Effect of central administration of QRFP(26) peptide on energy balance and characterization of a second QRFP receptor in rat.

The recently identified neuropeptide QRFP(26) is predominantly expressed in the hypothalamus and was suggested to play a role in the regulation of food intake following the observation of an acute orexigenic effect after central administration in mice. QRFP(26) exerts its effect via GPR103 and a newly identified receptor in mouse. The aim of our study was (a) to investigate the distribution of QRFP(26) and a newly discovered QRFP receptor mRNA in rat and (b) to further characterize the effects of central administration of QRFP(26) on energy balance in rats. QRFP(26) mRNA was detected in the retrochiasmatic nucleus, periventricular nucleus, arcuate nucleus and restricted areas of the lateral nucleus of the hypothalamus. We found an additional receptor with high homology for GPR103 in rat. This receptor increases inositol triphosphate production in transfected cells in presence of QRFP(26) and its mRNA was particularly enriched in ventral and posterior thalamic groups, anterior hypothalamus and medulla. When QRFP(26) (10 microg and 50 microg) was administered centrally before the start of the light phase both doses increased food intake for 2 h after injection without reaching statistical significance. QRFP(26) caused no changes in locomotor activity or energy expenditure. In summary, central QRFP(26) injection causes slight and transient hyperphagia in rats without changing any other energy balance parameters after 24 h. We conclude that QRFP(26) has limited impact on the central regulation of energy balance in rats and that its essential function remains to be clarified.

Orphanin FQ causes contractions via inhibiting purinergic pathway in the rat colon.

BACKGROUND & AIMS: We have previously shown that orphanin FQ (OFQ) preferentially stimulates muscle contraction in the rat colon. However, the mechanism of action of OFQ remains unclear. METHODS: We studied the effects of OFQ on muscle contractions and inhibitory junction potentials (IJPs) in rat colon. The site of action of OFQ was also investigated by in situ hybridization of OFQ receptors. RESULTS: OFQ (10(-10) to 10(-6) mol/L) caused circular muscle contractions that were blocked by tetrodotoxin (10(-7) mol/L), suggesting the contractions were nerve mediated. Suramin (a nonselective P(2)-purinoceptor antagonist; 10(-4) mol/L) and reactive blue 2 (a P(2Y)-purinoceptor antagonist; 3 x 10(-5) mol/L), but not pyridoxalphosphate-6-azophenyl-2',4' disulfonic acid (PPADS; a P(2X)-purinoceptor antagonist; 3 x 10(-5) mol/L), abolished OFQ-induced colonic contractions. Focal stimulation of interganglionic fiber tracts evoked biphasic IJPs in colonic circular muscle cells. Suramin and reactive blue 2 inhibited the peak amplitude of the IJP, whereas PPADS had no effect. Cumulative addition of OFQ (10(-10) to 10(-6 )mol/L) significantly inhibited the IJPs. In situ hybridization revealed that OFQ receptor messenger RNA was expressed in the colonic myenteric plexus but not in the smooth muscle cells, suggesting that the site of action of OFQ is neuronal. CONCLUSIONS: These results suggest that OFQ causes muscle contractions by inhibiting purinergic inhibitory motorneurons in the rat colon.

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting.

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.

Opioid peptide mRNA expression in the colon of the rat.

Since the discovery of opioid peptides, several immunohistochemical and radioimmunological studies have demonstrated their localization in the gastrointestinal tract without demonstrating the localization of their common precursor. The present study describes the distribution and the colocalization of proenkephalin and prodynorphin messenger RNAs (mRNAs) in the colon of rat by in situ hybridization. Proenkephalin and prodynorphin mRNAs were found in myenteric plexus, but not in the submucous plexus or in the mucosa. In myenteric plexus, the number of neurons expressing proenkephalin is 2.5 times greater than that of the neurons expressing only prodynorphin. Furthermore, double in situ hybridization histochemistry indicates that at least three groups of opioid neurons can be distinguished, those containing proenkephalin and prodynorphin mRNAs together, and those containing only proenkephalin mRNA or only prodynorphin mRNA.

Anatomy of an endogenous antagonist: relationship between Agouti-related protein and proopiomelanocortin in brain.

Agouti-related protein (AGRP) is a recently discovered orexigenic neuropeptide that inhibits the binding and action of alpha-melanocyte-stimulating hormone derived from proopiomelanocortin (POMC) at the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) and has been proposed to function primarily as an endogenous melanocortin antagonist. To better understand the interplay between the AGRP and melanocortin signaling systems, we compared their nerve fiber distributions with each other by immunohistochemistry and their perikarya distribution with MC3R and MC4R by double in situ hybridization. Although deriving from distinct cell groups, AGRP and melanocortin terminals project to identical brain areas. Both AGRP and melanocortin neurons selectively express the MC3R, which provides a neuroanatomical basis for a dual-input circuit with biological amplification and feedback inhibition. These studies highlight a broader complexity in POMC-mediated behavior in the brain.

Physiological and anatomical circuitry between Agouti-related protein and leptin signaling.

Agouti-related protein (AGRP) is an orexigenic neuropeptide that acts via central melanocortin receptors, and whose messenger RNA (mRNA) levels are elevated in leptin-deficient mice. Fasting associated with a decline in circulating leptin normally causes a 15-fold elevation of hypothalamic Agrp mRNA levels but has no effect in leptin-deficient mice. Chronic hyperleptinemia associated with the tubby and Cpe(fat) mutations has no effect on Agrp mRNA levels, but short term leptin administration causes a 17% reduction of Agrp mRNA levels in nonmutant mice and a 700% reduction in leptin-deficient mice. In young nonobese animals, melanocortin receptor blockade associated with the Ay mutation causes complete resistance to leptin-induced weight loss. Dual in situ hybridization reveals that Agrp-expressing neurons in the medial portion of the arcuate nucleus constitute a subpopulation different from Pomc-expressing neurons, and that a significant proportion of Agrp-expressing neurons (10-25%) coexpresses the leptin receptor, Lepr-b. Immunocytochemistry confirms distinct locations of AGRP- and POMC-expressing cell bodies, but reveals an overlapping distribution of their terminal fields in the arcuate nucleus, the paraventricular hypothalamus, and the dorsomedial hypothalamus. These results suggest that in the fed state, AGRP is normally suppressed by leptin, and that release of this suppression during fasting leads to increased ingestive behavior.

Functional significance of a newly discovered neuropeptide, orphanin FQ, in rat gastrointestinal motility.

BACKGROUND & AIMS: Orphanin FQ (OFQ) is a recently discovered neuropeptide that structurally resembles an opioid peptide. However, the functional role of OFQ in rat gastrointestinal tract remains unknown. METHODS: We investigated the effects of OFQ on contractions of muscle strips obtained from different regions of the gastrointestinal tract. Immunohistochemical studies were performed on rat colonic tissue using OFQ antibody. RESULTS: OFQ (10(-9) to 10(-7) mol/L) caused significant contractions in the rat colon but not in the stomach or small intestine. Tetrodotoxin, veratridine, and long-term serosal application of benzalkonium chloride completely abolished OFQ-induced colonic contractions without affecting myogenic contractions in response to carbachol. OFQ-induced contractions were not affected by naloxone, atropine, phentolamine, propranolol, methysergide, substance P antagonist, vasoactive intestinal polypeptide antagonist, apamin, and NG-nitro-L-arginine methyl ester. OFQ (10(-9) to 10(-7) mol/L) significantly reduced muscle contractions and 3H-acetylcholine release in response to electrical field stimulation in both the stomach and small intestine but not in the colon. OFQ-immunopositive neuronal fibers were found in the colonic myenteric plexus. CONCLUSIONS: These studies indicate that the mechanisms and sites of action of OFQ are region specific. OFQ inhibits cholinergic transmission in the stomach and small intestine, whereas OFQ stimulates colonic contraction possibly by inhibiting an inhibitory neural pathway within the myenteric plexus.

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide.

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.

Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract.

Several pharmacological and electrophysiological studies have shown that the opioid receptors are widely distributed in the gastrointestinal tract. Despite such consensus, there are conflicting findings regarding their effects in intestinal function, and their precise site of action remained unclear. The aim of the present study was therefore to delineate the cellular localization of mu and kappa opioid receptors in rat gastrointestinal tract using polyclonal antibodies generated to C-terminal end of the cloned mu (63 amino acids) and kappa (41 amino acids) receptors. The distribution of mu differs from that of kappa receptors within the gastrointestinal wall, with a greater abundance of mu receptor-like immunoreactive fibres in all intestinal layers. Numerous neurons expressing mu receptor-like proteins were found in the submucosal plexus with comparatively few in the myenteric plexus. In contrast, a higher number of neurons expressing kappa receptor-like immunoreactivity were visualized in the myenteric plexus with a small number in the submucosal plexus. A high number of immunopositive neurons were found in the myenteric plexus of the stomach and the proximal colon with both antibodies. In the submucosal and mucosal layers. mu receptor-immunoreactive fibres were more abundant and distributed around the crypts, blood vessels and lymphatic nodes. Interestingly, numerous mu and fewer kappa receptor-immunoreactive interstitial cells are localized in the region of myenteric plexus and at the internal border of the circular muscle. Finally, smooth muscle cells did not demonstrate any mu- nor kappa-receptor immunoreactivity. These findings suggest that in the rat gastrointestinal tract, mu and kappa opioid receptors may directly influence neuronal and interstitial cell activity. This appears not to be the case for the smooth muscle cells. In the muscular layers, the anatomical data point to mu receptor actions being mediated by nerve terminals, whereas kappa receptor effects may be mediated by both nerve terminals and somatodendritic synaptic mechanisms. In contrast, in the submucosal and mucosal layers, mu receptors predominate and are localized on both nerve terminals and somatodendritic synaptic elements.

Opioid receptor expression in the rat gastrointestinal tract: a quantitative study with comparison to the brain.

The present study was undertaken to analyze the expression of two opioid receptor genes (mu and kappa) in different gastrointestinal regions of the rat. A combination of mRNA quantification and immunohistochemical visualization was used to characterize their expression. Using naive animals, RNA was extracted from tissues and used in RNase protection assays: both receptor mRNAs were expressed in all investigated areas but displayed different expression profiles across the various regions of the digestive tract. Stomach and proximal colon appeared to have the highest expression levels of both receptors, whereas the lowest expression levels were found in the duodenum. Expression levels for both receptors were always lower in the gastrointestinal tract compared to the brain. However, the kappa-receptor expression in the proximal colon represented 40% of the amount found in the brain, which is almost 4 times as high as the respective mu-receptor expression. In contrast to smooth muscle cells, myenteric plexus perikarya of the rat stomach and colon were immunoreactive with antibodies raised against the C-termini of both kappa- and mu-opioid receptors. Numerous nerve fibers were also immunoreactive for both mu- and kappa-receptors and distributed in the longitudinal and circular muscle layers. Small perikarya immunoreactive for mu-receptor were localized around the myenteric plexus and at the submucosal border of the circular muscle, whereas only few perikarya were immunoreactive for the kappa-receptor. We conclude that at least in rat stomach and colon, mu- and kappa-opioid receptors may directly control neuronal communication but seem to have no direct influence on smooth muscle cells.

Distribution of enkephalin-like immunoreactivity in the cat digestive tract.

Immunohistochemical investigations were carried out to determine the pattern of distribution of methionine- and leucine-enkephalin-like materials in the cat pylorus, duodenum, ileum and proximal and distal colon. The present results indicate that leucine-enkephalin-like materials are less densely distributed than methionine-enkephalin-like materials, but that the two patterns of distribution show some similarities. Considerable regional differences exist however in the distribution of these enkephalin-like materials in the muscular layers. In the duodenum, ileum and proximal colon, the immunoreactivity was mainly confined to the myenteric plexus and the circular muscle layer, where it was present in nerve cell bodies and in numerous fibres. In the longitudinal muscle and submucous layers, a few immunoreactive fibres were observed which sometimes surrounded blood vessels. In the pylorus and the distal colon, however, numerous immunoreactive fibres were observed in the longitudinal and circular muscle layers; the immunoreactivity was detected in the cell bodies of numerous myenteric plexus neurons but those of only a few submucous plexus neurons. In addition, the pylorus tissues contained immunoreactive plexi which were localized either within the longitudinal muscle or between the serosa and the longitudinal muscle layer. These plexi were connected to the myenteric plexus by immunoreactive nerve strands. In all the small intestinal segments studied, numerous immunoreactive varicosities were present in the deep muscular plexus, in the inner part of the circular muscle layer. Our results suggest that in cats, the nervous control of external muscular layers mediated by enkephalins shows regional differences. In the pylorus and the distal colon, it involves both the longitudinal and circular muscle layers, whereas in other intestinal segments, only the circular muscle layer is involved.

Distribution of enkephalin immunoreactivity in sympathetic prevertebral ganglia and digestive tract of guinea-pigs and rats.

The aim of the present study was to determine the distribution of methionine-enkephalin (ME) and leucine-enkephalin (LE) immunoreactivity in the sympathetic prevertebral ganglia (coeliac plexus and inferior mesenteric ganglion) and in the myenteric plexus-muscular layer complex of the digestive tract in guinea-pigs and rats. This study was performed using the same immunological approaches including radioimmunoassays and HPLC characterization as those used previously on cats in order to be able to make inter-region and inter-species comparisons. In rat and guinea-pig prevertebral ganglia, the distributions of the enkephalin immunoreactivities were comparable and were characterized by a low ME/LE concentration ratio, of less than 1. In the digestive tract of rats, the enkephalin immunoreactivities were homogeneously distributed, whereas in guinea-pigs, they were found to be very low in the lower oesophageal sphincter and high in the duodenum. In both species, the ME/LE concentration ratio was around 2. The ME/LE concentration ratio determined in the present study in peripheral nervous structures was much lower than that determined previously in the rat brain. Radioimmunoassay and biochemical data might indicate that different mechanisms are responsible for the processing and/or degradation of enkephalins in the central and peripheral nervous systems. The present study provides further evidences that there are tissue- and species-dependent differences in the distribution of enkephalin immunoreactivities. These differences should be taken into consideration when dealing with the effects and the role of enkephalins in the nervous control of intestinal motility in mammals.

Changes in enkephalin immunoreactivity of sympathetic ganglia and digestive tract of the cat after splanchnic nerve ligation.

The aim of the present study was to analyze changes in the enkephalin immunoreactivity of sympathetic prevertebral ganglia coeliac plexus and inferior mesenteric ganglion) and intestinal tract (myenteric plexus and external muscle layers) in cats 2 days after left thoracic splanchnic nerve ligation, using radioimmunoassay and immunohistochemical techniques. Specific polyclonal antibodies directed against methionine- and leucine-enkephalin were used. The nerve ligation led to a considerable increase in the enkephalin immunoreactivity in the cranial part of the ligated nerves. This finding confirms the presence, in the cat, of an enkephalin output originating from thoracic spinal structures which are probably enkephalin-containing preganglionic neurons. In prevertebral ganglia the nerve ligation induced a marked decrease in the enkephalin immunoreactivity, which was probably due to the interruption of thoracic enkephalin efferents projecting towards both the coeliac plexus and the inferior mesenteric ganglion. In the digestive tract, the nerve ligation depressed the methionine-enkephalin immunoreactivity only in the gastro-duodenal region, and had no effect on the ileo-colonic region. The results of the present study add to the growing evidence that the sympathetic nervous system is involved in regulating the enteric enkephalinergic innervation, which is probably involved in controlling the intestinal motility.

Enkephalin-containing neurons in the inferior mesenteric ganglion projecting to the distal colon of cat: evidence from combined retrograde tracing by fluorescent microspheres and immunohistochemistry.

Retrograde tracing with rhodamine fluorescent microspheres combined with fluorescein immunolabelling of methionine-enkephalin showed the presence of enkephalin-like material in neurons of the inferior mesenteric ganglion (sympathetic prevertebral ganglion) projecting to the distal colon in cat. Two weeks after injecting the microspheres into the wall of the distal colon, the inferior mesenteric ganglion was dissected out and incubated for 24 hours in a colchicine-containing culture medium in order to facilitate the detection of enkephalins in the soma of ganglion neurons. It was observed that retrogradely labelled ganglion cells contained enkephalin-like immunoreactive material. These ganglion cells corresponded to enkephalin-like postganglionic neurons, the terminals of which were located inside the wall of the distal colon. These enkephalin-like neurons were numerous and scattered throughout the ganglion. Sometimes enkephalin-like immunoreactive fibers, probably originating from spinal preganglionic neurons, ran close to immunoreactive and non-immunoreactive retrogradely labelled ganglion cells. This suggests that enkephalin-like immunoreactive fibers may make synaptic connections with enkephalin-like and non-enkephalin-like postganglionic neurons projecting to the distal colon. The present study establishes for the first time the existence of an enkephalin-like postganglionic pathway to the digestive tract originating from a sympathetic prevertebral ganglion. This finding indicates that the enkephalinergic innervation of the cat digestive tract may have at least two possible sources: (i) the sympathetic prevertebral ganglia; and (ii) the enteric nervous ganglia.