Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization.

RF-amide peptides, a family of peptides characterized by a common carboxy-terminal Arg-Phe-NH2 motif, play various physiological roles in the brain including the modulation of neuroendocrine signaling. Neuropeptide FF (NPFF) receptors exhibit a high affinity for all RF-amide peptides, which suggests that the neurons expressing these NPFF receptors may have multiple functions in the brain. However, the distribution of the neurons expressing NPFF receptors in the rat brain remains poorly understood. This study aimed to determine the detailed histological distribution of mRNA that encodes the neuropeptide FF receptors (Npffr1 and Npffr2) in the rat brain using in situ hybridization. Neurons with strong Npffr1 expression were observed in the lateral septal nucleus and several hypothalamic areas related to neuroendocrine functions, including the paraventricular nucleus (PVN) and arcuate nucleus, whereas Npffr2-expressing neurons were observed mainly in brain regions involved in somatosensory pathways, such as several subnuclei of the thalamus. Npffr1 expression was observed in 70% of corticotropin-releasing hormone neurons, but in only a small population of oxytocin and vasopressin neurons in the PVN. Npffr1 expression was also observed in the dopaminergic neurons in the periventricular nucleus and the dorsal arcuate nucleus, and in the kisspeptin neurons in the anteroventral periventricular nucleus. These results suggest that NPFFR1-mediated signaling may be involved in neuroendocrine functions, such as in reproduction and stress response. In conjunction with a detailed histological map of NPFFRs, this study provides useful data for future neuroendocrine research.

Pigment epithelium-derived factor (PEDF) and PEDF-receptor in the adult mouse brain: Differential spatial/temporal localization pattern.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western-blotting, and also by enzyme-linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age-dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.

Role of RF-amid Related Peptide-3 (RFRP-3) in Inhibitory Effect of Orexin A on Reproductive Function in the Animal Model of Male Wistar Rats.

AIMS: The aim of the present study is to examine the orexin A (OXA) signaling can leave any impact on the hypothalamic-pituitary-gonadal (HPG) axis and this impact can be relayed through the pathway of RF amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibitory hormone)/G-protein coupled receptor (GPR)-147 (RFRP-3 receptor) as a novel target for controlling of HPG axis in the male rats. MATERIALS AND METHODS: Male rats were categorized randomly into experimental groups including control vehicle, OXA, and its antagonists' group and went through to surgical cannulation into the third ventricle. After the intracerebroventricular injection of each solution, blood samples were collected for measurements of the LH and testosterone using radioimmunoassay method. Hypothalamus of the animals were isolated for analysis of the relative expression of Rfrp-3/Gpr-147 along with Gnrh gene by Real time-PCR. Also, in the different cohort of animal sexual behavior test was done. RESULTS: It was shown that OXA significantly decreases the mean serum level of the LH and testosterone and, at the same time, its antagonists neutralize this impact. Moreover, we demonstrated that OXA has reduced the hypothalamic gene expression of Gnrh and increased the expression of Rfrp-3 and Gpr-147 genes. While OXA antagonists neutralize this impact. CONCLUSIONS: The results of this study are related to the impact of orexin on the HPG axis. It is recommended that RFRP-3/GPR-147 system as the interneural pathway relay the data of orexin to the neurons of GnRH.

Growth hormone-releasing hormone receptor mediates cytokine production in ciliary and iris epithelial cells during LPS-induced ocular inflammation.

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1ß in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1ß, IL-6, and iNOS and increased secretion of IL-1ß and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1ß and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.

Upregulation of Mas-related G Protein coupled receptor X2 in asthmatic lung mast cells and its activation by the novel neuropeptide hemokinin-1.

Hemokinin-1 (HK-1) is a novel neuropeptide produced by human bronchial cells and macrophages and causes contraction of human bronchi ex vivo. It is also generated by antigen/IgE-activated murine mast cells (MCs) and contributes to experimental chronic allergic airway inflammation via the activation of the neurokinin receptor-1 (NK-1R) expressed on murine MCs. We found elevated MC numbers in the lungs of individuals who died from asthma (asthma) when compared to lungs of individuals who died from other causes (non-asthma). Mas-related G Protein coupled receptor X2 (MRGPRX2) is a novel G-protein coupled receptor (GPCR) that is expressed predominantly on human MCs. We detected low level of MRGPRX2 in non-asthma lung MCs but its expression was significantly upregulated in asthma lung MCs. HK-1 caused degranulation in a human MC line (LAD2) and RBL-2H3 cells stably expressing MRGPRX2 and this response was resistant to inhibition by an NK-1R antagonist. However, knockdown of MRGPRX2 in LAD2 cells resulted in substantial inhibition of HK-1-induced degranulation. These findings suggest that while HK-1 contributes to the development of experimental asthma in mice via NK-1R on murine MCs the effect of this neuropeptide on human bronchoconstriction likely reflects the activation of MRGPRX2 on lung MCs. Thus, development of selective MRGPRX2 antagonists could serve as novel target for the modulation of asthma.

Photoperiod regulate gonad development via kisspeptin/kissr in hypothalamus and saccus vasculosus of Atlantic salmon (Salmo salar).

Atlantic salmon exhibit seasonal reproduction. However, the mechanisms governing this are still unclear. Generally speaking, kisspeptin has been recognized as a regulator of reproduction. Here, we report a relationship between kisspeptin, GnRH and photoperiod in Atlantic salmon. The results demonstrated that the expression of the Atlantic salmon kisspeptin-receptor (skissr) was not always consistent with the expression pattern of Atlantic salmon GnRH3 (sGnRH3) during all developmental processes. Kisspeptin may exert its influence primarily in the early and later stages of gonad development by promoting the secretion of sGnRH3. Meanwhile, the expression levels of kissr were higher in fish with gonads at stage II and stage V under the long-day photoperiod regime than under the short-day regime. In addition, both skissr and sGnRH3 were also expressed in the saccus vasculosus (SV), an organ only found in fish. The SV might be a seasonal sensor regulating reproduction in addition to the hypothalamus (Hyp).

Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.

Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.

Somatic and Neuroendocrine Changes in Response to Chronic Corticosterone Exposure During Adolescence in Male and Female Rats.

Prolonged stress and repeated activation of the hypothalamic-pituitary-adrenal axis can result in many sex-dependent behavioural and metabolic changes in rats, including alterations in feeding behaviour and reduced body weight. In adults, these effects of stress can be mimicked by corticosterone, a major output of the hypothalamic-pituitary-adrenal axis, and recapitulate the stress-induced sex difference, such that corticosterone-treated males show greater weight loss than females. Similar to adults, chronic stress during adolescence leads to reduced weight gain, particularly in males. However, it is currently unknown whether corticosterone mediates this somatic change and whether additional measures of neuroendocrine function are affected by chronic corticosterone exposure during adolescence in a sex-dependent manner. Therefore, we examined the effects of non-invasively administered corticosterone (150 or 300 µg/ml) in the drinking water of male and female rats throughout adolescent development (30-58 days of age). We found that adolescent animals exposed to chronic corticosterone gain significantly less weight than controls, which may be partly mediated by the effects of corticosterone on food consumption, fluid intake and gonadal hormone function. Our data further show that, despite similar circulating corticosterone levels, males demonstrate a greater sensitivity to these changes than females. We also found that Npy1 and Npy5 receptor mRNA expression, genes implicated in appetite regulation, was significantly reduced in the ventral medial hypothalamus of corticosterone-treated males and females compared to controls. Finally, parameters of gonadal function, such as plasma sex steroid concentrations and weight of reproductive tissues, were reduced by adolescent corticosterone treatment, although only in males. The data obtained in the present study indicate that chronic corticosterone exposure throughout adolescent development results in significant and sex-dependent somatic and neuroendocrine changes, and the results also provide an experimental framework for further investigating the impact of corticosterone on metabolic and neuroendocrine function during adolescence.

GHRH Receptor Expression in Malignant Mixed Müllerian Tumors: A Potentially Targetable Biopredictor.

Malignant mixed Müllerian tumors (MMMTs) are aggressive malignant neoplasms with a high recurrence rate and poor prognosis. Despite advances in adjuvant therapies in recent years, the prognosis of these tumors has not improved. Growth hormone-releasing hormone (GHRH) is produced by a variety of malignant tumors and acts as a growth factor in an autocrine/paracrine manner. Its function requires the presence of its receptors to exert its effects on neoplastic cells. In this study, we evaluated the expression of GHRH receptors (GHRH-R) in a group of MMMTs. Thirty-one examples of MMMTs from endometrium, ovary, uterine tube, and pelvic peritoneum were retrieved from the files of Department of Pathology at the University of Miami, Jackson Memorial Hospital. Immunohistochemistry for GHRH-R was performed on paraffin sections and the staining results were evaluated separately in both epithelial and mesenchymal components of each tumor. The presence of pituitary type growth hormone-releasing hormone receptor mRNA and that of its biologically active splice variant were also evaluated by RT-PCR in 6 of the tumors. Positive immunohistochemical reaction for GHRH-R was detected in 30 tumors (96%). The epithelial and sarcomatous components were positive in 30 (96%), whereas one endometrial tumor was negative in both components. The mRNA for GHRH-R and its splice variant was found in all 6 tested tumors. This study shows that GHRH-R is expressed by the majority of MMMTs in both epithelial and mesenchymal components. This finding could potentially serve as a basis for therapeutic approaches using synthetic peptide antagonists of GHRH-R that have shown significant efficacy with minimal side effects in experimental models.

[Role of the skin expression of neuropeptides, neurotrophins and their receptors in the pathogenesis of dermatoses].

OBJECTIVE: to define the role of neurotransmitters and their receptors in the development of itch and in the maintenance of a skin inflammatory response in patients with psoriasis and atopic dermatitis. MATERIAL AND METHODS: Skin biopsy specimens from 30 patients with psoriasis and 30 patients with atopic dermatitis were investigated by histological, immunoperoxidase, and indirect immunofluorescence assays. The investigators determined the expression of protein gene product 9.5 (PGP9.5), amphiregulin, semaphorin 3A, calcitonin gene-related peptide (CGRP) and its receptor (CGRP-R), nerve growth factor (NGF) and its receptor TrkA, and substance P (SP) and its receptor SP-R. The indirect immunofluorescence assay was used for quantitative analysis. The findings were statistically analyzed using a Statistica 10 program. RESULTS: Immunoperoxidase examination of the skin biopsy specimens from patients with atopic dermatitis and psoriasis revealed enhanced expression of amphiregulin, NGF, and PGP9.5, appearance of positively stained epidermal nerve fibers, and decreased expression of the nerve reduction factor semaphorin 3A in all cases. Some patients with atopic dermatitis and psoriasis showed increased expression of CGRP and CGRP-R, SP, SP-R, and TrkA. A pronounced inflammatory response was generally observed in these cases. CONCLUSION: The investigation performed suggests that atopic dermatitis and psoriasis are characterized by a larger number of epidermal nerve fibers and by a direct correlation between this indicator, disease severity, and itch intensity. The production of neuropeptides and neurotrophins is closely related to the development of a skin inflammatory response irrespective of its cause and dysregulation of these processes is likely to favor the body's sensitization and the chronic pattern the course of diseases.

Neuroleptics Affect Neuropeptide S and NPSR mRNA Levels in the Rat Brain.

Neuropeptide S (NPS) has a multidirectional regulatory activity, especially when considered as a potent endogenous anxiolytic factor. Accumulating data suggests that neuroleptics affect peptidergic signaling in various brain structures. However, there is no information regarding the influence of treatment with antipsychotics on brain NPS expression. In the current study, we assessed the NPS and NPS receptor (NPSR) mRNA levels in the brains of rats shortly and chronically treated with chlorpromazine and olanzapine using quantitative real-time PCR. Both single-dose and long-term (4 months) olanzapine treatment led to the upregulation of NPS expression in the rat hypothalamus. It supports the hypothesis that NPS is involved in the dopamine-dependent anxiolytic actions of selected neuroleptics and possibly also in the pathophysiology of mental disorders. On the other hand, NPSR expression decreased after single-dose and chronic chlorpromazine administration in the hypothalamus, as well as after chronic olanzapine and chlorpromazine administration in the striatum and hippocampus. These results cast a new light on the pharmacology of antipsychotics and contribute to a better understanding of the mechanisms responsible for their action. Furthermore, our findings underline the complex nature of potential interactions between dopamine receptors and brain peptidergic pathways, which has potential clinical applications.

Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and ß2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

Expression of Receptors for Pituitary-Type Growth Hormone-Releasing Hormone (pGHRH-R) in Human Papillary Thyroid Cancer Cells: Effects of GHRH Antagonists on Matrix Metalloproteinase-2.

Papillary thyroid cancer (PTC) is the most prevalent of all endocrine cancers. In recent studies, the presence of receptors for pituitary-type growth hormone-releasing hormone (pGHRH-R) has been demonstrated in various human cancers, including human prostate, brain, and other cancer lines. Thyroid malignancies, however, have not yet been investigated in this regard. In this study, we found that pGHRH-R and its functional splice variant, SV1, are present in normal thyroid and PTC cells. We also treated seven normal and PTC tumor thyroid cells in vitro with a GHRH antagonist, MIA-602, to compare its anti-proliferation and anti-invasion potential against vehicle-treated cells. We found that treatment with GHRH antagonist increases the expression of SV1 and pGHRH-R in tumor cells compared to tumor cells exposed to vehicle only, a response which may alter the sensitivity of signaling kinases within the cells. GHRH antagonist treatment of tumor cells also reduced activity of the tumor invasion marker, matrix metalloproteinase (MMP)-2, compared to tumor cells exposed to vehicle only. The expression of pGHRH-R and SV1, as well as MMP-2 activity, in normal thyroid cells remained unaffected by GHRH antagonist treatment. Similarly, cell proliferation rates for tumor or normal thyroid cells were not affected by GHRH antagonist treatment. Our findings have important implications for the therapeutic use of GHRH antagonist in cases of aggressive PTC refractory to conventional treatment modalities, and in which protein expression and MMP-2 activity in normal thyroid tissue is left unaltered.

Effects of intrathecal opioids combined with low-dose naloxone on motilin and its receptor in a rat model of postoperative pain.

AIMS: To investigate the effects of intrathecal morphine and fentanyl combined with low-dose naloxone on the expression of motilin and its receptor in a rat model of postoperative pain. MAIN METHODS: An intrathecal catheter was implanted, and saline, opioids (morphine and fentanyl) and naloxone were intrathecally administered 7 days later. An incisional pain model was established to induce pain behaviors in rats by unilateral plantar incision. Thermal hyperalgesia and mechanical allodynia were measured by using a radiant heat and electronic Von Frey filament, respectively. The expression of motilin in the hippocampus, stomach, duodenum, and plasma was determined by ELISA; and the expression of motilin receptor in the hippocampus was detected by Western blot assay. KEY FINDINGS: Motilin and its receptor were detected in the hippocampus. Acute incisional pain increased the motilin expression in the hippocampus and duodenum, while decreasing its expression in the gastric body and plasma. Postoperative analgesia with morphine+fentanyl upregulated the expression of motilin in the hippocampus; however, motilin was downregulated in peripheral sites. Naloxone at 1 ng/kg restored motilin to baseline levels. Acute pain, morphine+fentanyl, and naloxone all induced the expression of motilin receptor in the hippocampus. SIGNIFICANCE: Acute pain, postoperative analgesia with opioids, and naloxone significantly impacted the expression of hippocampal and peripheral motilin. Variation trends in all sites were not identical. Intrathecal injection of low-dose naloxone upregulated paw withdrawal thermal latency and enhanced the analgesic effects of opioids. The findings presented here provide a new basis for central and peripheral regulations in GI motility, clinical postoperative analgesia, and management of analgesic complications.

Temporal gene expression profile after acute electroconvulsive stimulation in the rat.

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.

Differential immunostaining of various types of breast carcinomas for growth hormone-releasing hormone receptor - Apocrine epithelium and carcinomas emerging as uniformly positive.

Different classes of breast cancers were explored for their positivity for growth hormone-releasing hormone receptors (GHRH-R) in this pilot study, as no systematic evaluation of such tumors has been performed to date. Seventy-two small primary breast carcinomas were evaluated for GHRH-R expression by immunohistochemistry using a polyclonal antibody and a cutoff value of 10% staining. GHRH-R positivity was detected in 58% of all cases, 20/23 (87%) of invasive lobular carcinomas (ILC) and 22/46 (48%) of invasive ductal carcinomas (IDC). GHRH-R positivity was more frequent in grade 2 tumors (86%), as compared to grade 1 (18%) or grade 3 (47%) cancers. GHRH-R expression was not associated with mitotic scores, the Ki-67 labeling indices or nodal status. IDCs with casting-type calcifications on the mammogram showed positivity for GHRH-R in 9/12 (75%) cases. Most importantly, apocrine epithelium, and all 10 apocrine carcinomas added later to the study were GHRH-R-positive. These preliminary results suggest a greater than average GHRH-R expression in ILCs and IDCs associated with casting-type calcifications on the mammogram. Apocrine carcinomas seem uniformly positive for GHRH-R. Whether these findings could indicate a potential role of GHRH-antagonists in targeted treatment of these types of breast cancer requires further studies.

Effects of changes in energy homeostasis and exposure of noxious insults on the expression of orexin (hypocretin) and its receptors in the brain.

This review summarizes data regarding the brain expression of the orexin (hypocretin) system including: prepro-orexin (PPO), orexin A (OxA), orexin B (OxB) and the two orexin receptors 1 and 2 (OxR1, OxR2). Clinical data is limited to OxA and OxB in cerebral spinal fluid and serum/plasma, thus necessitating the development of animal models to undertake mechanistic studies. We focus on changes in animal models that were either exposed to a regime of altered sleep, metabolic energy homeostasis, exposed to drugs and noxious insults. Many more expressional studies are available for PPO, OxA and OxB levels, compared to studies of the receptors. Interestingly, the direction and pattern of change for PPO, OxA and OxB is inconsistent amongst studies, whereas for the receptors, there tends to be increased expression for both OxR1 and OxR2 after alterations in energy homeostasis, and an increased expression after noxious insults or exposure to some drugs. The clinical implications of these results from animal models are discussed in light of the findings from human studies, and future research directions are suggested to fill knowledge gaps with regard to the orexin system, particularly during early brain development.

A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression.

Leptin, ghrelin and neuropeptide W (NPW) modulate vagal afferent activity, which may underlie their appetite regulatory actions. High fat diet (HFD)-induced obesity induces changes in the plasma levels of these peptides and alters the expression of receptors on vagal afferents. We investigated homologous and heterologous receptor regulation by leptin, ghrelin and NPW. Mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Nodose ganglia were cultured overnight in the presence or absence of each peptide. Leptin (LepR), ghrelin (GHS-R), NPW (GPR7) and cholecystokinin type-1 (CCK1R) receptor mRNA, and the plasma leptin, ghrelin and NPW levels were measured. SLD: leptin reduced LepR, GPR7, increased GHS-R and CCK1R mRNA; ghrelin increased LepR, GPR7, CCK1R, and decreased GHS-R. HFD: leptin decreased GHS-R and GPR7, ghrelin increased GHS-R and GPR7. NPW decreased all receptors except GPR7 which increased with HFD. Plasma leptin was higher and NPW lower in HFD. Thus, HFD-induced obesity disrupts inter-regulation of appetite regulatory receptors in vagal afferents.

ß-Defensins activate human mast cells via Mas-related gene X2.

Human ß-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells in vitro and cause increased vascular permeability in rats in vivo. In this study, we sought to determine whether hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells (BMMC) in vitro and had no effect on vascular permeability in vivo. By contrast, these peptides induced sustained Ca(2+) mobilization and substantial degranulation in human mast cells, with hBD3 being more potent. Pertussis toxin (PTx) had no effect on hBD-induced Ca(2+) mobilization, but La(3+) and 2-aminoethoxydiphenyl borate (a dual inhibitor of inositol 1,4,5-triphosphate receptor and transient receptor potential channels) caused substantial inhibition of this response. Interestingly, degranulation induced by hBDs was substantially inhibited by PTx, La(3+), or 2-aminoethoxydiphenyl borate. Whereas human mast cells endogenously express G protein-coupled receptor, Mas-related gene X2 (MrgX2), rat basophilic leukemia, RBL-2H3 cells, and murine BMMCs do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD-induced degranulation, but had no effect on anaphylatoxin C3a-induced response. Furthermore, ectopic expression of MrgX2 in RBL-2H3 and murine BMMCs rendered these cells responsive to hBDs for degranulation. This study demonstrates that hBDs activate human mast cells via MrgX2, which couples to both PTx-sensitive and insensitive signaling pathways most likely involving Gαq and Gαi to induce degranulation. Furthermore, murine mast cells are resistant to hBDs for degranulation, and this reflects the absence of MrgX2 in these cells.

Morphine dependence is associated with changes in neuropeptide S receptor expression and function in rat brain.

Neuropeptide S (NPS) is a newly identified ligand for the previously discovered G-protein coupled receptor 154 now named NPSR. Recently, it has been found that NPSR gene expression is altered during ethanol withdrawal. In this study we tried to elucidate if NPSR gene expression is modified in response to morphine withdrawal and its protracted abstinence. To induce opioid dependence Wistar rats were treated for 7 days with morphine. Twelve hours and 7 days after the last morphine administration brains were removed and the expression of NPSR mRNA was analyzed by in situ hybridization (ISH). Successful induction of opioid dependence was confirmed by the naloxone-precipitated withdrawal test 2 h after the last morphine administration. Moreover, 7 days after the last morphine dose animals were checked for signs of anxiety and for intracerebroventricular (ICV) NPS (0.3 and 1.0 nmol) induced anxiolytic effects by elevated plus maze (EPM). Results showed that in morphine treated rats strong somatic signs of naloxone-precipitated withdrawal occurred. ISH data revealed changes in NPSR gene expression in the ventral tegmental area as well as in the basolateral amygdaloid and bed nucleus of stria terminalis at 12 h and 7 days into abstinence, respectively. At 7 days into abstinence post dependent animals showed higher levels of anxiety than controls which were significantly attenuated by NPS. These results demonstrated that morphine dependence induction led to (i) changes in NPSR mRNA expression; (ii) increased anxiety; and (iii) more potent anxiolytic-like effect of NPS.