immunoblotting experiments showed one band of 16 kDa corresponding to prepro‐orexin, and one band of 38 kDa corresponding to the OX2R receptor. Immunohistochemical localization of OXA and OXB was observed in the enteric nervous system throughout the gastroenteric tract of both species. OXA and OXB immunoreactive cells were found in the gastric and intestinal regions of sea bass, and were mainly found in the basal region of folds in intestinal bulb, and in the midgut and hindgut of goldfish. The distribution of OX2R was mainly detected in the mucosa of the gastroenteric tract of sea bass and goldfish. This distribution suggests an endocrine action of OXA and OXB in the gastrointestinal tract as well as involvement in the peripheral control of food intake and digestive processes in both species. This study might also serve to determine the productive factors in breeding and as a baseline for future experimental studies on the regulation of the gastroenteric functions in non‐mammalian vertebrates. Anat Rec, 299:1121–1129, 2016. © 2016 Wiley Periodicals, Inc.     

Merkel cells, a new localization of prepro-orexin and orexin receptors

Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.     

Immunohistochemical localization of orexin A and orexin type 2 receptor-positive cells in the placenta of dogs

The aim of the present study was to examine the presence and distribution of cells that express immunopositivity for orexin A (OXA) and its type 2 receptor (OX2R) in the dog placenta toward the end of pregnancy using immunohistochemical techniques. In the placental fetal portion, a few OXA and OX2R-positive cells were seen scattered in the outermost coating layer of chorionic villi and in the trophoblastic protrusions. Closer to the maternal portion, immunopositive labeling for both peptides was visible in the glandular epithelia and that for OXA also in the endothelium of the capillaries. These observations allow us to hypothesize that the canine placenta may be not only a source of orexin A, but also its target, and that orexin A may play an important role in controlling the function of this important organ for normal fetal development.     

Effects of orexin (hypocretin) on GIRK channels

Orexins (hypocretins) are recently discovered excitatory transmitters implicated in arousal and sleep. Yet, their ionic and signal transduction mechanisms have not been fully clarified. Here we show that orexins suppress G-protein-coupled inward rectifier (GIRK) channel activity, and this suppression is likely to lead to neuronal excitation. Cultured neurons from the locus coeruleus (LC) and the nucleus tuberomammillaris (TM) were used, as well as HEK293A cells transfected with GIRK1 and 2, either human orexin receptor type 1 (OX1R) or type 2 (OX2R), mu opioid receptor and GFP cDNAs. In GTPgammaS-loaded cells, orexin A (OXA, 3 microM) inhibited GIRK currents that had previously been activated by somatostatin (in LC cells), nociceptin (TM cells), or the mu opioid agonist DAMGO (HEK cells). In guanosine triphosphate (GTP)-loaded HEK cells, in which GIRK currents were not preactivated, OXA induced a biphasic response through both types of orexin receptors: an initial current increase and a subsequent decrease to below resting levels. Current-voltage (I-V) relationships revealed that both the OXA-induced and suppressed currents are inwardly rectifying with reversal potentials around EK. The OXA-induced initial current was partially pertussis toxin (PTX) sensitive and partially PTX insensitive, whereas the OXA-suppressed current was PTX insensitive. These data suggest that orexin receptors couple with more than one type of G-protein, including PTX-sensitive (such as Gi/o) and PTX-insensitive (such as Gq/11) G-proteins. The modulation of GIRK channels by orexins may be one of the cellular mechanisms for the regulation of brain nuclei (e.g., LC and TM) that are crucial for arousal, sleep, and appetite.     

出生前尼古丁暴露增加新生大鼠下丘脑orexin A及延髓内orexin 1型受体表达

流行病学调查显示,出生前暴露于烟雾环境是新生儿猝死综合征发生的首位原因,尼古丁是香烟烟雾中最主要的影响胎儿神经系统发育的成分。为了观察出生前尼古丁暴露对新生大鼠下丘脑orexin A(OXA)及延髓内orexin 1型受体(OX1R)表达的影响,本实验将20只雌性成年大鼠随机均分为二组,怀孕后第5 d开始每天分别皮下注射尼古丁6 mg/kg(模型组)或等量的生理盐水(对照组),直至分娩。随机选取模型组和对照组所产的新生大鼠(1～3 d),采用免疫组织化学方法和图像分析技术,观察新生鼠下丘脑内OXA及延髓内OX1R阳性神经元的分布情况。结果显示:两组新生大鼠下丘脑内OXA免疫阳性细胞均有表达,且都主要存在于下丘脑背内侧区与穹窿周围,模型组的新生大鼠OXA免疫阳性细胞的相对光密度(ROD)值高于对照组(P<0.05)。延髓内OX1R免疫阳性细胞在两组内均有广泛分布,主要分布在腹外侧区和舌下神经核。在这两个区域,模型组新生鼠的OX1R免疫阳性细胞的ROD值均高于对照组(P<0.001)。以上结果表明,出生前尼古丁暴露的新生大鼠,下丘脑OXA及延髓内OX1R的表达均上调,提示出生前尼古丁暴露改变了新生大鼠脑内OXA系统递质的释放和突触传递,这意味着脑内orexin系统参与出生前尼古丁暴露导致的各种疾患。     

Localization of the orexin system in the gastrointestinal tract of fallow deer

The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine.     

Orexin 1 receptor in the seminiferous tubules of boar testis: An immunohistochemical study

Orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R) are two G-protein-coupled receptors that bind their ligands, orexin A (OXA) and B (OXB), with different affinities. The male genital system represents an important target for OXA, which appears to play a role in the control of steroidogenesis and germ cell development in the testis. It is known that among domestic breeding animals, in the boar the number of Leydig cells is very high and OXA appears to have stimulatory activity on testosterone production. In this study, we aimed to evaluate the presence of OX₁R in the boar testis in order to extend our knowledge concerning the distribution and a potential functional role of the orexinergic system in the male reproductive tract of farm animals. The presence of OX₁R immunopositive cells in seminiferous tubules of the boar testis enables us to hypothesize a possible role of OXA on male germ cells cycle in pig. Further investigations, involving functional and ultrastructural analysis, may contribute to our understanding of the role of orexins in the boar genital system.     

Orexin and adiponectin in high fat diet–induced insulin resistance

Orexin A (OXA) is a hypothalamic neuropeptide with both central and peripheral activities on insulin signaling and energy balance. Adiponectin is an adipocytokine which regulates metabolisms of lipid and glucose via its receptors AdipoR1 and AdipoR2. This study investigated immunohistochemical changes in expressions of OXA, its receptor OX1R in pancreas and AdipoR1 in skeletal muscle with a high fat diet (HFD)-induced insulin resistance (IR). Standard 45% fat and 60% fat diets were administered to Wistar rats for 3 and 8 w. OXA expression increased with both 3- and 8-w 45% fat diets. OX1R expression also increased with a 3-w 45% fat diet, but decreased with the 3-w 60% fat diet. OXA, OX1R, and AdipoR1 expressions decreased with a 8-w 60% fat diet. An early adaptation in context of OXA was observed in pancreas β-cell to HFD-induced IR. OXA, OX1R, and AdipoR1 expressions decreased with either the higher amount or longer duration of HFD consumption.     

Promotion of sleep by suvorexant-a novel dual orexin receptor antagonist

Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX(1)R and OX(2)R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX(2)R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.     

Orexins/hypocretins and orexin receptors in apoptosis: a mini-review

An unexpected and fascinating aspect of the neuropeptides orexins has recently emerged when it was shown that orexins acting at orexin receptors OX1R or OX2R induce dramatic apoptosis resulting in massive reduction in cell growth in various cancer cell lines. This mini-review will provide the reader with recent findings related to the proapoptotic actions of orexins and the entirely novel mechanism whereby the seven membrane-spanning G-protein-coupled receptor (GPCR) OX1R triggers apoptosis. Recent data show that orexins induce tyrosine phosphorylation of the tyrosine-based motifs – immunoreceptor tyrosine-based inhibitory motif and immunoreceptor tyrosine-based switch motif – in OX1R. These phosphorylations result in the recruitment and activation of the phosphotyrosine phosphatase SHP-2 and subsequent cytochrome c-mediated mitochondrial apoptosis. Finally, this mini-review will also speculate on: (1) the potential importance of tyrosine-based motifs in the large family of GPCRs; (2) the interest of orexin receptors as therapeutic targets in cancer therapy; (3) the possible role of orexin receptor-mediated apoptosis in physiology and pathophysiology in the brain (neurodevelopment, neurodegenerative diseases) and in the periphery.     

大鼠发情周期下丘脑中食欲素及其受体的变化

目的 观察大鼠发情周期各期下丘脑中食欲素及其受体(OX1R、OX2R)表达的变化,探讨食欲素对发情周期的可能调节作用.方法 通过竞争性逆转录多聚酶链式反应 (competitive RT-PCR)方法检测大鼠下丘脑中食欲素前体(Prepro-Orexin)、食欲素-A、OXIR和OX2R在发情周期各期的表达.结果 发情前期的OX1R mRNA表达水平明显高于发情后期,而prepro-OX和OX2R mRNA的表达各期间没有明显的不同.结论 食欲素可能通过结合OX1R调节促性腺激素释放激素和/或黄体生成素的分泌而参与排卵的发生.     

Histochemical studies of epithelial cell glycoconjugates in atrophic, metaplastic, hyperplastic, and neoplastic canine prostate

The nature and distribution of lectin receptors were studied in normal, atrophic, metaplastic, hyperplastic, and neoplastic epithelium of canine prostate. Results were compared with prostatic epithelium of castrated dogs treated for 2 weeks with estradiol-17 beta 17-cyclopentylpropionate, 5 alpha-adrostane-3 alpha,17 beta-diol dipropionate, or 5 alpha-dihydrotestosterone. Eight biotinylated lectins were used as histochemical probes and avidin-biotin-peroxidase complex served as the visualant. Receptors for Ulex europaeus agglutinin-I were present in atrophic prostatic epithelium. Receptors for U. europaeus agglutinin-I, wheat, germ agglutinin, Dolichos biflorus agglutinin, and soybean agglutinin were present in epithelium that had undergone squamous metaplasia. Binding of peanut agglutinin receptors was present, to a limited extent, in squamous epithelium and was increased after they were unmasked (sialic acid residues cleaved with neuraminidase). In glandular cells of normal canine prostate and in benign prostatic hyperplasia, receptor sites were stained with Ricinus communis agglutinin-I, Concanavalia ensiformis agglutinin wheat germ agglutinin, and U. europaeus agglutinin-I. The basal cells in these tissues did not bind lectins. Prostatic carcinoma cells demonstrated receptors for wheat germ agglutinin and U. europaeus agglutinin-I. Responding and atrophic acini were present in prostates of castrated dogs treated with estradiol-17 beta 17-cyclopentylpropionate. Glandular cells of atrophic acini exhibited lectin receptor profiles similar to counterparts in castrated-untreated dogs. However, glandular cells responding to estrogen exhibited staining of free and cryptic peanut agglutinin receptor sites. Glandular cells of castrated dogs treated with 5 alpha-androstane 3 alpha,17 beta-diol dipropionate and 5 alpha-dihydrotestosterone have a pattern of lectin receptors similar to that found in normal and hyperplastic epithelium. Our studies show significant differences in lectin-binding patterns in the epithelium of atrophic, metaplastic, hyperplastic, and neoplastic canine prostate. They also demonstrate that the species of carbohydrate residues present in the glandular cells can be modified with sex hormones.     

Ultrastructural localization of orexin-1 receptor in pre- and post-synaptic neurons in the rat arcuate nucleus

The distribution and ultrastructural localization of the orexin-1 receptor (OX(1)R) in the rat arcuate nucleus were studied using immunocytochemical techniques. OX(1)R-containing neurons were found throughout the nucleus, but were concentrated in its posterior region. Both OX(1)R-positive perikarya and dendrites were found to receive synapses from other unknown axon terminals. In addition, a small number of OX(1)R-positive axon terminals were observed. These OX(1)R-immunoreactive axon terminals were often found to make synapses on unidentified immunonegative dendritic processes. The present results indicate that orexin may act on food intake regulating neurons through both pre- and post-synaptic OX(1)R.     

增食欲素系统在胰岛素抵抗大鼠胰腺组织中的表达及意义

目的：研究高脂肪膳食诱导的胰岛素抵抗（IR）大鼠胰腺组织中增食欲素（orexin）及其受体的变化规律。方法:IR大鼠模型采用高脂肪膳食诱导并经钳夹技术证实。采用微型血糖仪和化学发光免疫分析法测定全血葡萄糖和血清胰岛素的变化。应用实时定量PCR和Western 印迹杂交分析检测胰腺组织中orexin及orexin受体（OXR）mRNA和蛋白质在IR状态下的表达改变。结果:高脂肪膳食喂养4周后，实验组大鼠葡萄糖输注率由对照组(12.5±0.6) mg·kg-1·min-1下降至(7.6±0.4) mg·kg-1·min-1。实验组大鼠的体重、全血葡萄糖、血清胰岛素水平高于对照组，分别为26％、25％和30％（P<0.05）；高脂肪膳食诱导的IR大鼠胰腺组织中orexin mRNA和蛋白质的表达比对照组减少约71%和63％，而OX1R mRNA和蛋白质的表达比对照组降低约49％和67％（P<0.05），OX2R mRNA和蛋白质的表达改变不明显。结论:高脂肪膳食可诱导大鼠体内产生IR，并抑制胰腺中orexin及其OX1R基因的转录和翻译。Orexin系统可能是参与调节脂肪－胰岛轴的主要因素。     

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.     

妊娠、分娩以及泌乳期大鼠下丘脑中食欲素及其受体表达的变化

目的 观察大鼠下丘脑中食欲素(Orexin)及其受体(OX1R、OX2R)在妊娠、分娩以及泌乳期表达的变化,以探讨Orexin与生殖功能之间的关系.方法 通过竞争性逆转录多聚酶链式反应(Competitive RT-PCR)和免疫组织化学方法测定大鼠下丘脑中Orexin前体(Prepro-OX)、Orexin-A、OX1R和OX2R在妊娠、分娩以及泌乳期的表达.结果 Orexin-A和OX1R阳性神经元主要分别存在于大鼠下丘脑外侧区(LHA)以及妊娠和泌乳期大鼠的下丘脑室旁核 (PVN)和视上核(SON).泌乳第1d的Prepro-Orexin mRNA和OX1R的表达水平明显高于妊娠后期和泌乳期.OX2R的表达在生殖各时期之间没有明显改变.结论 Orexin可能参与大鼠泌乳早期的生殖功能调节,下丘脑的PVN及SON可能是其重要的作用部位.     

Characterization and application of two novel monoclonal antibodies against human OX40: costimulation of T cells and expression on tumor as well as normal gland tissues

OX40, a membrane-bound molecule of the tumor-necrosis-factor-receptor superfamily, is a critical costimulatory receptor during the immune response. Here, we newly generated two specific mouse antihuman OX40 monoclonal antibodies (mAbs) (2G2 and 1F7), whose specificities are quite different from the available OX40 mAb (ACT35) by competition assay. It was also found that both mAbs could enhance the proliferation, activation and differentiation of T lymphocytes primed by anti-CD3 mAb. These results evidenced that both were functional antihuman OX40 mAbs. Furthermore, stained by 2G2 and 1F7, FCM and immunohistochemistry detected the constitutive expression of OX40 on tumor cell lines from epithelium, breast cancer and glioma tissues. Meanwhile, the non-tumor tissues (thyroid gland, stomach gland) were also found OX40 expression. These results suggested that OX40 is not only expressed in activated T cells, but also in some tumors as well as normal gland tissues. Such expression pattern indicated that OX40 may be a valuable surface antigen in unveiling its expression and function outside the immune system. Briefly, these novel antibodies may contribute to the evaluation of the mechanism of tumor metastasis and eventually shed light on further study of tumor immunotherapy and autoimmune diseases.