The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Cell-selective modulation of the Drosophila neuromuscular system by a neuropeptide

Neuropeptides can modulate physiological properties of neurons in a cell-specific manner. The present work examines whether a neuropeptide can also modulate muscle tissue in a cell-specific manner, using identified muscle cells in third instar larvae of fruit flies. DPKQDFMRFa, a modulatory peptide in the fruit fly Drosophila melanogaster, has been shown to enhance transmitter release from motor neurons and to elicit contractions by a direct effect on muscle cells. We report that DPKQDFMRFa causes a nifedipine-sensitive drop in input resistance in some muscle cells (6 and 7) but not others (12 and 13). The peptide also increased the amplitude of nerve-evoked contractions and compound excitatory junctional potentials (EJPs) to a greater degree in muscle cells 6 and 7 than 12 and 13. Knocking down FMRFa receptor (FR) expression separately in nerve and muscle indicate that both presynaptic and postsynaptic FR expression contributed to the enhanced contractions, but EJP enhancement was due mainly to presynaptic expression. Muscle-ablation showed that DPKQDFMRFa induced contractions and enhanced nerve-evoked contractions more strongly in muscle cells 6 and 7 than cells 12 and 13. In situ hybridization indicated that FR expression was significantly greater in muscle cells 6 and 7 than 12 and 13. Taken together, these results indicate that DPKQDFMRFa can elicit cell-selective effects on muscle fibres. The ability of neuropeptides to work in a cell-selective manner on neurons and muscle cells may help explain why so many peptides are encoded in invertebrate and vertebrate genomes.

La modulation transcriptionnelle du neuropeptide enképhaline par les récepteurs nucléaires Nur77 et RXRγ

Certains neuropeptides (enképhaline et neurotensine) sont des modulateurs du système dopaminergique. Chez les rongeurs, le traitement avec l’antipsychotique typique halopéridol (antagoniste des récepteurs D2), augmente fortement leurs niveaux d’ARNm dans le striatum, une structure centrale du système dopaminergique qui contrôle l’activité locomotrice. Comme l’halopéridol est associé avec de nombreux effets secondaires moteurs, on peut penser que la modulation des neuropeptides est possiblement un mécanisme d’adaptation visant à rétablir l’homéostasie du système dopaminergique après le blocage des récepteurs D2. Cependant, le mécanisme moléculaire de cette régulation transcriptionnelle n’est pas bien compris. Nur77 est un facteur de transcription de la famille des récepteurs nucléaires orphelins qui agit en tant que gène d’induction précoce. Le niveau de son ARNm est aussi fortement augmenté dans le striatum suivant un traitement avec halopéridol. Plusieurs évidences nous suggèrent que Nur77 est impliqué dans la modulation transcriptionnelle des neuropeptides. Nur77 peut former des hétérodimères fonctionnels avec le récepteur rétinoïde X (RXR). En accord avec une activité transcriptionnelle d’un complexe Nur77/RXR, l’agoniste RXR (DHA) réduit tandis que l’antagoniste RXR (HX531) augmente les troubles moteurs induits par un traitement chronique à l’halopéridol chez les souris sauvages tandis que ces ligands pour RXR n’ont aucun effet chez les souris Nur77 nulles. Nos travaux ont révélé que l’antagoniste RXR (HX531) réduit l’augmentation des niveaux d’enképhaline suivant un traitement chronique avec l’halopéridol. Nous avons ensuite démontré la liaison in vitro de Nur77 sur un élément de réponse présent dans le promoteur proximal de la proenképhaline, le peptide précurseur de l’enképhaline. Ces résultats supportent l’hypothèse que Nur77, en combinaison avec RXR, pourrait participer à la régulation transcriptionnelle des neuropeptides dans le striatum et donc contribuer à la neuroadaptation du système dopaminergique suivant un traitement aux antipsychotiques typiques.

Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide mRNA concentrations in growing wethers

The objectives of the present study were 1) to evaluate the effects of supplemental fat and ME intake on plasma concentrations of glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide, ghrelin, and oxyntomodulin; and 2) to determine the association of these peptides with DMI and the hypothalamic concentration of mRNA for the following neuropeptides: neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC). In a completely randomized block design with a 2 x 2 factorial arrangement of treatments, 32 pens with 2 wethers each were restricted-fed (2.45 Mcal/lamb per day) or offered diets ad libitum (n = 16) with or without 6% supplemental fat (n = 16) for a period of 30 d. Dry matter intake was measured daily. On d 8, 15, 22, and 29, BW was measured before feeding, and 6 h after feeding, blood samples were collected for plasma measurement of insulin, GLP-1, CCK, ghrelin, glucose-dependent insulinotropic polypeptide, oxyntomodulin, glucose, and NEFA concentrations. On d 29, blood was collected 30 min before feeding for the same hormone and metabolite analyses. At the end of the experiment, wethers were slaughtered and the hypothalami were collected to measure concentrations of NPY, AgRP, and POMC mRNA. Offering feed ad libitum (resulting in greater ME intake) increased plasma insulin and NEFA concentrations (P = 0.02 and 0.02, respectively) and decreased hypothalamic mRNA expression of NPY and AgRP (P = 0.07 and 0.02, respectively) compared with the restricted-fed wethers. There was a trend for the addition of dietary fat to decrease DMI (P = 0.12). Addition of dietary fat decreased insulin and glucose concentrations (P < 0.05 and 0.01, respectively) and tended to increase hypothalamic mRNA concentrations for NPY and AgRP (P = 0.07 and 0.11, respectively). Plasma GLP-1 and CCK concentrations increased in wethers offered feed ad libitum compared with restricted-fed wethers, but the response was greater when wethers were offered feed ad libitum and had supplemental fat in the diet (fat x intake interaction, P = 0.04). The prefeeding plasma ghrelin concentration was greater in restricted-fed wethers compared with those offered feed ad libitum, but the concentrations were similar 6 h after feeding (intake x time interaction, P < 0.01). Supplemental dietary fat did not affect (P = 0.22) plasma ghrelin concentration. We conclude that insulin, ghrelin, CCK, and GLP-1 may regulate DMI in sheep by regulating the hypothalamic gene expression of NPY, AgRP, and POMC.

Effects of glucose, propionate and splanchnic hormones on neuropeptide mRNA concentrations in the ovine hypothalamus

The capacity for glucose, propionate or hormones of splanchnic origin to influence appetite by directly regulating the expression of neuropeptides in the feeding centres of the hypothalamus of the ruminant is not described. Therefore, our objective was to measure the direct effect of metabolites (glucose and propionate) or hormones [insulin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and polypeptide YY (PYY)] on hypothalamic mRNA concentrations for neuropeptide Y (NPY), agouti-related peptide (AgRP) and proopiomelanocortin (POMC) following in vitro incubation. Hypothalamic tissue from 4- to 5-month-old lambs was obtained at slaughter and immediately incubated in culture media for 2 h at 36 °C. Treatments included a control Dulbecco’s modified Eagle medium (DMEM) containing 1 mm glucose or DMEM with the following additions: 10 mm glucose, 1 mm propionate, 1 nm insulin, 120 pm GLP-1, 100 pm PYY, 80 pm CCK or 10 mm glucose plus 1 nm insulin. The abundance of mRNA for NPY, AgRP and POMC was measured using quantitative reverse transcriptase PCR. Fisher’s protected LSD test was used to compare changes in relative mRNA concentrations for the hypothalamus incubated in the control media vs. the rest of the treatments. The media containing glucose plus insulin increased POMC mRNA concentration (p < 0.05), but did not affect NPY or AgRP mRNA concentration. There were no effects observed for the other treatments (p > 0.20). Results of the present study are consistent with the concept that effects of propionate on feed intake in ruminants is not mediated through direct effects on the hypothalamus, and that insulin is required for an effect of glucose on hypothalamic POMC expression.

Effect on components of the intestinal microflora and plasma neuropeptide levels of feeding Lactobacillus delbrueckii, Bifidobacterium lactis, and inulin to adult and elderly rats

The aim of this study was to compare the effects of the mixture of Lactobacillus delbrueckii subsp. rhamnosus strain GG, Bifidobacterium lactis Bb12, and inulin on intestinal populations of lactobacilli, bifidobacteria, and enterobacteria in adult and elderly rats fed the same (in quality and quantity) diet. The portal plasma levels of two neuropeptides, neuropeptide Y (NPY) and peptide YY (PYY), were also evaluated to assess the physiological consequences of the synbiotic treatment for the gastrointestinal (GI) tracts of rats of different ages. Adult (n = 24) and elderly (n = 24) male rats were fed the AIN-93 M maintenance diet. After 2 weeks of adaptation, the diet of 12 rats of each age group was supplemented with 8% inulin and with strains GG and Bb12 to provide 2.2 x 10(9) CFU of each strain g(-1) of the diet. Blood and different regions of the GI tract were sampled from all rats after 21 days of the treatment. Treatment with the mixture of strain GG, strain BB12, and inulin induced significantly different changes in the numbers of lactobacilli, bifidobacteria, and enterobacteria of the stomach, small intestine, cecum, and colon microflora. Moreover, the GG, BB12, and inulin mixture increased the concentrations of NPY and PYY for adult rats. For the elderly animals, the PYY concentration was not changed, while the NPY concentration was decreased by treatment with the GG, BB12, and inulin mixture. The results of the present study indicate that the physiological status of the GI tract, and not just diet, has a major role in the regulation of important groups of the GI bacteria community, since even the outcome of the dietary modification with synbiotics depends on the ages of the animals.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.

Brain neuropeptide Y and CCK and peripheral adipokine receptors : temporal response in obesity induced by palatable diet

Objective:
Palatable food disrupts normal appetite regulation, which may contribute to the etiology of obesity. Neuropeptide Y (NPY) and cholecystokinin play critical roles in the regulation of food intake and energy homeostasis, while adiponectin and carnitine palmitoyltransferase (CPT) are important for insulin sensitivity and fatty acid oxidation. This study examined the impact of short- and long-term consumption of palatable high-fat diet (HFD) on these critical metabolic regulators.

Methods:
Male C57BL/6 mice were exposed to laboratory chow (12% fat), or cafeteria-style palatable HFD (32% fat) for 2 or 10 weeks. Body weight and food intake were monitored throughout. Plasma leptin, hypothalamic NPY and cholecystokinin, and mRNA expression of leptin, adiponectin, their receptors and CPT-1, in fat and muscles were measured.

Results:
Caloric intake of the palatable HFD group was 2&ndash;3 times greater than control, resulting in a 37% higher body weight. Fat mass was already increased at 2 weeks; plasma leptin concentrations were 2.4 and 9 times higher than control at 2 and 10 weeks, respectively. Plasma adiponectin was increased at 10 weeks. Muscle adiponectin receptor 1 was increased at 2 weeks, while CPT-1 mRNA was markedly upregulated by HFD at both time points. Hypothalamic NPY and cholecystokinin content were significantly decreased at 10 weeks.

Conclusion:
Palatable HFD induced hyperphagia, fat accumulation, increased adiponectin, leptin and muscle fatty acid oxidation, and reduced hypothalamic NPY and cholecystokinin. Our data suggest that the adaptive changes in hypothalamic NPY and muscle fatty acid oxidation are insufficient to reverse the progress of obesity and metabolic consequences induced by a palatable HFD.

Noradrenaline, but not neuropeptide Y, is elevated in cerebrospinal fluid from the third cerebral ventricle following audiovisual stress in gonadectomised rams and ewes

There are sex differences in the activation of the hypothalamo-pituitary-adrenal axis in response to stress, but the source of these differences is unknown. The hypothalamo-pituitary-adrenal axis is regulated by corticotropin-releasing hormone and arginine-vasopressin neurones located in the paraventricular nucleus and these, in turn, are regulated by neural systems that include afferent noradrenergic and neuropeptide Y (NPY)-producing neural pathways. We tested the hypothesis that concentrations of noradrenaline and NPY will be elevated in cerebrospinal fluid (CSF) sampled from the third cerebral ventricle in response to stress, and these responses will differ in males and females. We collected concurrent samples of CSF (1 ml) from the third ventricle and blood (5 ml) from the jugular vein from gonadectomised rams (n = 7) and ewes (n = 5) at 10-min intervals for 3 h. This procedure was conducted on a day when no stress was imposed and on a day when audiovisual stress was imposed for 5 min after 1 h of sampling. Following the audiovisual stress, plasma concentrations of cortisol and CSF concentrations of noradrenaline were elevated (p < 0.05), but CSF concentrations of NPY did not change. Adrenaline was not detected in samples of CSF. The rise in plasma cortisol following the stress was greater (p < 0.05) in ewes than in rams, but there were no sex differences in the rise in noradrenaline. Basal concentrations of NPY in the CSF were higher (p < 0.05) in rams than in ewes. We conclude that the sex differences in the stress-induced activity of the hypothalamo-pituitary-adrenal axis in sheep are not likely to be due to differences in the level of noradrenergic and/or NPY input to the hypothalamus.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship between levels of neuropeptide Substance P in periodontal disease and chronic pain: a literature review

The aim of the current review was to investigate the relationship between levels of neuropeptide Substance P in periodontal disease and chronic pain. Substance P is a neuropeptide that is directly related with pain. In periodontal disease, it is expressed during the inflammatory process, and is one of the factors responsible for bone resorption. Studies have shown that Substance P levels are highest in the gingival crevicular fluid from sites with active periodontal disease and bone loss. The persistence of these substances could be sufficient to stimulate neurogenic inflammation in susceptible tissues, and cause pain. The scientific literature shows that Substance P expressed during periodontal disease can be a risk factor for patients with systemic inflammatory pathologies, such as chronic arthritis or rheumatoid arthritis. Additional research is needed to confirm the participation of this substance in the origin of some types of chronic pain.