Effects Of Neuromedin Research Articles

Effects of neuromedin U-8 on stress responsiveness and hypothalamus-pituitary-adrenal axis activity in male C57BL/6J mice

Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure.

Effect of Neuromedin S (NMS) on Growth Hormone and its Relationship with NMS Induced Testosterone Secretion in Male Rhesus Monkeys (Macaca mulatta)

Effect of Neuromedin S (NMS) on Growth Hormone and its Relationship with NMS Induced Testosterone Secretion in Male Rhesus Monkeys (Macaca mulatta)

Effect of neuromedin U on allergic airway inflammation in an asthma model.

Asthma is a major inflammatory airway disease with high incidence and mortality rates. The Global Initiative for Asthma released a report called ‘The Global Burden of Asthma’ in 2004. However, the specific pathogenesis of asthma remains unclear. An increasing number of studies have demonstrated that neuromedin U (NMU) plays a pleiotropic role in the pathogenesis of asthma. NMU is a highly structurally conserved neuropeptide that was first purified from porcine spinal cord and named for its contractile effect on the rat uterus. NMU amplifies type 2 innate lymphoid cell (ILC2)-driven allergic lung inflammation. The NMU receptors (NMURs), designated as NMUR1 and NMUR2, belong to the G protein-coupled receptor family. NMUR1 has also been found in immune cells, including ILC2s, mast cells and eosinophils. In view of the important roles of NMU in the pathogenesis of asthma, the present review evaluates the potential mechanisms underlying the impact of NMU on asthma and its association with asthma therapy.

The effects of neuromedin S on the hypothalamic-pituitary-testicular axis in male pigs in vitro

Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R, LH and FSH mRNAs in anterior pituitary cells, and NMU2R, STAR, P450 and 3β-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction.

Small-Molecule Neuromedin U Receptor 2 Agonists Suppress Food Intake and Decrease Visceral Fat in Animal Models.

Obesity is a growing public health concern, with 37.5% of the adult population in need of therapeutics that are more efficacious with a better side effect profile. An innovative target in this regard is neuromedin U, a neuropeptide shown to suppress food intake and attenuate weight gain in animal models. These effects of neuromedin U on feeding behavior are thought to be related to agonism at the centrally expressed neuromedin U receptor 2 (NMUR2). As peptides present unique challenges that limit their therapeutic potential, the discovery of small‐molecule NMUR2 agonists is needed to validate the targets therapeutic value, but to date, none have been evaluated in any animal model of disease. We therefore assessed two small‐molecule NMUR2 agonists for their in vitro signaling and their in vivo efficacy. The NMUR2 agonists were synthesized and both NMUR2 agonists, NY0116 and NY0128, decreased cAMP while stimulating calcium signaling in stably expressing NMUR2 HEK293 cells. When small‐molecule NMUR2 agonists were tested in vivo, acute administration significantly decreased high‐fat diet consumption. Repeated administration of the compounds decreased body weight and more specifically, decreased the percentage of visceral adipose tissue (VAT) in obese mice. These results have confirmed small‐molecule NMUR2 agonists are efficacious in animal models to decrease fat content, food intake, and body weight, suggesting NMUR2 is a promising therapeutic target for metabolic disorders.

Effects of neuromedin B on steroidogenesis, cell proliferation and apoptosis in porcine Leydig cells.

Neuromedin B (NMB), a mammalian bombesin-related peptide, has numerous physiological functions, including regulating hormone secretions, cell growth, and reproduction, by binding to its receptor (NMBR). In this study, we investigated the effects of NMB on testosterone secretion, steroidogenesis, cell proliferation, and apoptosis in cultured primary porcine Leydig cells. NMBR was mainly expressed in the Leydig cells of porcine testes, and a specific dose of NMB significantly promoted the secretion of testosterone in the primary Leydig cells; moreover, NMB increased the expression of mRNA and/or proteins of NMBR and steroidogenic mediators (steroidogenic acute regulatory (STAR), CYP11A1, and HSD3B1) in the Leydig cells. In addition, specific doses of NMB promoted the proliferation of Leydig cells and increased the expression of proliferating cell nuclear antigen and Cyclin B1 proteins, while suppressing Leydig cell apoptosis and decreasing BAX and Caspase-3 protein expression. These results suggest that the NMB/NMBR system might play an important role in regulating boar reproductive function by modulating steroidogenesis and/or cell growth in porcine Leydig cells.

Possible Role of Neuromedin S (NMS) in Male Reproduction: Effect of NMS on Adipokines Secretion in Male Rhesus Monkeys (Macaca mulatta)

Possible Role of Neuromedin S (NMS) in Male Reproduction: Effect of NMS on Adipokines Secretion in Male Rhesus Monkeys (Macaca mulatta)

1071 Effect of neuromedin u on pig immune regulation

1071 Effect of neuromedin u on pig immune regulation

Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro.

Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

Connective Tissue Growth Factor–Mediated Upregulation of Neuromedin U Expression in Trabecular Meshwork Cells and its Role in Homeostasis of Aqueous Humor Outflow

Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated the effects of Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes in gene expression profile in human trabecular meshwork (HTM) cells. CTGF levels were quantified by immunoblotting and ELISA. CTGF-induced changes in gene expression, actin cytoskeleton, myosin light chain (MLC) phosphorylation, and extracellular matrix (ECM) proteins were evaluated in trabecular meshwork (TM) cells by cDNA microarray, q-PCR, fluorescence microscopy, and immunoblot analyses. The effects of neuromedin U (NMU) on aqueous humor (AH) outflow were determined in enucleated porcine eyes. Expression of a constitutively active form of RhoA (RhoAV14), activation of Rho GTPase by bacterial toxin, or inhibition of Rho kinase by Y-27632 in HTM cells led to significant but contrasting changes in CTGF protein levels that were detectable in cell lysates and cell culture medium. Stimulation of HTM cells with CTGF for 24 hours induced actin stress fiber formation, and increased MLC phosphorylation, fibronectin, and laminin levels, and NMU expression. NMU independently induced actin stress fibers and MLC phosphorylation in TM cells, and decreased AH outflow facility in perfused porcine eyes. These data revealed that CTGF influences ECM synthesis, actin cytoskeletal dynamics, and contractile properties in TM cells, and that the expression of CTGF is regulated closely by Rho GTPase. Moreover, NMU, whose expression is induced in response to CTGF, partially mimics the effects of CTGF on actomyosin organization in TM cells, and decreases AH outflow facility, revealing a potentially important role for this neuropeptide in the homeostasis of AH drainage.

Feeding and drinking response following central administration of neuromedin S in chicks

Neuromedin S (NMS) is recognized as an anorexigenic peptide in the brain of mammals. In chicks ( Gallus gallus), however, the effect of NMS has not been investigated. Therefore, the purpose of the present study was to investigate whether intracerebroventricular (ICV) injection of NMS affected feeding and drinking behavior in chicks. The injection of NMS (0.01–1 nmol) significantly decreased food intake under both ad libitum and food deprivation-induced feeding conditions. However, NMS did not affect water deprivation-induced drinking behavior. ICV injection of NMS stimulated voluntary locomotion and wing-flapping behavior. In addition, we found that those effects of NMS might be related to the hypothalamus–pituitary–adrenal axis because ICV injection of NMS stimulated corticosterone release. The present study suggests that central NMS functions an anorexigenic factor in chicks.

Expression of NMS and NMU2R in the pig reproductive axis during the estrus cycle and the effect of NMS on the reproductive axis in vitro

Evidence has revealed that neuromedin S (NMS) and neuromedin U-receptor type-2 (NMU2R) mRNAs are expressed in the central nervous system and reproductive organs. Previous data indicated that variation of NMS and NMU2R was due to the phases of the adult rat hypothalamus estrus cycle. However, the expression and function of NMS in the pig reproductive axis remains unexplored. In this study, 16 virginal gilts were classified into four groups: proestrus, estrus, diestrus 1, and diestrus 2; the expression of NMS and NMU2R in the cyclic pig hypothalamus–pituitary–ovary axis was studied by reverse transcriptaion-polymerase chain reaction (RT-PCR), and the effect of NMS on the reproductive axis in vitro was detected by radioimmunoassay (RIA). The cloned pig NMS and NMU2R sequences were 82% and 90.2% identical to those of the corresponding human homologues, respectively. RT-PCR showed that NMS and NMU2R mRNA expression in the hypothalamus and pituitary changed with the estrus cycle, i.e., with the highest level in the proestrus group and the lowest in the estrus group. In the ovary, NMS and NMU2R expression was highest in the diestrus 2 group and the lowest in the proestrus group. In the in vitro study, different concentrations of NMS induced the release of gonadotropin-releasing hormone, luteinizing hormone, and estradiol at different levels of the reproductive axis. Taken together, the expression pattern of NMS during the estrus cycle and its role in reproductive hormones in vitro provide novel evidences of the potential roles of NMS in the regulation of pig reproduction.

Effect of neuromedin on activity of lactate dehydrogenase in mitochondrial fraction of the brain in rats with thermal injury

We studied enzyme systems (lactate dehydrogenase) of mitochondria in cerebral nerve cells in experimental encephalopathy developing after thermal injury. In animals receiving neuromedin at the early terms after injury, the ratio of forward to reverse lactate dehydrogenase reactions significantly increased over the first day after injury and returned to normal on day 7.

Effect of neuromedin S on feeding regulation in the Japanese quail

Neuromedin S (NMS) was recently isolated from the brains of humans, mice and rats as an endogenous ligand for the orphan G protein-coupled receptors FM-3 and FM-4, which have been identified as neuromedin U (NMU) receptors 1 and 2, respectively. To investigate the role of NMS in avian species, we elucidated the effect of intracerebroventricular (i.c.v.) administration of rat NMS on food intake, body weight, body temperature and gross locomotor activity in adult Japanese quails. NMS significantly decreased food intake (and consequently body weight) in a time-dependent manner during 12-h light period, but increased both body temperature and gross locomotor activity. On the other hand, i.c.v. injection of rat NMU showed the reverse effects of NMS in Japanese quail. These results suggest that NMS may play an important role in regulating food intake and sympathetic nerve activity in the Japanese quail.

Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions

We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1nmol/3μl) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor.

Neuromedin U depolarizes rat hypothalamic paraventricular nucleus neurons in vitro by enhancing IH channel activity.

The effect of neuromedin U (NMU) on rat paraventricular nucleus (PVN) neurons was examined using whole cell patch-clamp recordings. Under current-clamp, 31% of PVN parvocellular neurons (n = 243) were depolarized by 100 nM NMU, but magnocellular neurons were not affected. NMU (10 nM to 1 microM) resulted in increased basal firing rate and depolarization in a dose-dependent manner with an EC50 of 70 nM. NMU-induced depolarization was unaffected by co-perfusion with 0.5 microM TTX + 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) + 10 microM bicuculline. Extracellular application of 70 microM ZD 7288 completely inhibited NMU-induced depolarization. Under voltage-clamp, 1 microM NMU produced negligible inward current but did increase the hyperpolarization-activated current (IH) at step potentials less than -80 mV. The effects of NMU on IH were voltage-dependent, and NMU shifted the IH conductance-voltage relationship (V1/2) by about 10.8 mV and enhanced IH kinetics without changing the slope constant (k). Extracellular application of 70 microM ZD 7288 or 3 mM Cs+ blocked IH and the effects of NMU in voltage-clamp. These results suggest that NMU selectively depolarizes the subpopulation of PVN parvocellular neurons via enhancement of the hyperpolarization-activated inward current.

Amylase secretion from dispersed human pancreatic acini: neither cholecystokinin a nor cholecystokinin B receptors mediate amylase secretion in vitro.

In humans, cholecystokinin (CCK) stimulates pancreatic secretion, and CCK-A receptor antagonists prevent it in vivo. However, the human pancreas has been reported to express mainly CCK-B receptors. To elucidate this discrepancy. We prepared dispersed acini from human pancreas and examined whether various doses of CCK stimulated the release of amylase, in comparison with the effects of neuromedin C, carbamylcholine, and secretin. Human pancreatic acini did not respond to any dose of CCK or secretin. Amylase release was stimulated by carbamylcholine and neuromedin C dose-dependently and was inhibited by respective antagonists. The localizations of CCK receptors in the human duodenum were determined. High concentrations of CCK-A receptors were detected in the mucosa as well as in smooth muscle of the duodenum by microautoradiography. In conclusion, human pancreatic acinar cells are responsible for carbamylcholine and neuromedin C but not for secretin. Neither CCK-A nor CCK-B receptor mediates amylase release from human pancreatic acini in vitro. Pancreatic secretion in humans in vivo may be regulated indirectly by CCK (via CCK-A receptors).

Nonpeptide neuromedin B receptor antagonists inhibit the proliferation of C6 cells

The ability of nonpeptide antagonists to interact with neuromedin B receptors on C6 cells was investigated. 2-[3-(2,6-Diisopropyl-phenyl)-ureido]3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-proprionate (PD165929), 3-(1 H-indol-3-yl)-2-methyl-2-[3(4-nitro-phenyl)-ureido]- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide (PD168368) and 3-(1 H-indol-3-yl)- N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(4-mitro-phenyl)-ureido]-propionamide (PD176252) inhibited ( 125I-Tyr 0)neuromedin B binding with IC 50 values of 2000, 40 and 50 nM, respectively. Because neuromedin B is a G-protein coupled serpentine receptor, the effects of neuromedin B antagonists on second messenger production and proliferation were investigated. PD168368 inhibited the ability of 10 nM neuromedin B to cause elevation of cytosolic Ca 2+, whereas it had no effect on basal cytosolic Ca 2+. PD168368 inhibited the ability of 100 nM neuromedin B to cause elevation of c- fos mRNA. Also, PD168368 in a dose-dependent manner inhibited the ability of 100 nM neuromedin B to cause phosphorylation of focal adhesion kinase. Using a [3-(4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2 H-tetrazolium bromide] assay, the order of antagonist potency to inhibit C6 proliferation was PD168368=PD176252>PD165929. Also, 1 μM PD168368 and PD176252 significantly inhibited colony number using a proliferation assay in vitro. PD168368 significantly inhibited C6 xenograft growth in nude mice in vivo. These results indicate that PD168368 is a C6 cell neuromedin B receptor antagonist, which inhibits proliferation.

Effects of neuromedins and related peptides on the body temperature of rats.

Neuromedin B (NMB) and neuromedin C (NMC) are peptides found in the mammalian central nervous system, and their concentrations are particularly high in the hypothalamus. The amino acid sequences of these peptides are similar to that of bombesin, which is known to induce marked hypothermia not only in amphibians but also in mammals. The effect of neuromedins and related peptides on the body temperature regulation was examined in the rat under a thermoneutral environment. The findings indicated that NMC caused moderate hypothermia following intracerebroventricular administration, while the effect of NMB was meager, suggesting that these two neuropeptides possess different physiological functions in the brain.

Neuromedin B stimulates arachidonic acid release, c-fos gene expression, and the growth of C6 glioma cells

The effects of neuromedin B (NMB) on C6 glioma cells were investigated. NMB bound with high affinity (IC50 = 1 nM) to C6 cells whereas BN and GRP were less potent (IC50 = 40 and 100 nM). NMB (1 nM) elevated cytosolic Ca2+ in individual C6 cells and the increase in cytosolic Ca2+ was reversed by 1 microM [D-Arg1, D-Pro2,D-Trp7.9,Leu11]substance P [APTTL]SP, a broad spectrum antagonist. NMB stimulated [3H]arachidonic acid release from C6 cells and the increase in [3H]arachidonic acid release was reversed [APTTL]SP. NMB increased transiently c-fos gene expression in C6 cells. NMB increased the number of C6 colonies in soft agar and the increase in growth caused by NMB was reversed by [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of C6 cells.