Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon.

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19-22% and 13-17%, respectively, P < 0.001-0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62-85%), whereas 18-38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33-36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

Clinical and Pathological Features of Severe Gut Dysmotility.

Severe gut motility disorders are characterized by ineffective propulsion of intestinal contents. As a result, patients often develop extremely uncomfortable symptoms, ranging from nausea and vomiting along with alterations of bowel habits, up to radiologically confirmed subobstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility due to morphological and functional alterations of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), interstitial cells of Cajal (ICCs) (mesenchymopathy), and smooth muscle cells (myopathy). In this chapter, we highlight some molecular mechanisms of CIPO and review the clinical phenotypes and the genetics of the different types of CIPO. Specifically, we will detail the role of some of the most representative genetic mutations involving RAD21, LIG3, and ACTG2 to provide a better understanding of CIPO and related underlying neuropathic or myopathic histopathological abnormalities. This knowledge may unveil targeted strategies to better manage patients with such severe disease.

Evidence of enteric angiopathy and neuromuscular hypoxia in patients with mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by thymidine phosphorylase (TP) enzyme defect. As gastrointestinal changes do not revert in patients undergone TP replacement therapy, one can postulate that other unexplored mechanisms contribute to MNGIE pathophysiology. Hence, we focused on the local TP angiogenic potential that has never been considered in MNGIE. In this study, we investigated the enteric submucosal microvasculature and the effect of hypoxia on fibrosis and enteric neurons density in jejunal full-thickness biopsies collected from patients with MNGIE. Orcein staining was used to count blood vessels based on their size. Fibrosis was assessed using the Sirius Red and Fast Green method. Hypoxia and neoangiogenesis were determined via hypoxia-inducible-factor-1α (HIF-1α) and vascular endothelial cell growth factor (VEGF) protein expression, respectively. Neuron-specific enolase was used to label enteric neurons. Compared with controls, patients with MNGIE showed a decreased area of vascular tissue, but a twofold increase of submucosal vessels/mm2 with increased small size and decreased medium and large size vessels. VEGF positive vessels, fibrosis index, and HIF-1α protein expression were increased, whereas there was a diminished thickness of the longitudinal muscle layer with an increased interganglionic distance and reduced number of myenteric neurons. We demonstrated the occurrence of an angiopathy in the GI tract of patients with MNGIE. Neoangiogenetic changes, as detected by the abundance of small size vessels in the jejunal submucosa, along with hypoxia provide a morphological basis to explain neuromuscular alterations, vasculature breakdown, and ischemic abnormalities in MNGIE.NEW & NOTEWORTHY Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is characterized by a genetically driven defect of thymidine phosphorylase, a multitask enzyme playing a role also in angiogenesis. Indeed, major gastrointestinal bleedings are life-threatening complications of MNGIE. Thus, we focused on jejunal submucosal vasculature and showed intestinal microangiopathy as a novel feature occurring in this disease. Notably, vascular changes were associated with neuromuscular abnormalities, which may explain gut dysfunction and help to develop future therapeutic approaches in MNGIE.

Quantitative analysis of enteric neurons containing choline acetyltransferase and nitric oxide synthase immunoreactivities in the submucosal and myenteric plexuses of the porcine colon.

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals' intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39-44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10-12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

Enteric neuron density correlates with clinical features of severe gut dysmotility.

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16-77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47-73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn's posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance (P = 0.0005) along with a decrease in the number of myenteric (P < 0.00001) and submucosal (P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range.NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

Insights into the Role of Opioid Receptors in the GI Tract: Experimental Evidence and Therapeutic Relevance.

Opioid drugs are prescribed extensively for pain treatment but when used chronically they induce constipation that can progress to opioid-induced bowel dysfunction. Opioid drugs interact with three classes of opioid receptors: mu opioid receptors (MORs), delta opioid receptors (DOR), and kappa opioid receptors (KORs), but opioid drugs mostly target the MORs. Upon stimulation, opioid receptors couple to inhibitory Gi/Go proteins that activate or inhibit downstream effector proteins. MOR and DOR couple to inhibition of adenylate cyclase and voltage-gated Ca2+ channels and to activation of K+ channels resulting in reduced neuronal activity and neurotransmitter release. KORs couple to inhibition of Ca2+ channels and neurotransmitter release. In the gastrointestinal tract, opioid receptors are localized to enteric neurons, interstitial cells of Cajal, and immune cells. In humans, MOR, DOR, and KOR link to inhibition of acetylcholine release from enteric interneurons and motor neurons and purine/nitric oxide release from inhibitory motor neurons causing inhibition of propulsive motility patterns. MOR and DOR activation also results in inhibition of submucosal secretomotor neurons reducing active Cl- secretion and passive water movement into the colonic lumen. Together, these effects on motility and secretion account for the constipation caused by opioid receptor agonists. Tolerance develops to the analgesic effects of opioid receptor agonists but not to the constipating actions. This may be due to differences in trafficking and downstream signaling in enteric nerves in the colon compared to the small intestine and in neuronal pain pathways. Further studies of differential opioid receptor desensitization and tolerance in subsets of enteric neurons may identify new drug or other treatment strategies of opioid-induced bowel dysfunction.

Prucalopride exerts neuroprotection in human enteric neurons.

Serotonin (5-hydroxytryptamine, 5-HT) and its transporters and receptors are involved in a wide array of digestive functions. In particular, 5-HT4 receptors are known to mediate intestinal peristalsis and recent data in experimental animals have shown their role in neuronal maintenance and neurogenesis. This study has been designed to test whether prucalopride, a well-known full 5-HT4 agonist, exerts protective effects on neurons, including enteric neurons, exposed to oxidative stress challenge. Sulforhodamine B assay was used to determine the survival of SH-SY5Y cells, human enteric neurospheres, and ex vivo submucosal neurons following H2O2 exposure in the presence or absence of prucalopride (1 nM). Specificity of 5-HT4-mediated neuroprotection was established by experiments performed in the presence of GR113808, a 5-HT4 antagonist. Prucalopride exhibited a significant neuroprotective effect. SH-SY5Y cells pretreated with prucalopride were protected from the injury elicited by H2O2 as shown by increased survival (73.5 ± 0.1% of neuronal survival vs. 33.3 ± 0.1%, respectively; P < 0.0001) and a significant reduction of proapoptotic caspase-3 and caspase-9 activation in all neurons tested. The protective effect of prucalopride was reversed by the specific 5-HT4 antagonist GR113808. Prucalopride promotes a significant neuroprotection against oxidative-mediated proapoptotic mechanisms. Our data pave the way for novel therapeutic implications of full 5-HT4 agonists in gut dysmotility characterized by neuronal degeneration, which go beyond the well-known enterokinetic effect.

Chronic constipation in Parkinson's disease: clinical features and molecular insights on the intestinal epithelial barrier.

Background: Chronic constipation (CC) is a severe symptom in Parkinson's disease (PD), with an unclear pathogenesis. Abnormalities of the enteric nervous system (ENS) and/or intestinal epithelial barrier (IEB) may be pathophysiologically relevant in PD patients with CC. We investigated possible molecular changes of the IEB in PD/CCs compared with CCs and controls. Methods: Twelve PD/CCs (2 female, age range 51-80 years), 20 CCs (15 female, age range 27-78 years), and 23 controls (11 female, age range 32-74 years) were enrolled. Ten PD/CCs and 10 CCs were functionally characterized by anorectal manometry (AM) and transit time (TT). Colon biopsies were obtained and assessed for gene and protein expression, and localization of IEB tight junction markers claudin-4 (CLDN4), occludin-1 (OCCL-1), and zonula occludens-1 (ZO-1) by RT-qPCR, immunoblot and immunofluorescence labeling. Results: PD/CCs were clustered in 2 functional categories: patients with delayed TT and altered AM (60%), and a second group showing only modifications in AM pattern (40%). Gene expression of CLDN4, OCCL-1 and ZO-1 was higher in PD/CCs than controls (P<0.05). Conversely, PD/CCs showed a trend to decrease (P>0.05) in CLDN4 and OCCL-1 protein levels than controls, whereas ZO-1 protein was comparable. In PD/CCs compared with controls, decreasing tendency of vasoactive intestinal polypeptide mRNA, protein and immunoreactive fiber density were observed, although the difference was not statistically significant. Conclusion: Transit and anorectal dysfunctions in PD/CCs are associated with difference in ZO-1, OCCL-1 and CLDN4 expression, thus supporting the role of an altered IEB as a contributory mechanism to possible neuronal abnormalities.

Expression and regulation of α-transducin in the pig gastrointestinal tract.

Taste signalling molecules are found in the gastrointestinal (GI) tract suggesting that they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signalling molecule, α-transducin (Gαtran ), throughout the pig GI tract and the peptide content of Gαtran cells. The highest density of Gαtran -immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gαtran -IR cells contained chromogranin A. In the stomach, many Gαtran -IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gαtran -IR and Gαgust -IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in Gαtran -IR cells in the cardiac mucosa (29.3 ± 0.8 versus 64.8 ± 1.3, P < 0.05), pylorus (98.8 ± 1.7 versus 190.8 ± 1.9, P < 0.0 l), caecum (8 ± 0.01 versus 15.5 ± 0.5, P < 0.01), descending colon (17.8 ± 0.3 versus 23 ± 0.6, P < 0.05) and rectum (15.3 ± 0.3 versus 27.5 ± 0.7, P < 0.05). Refeeding restored the control level of Gαtran -IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, Gαtran -IR cells were significantly reduced after refeeding, whereas Gαtran -IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting.

Ligand-induced µ opioid receptor internalization in enteric neurons following chronic treatment with the opiate fentanyl.

Morphine differs from most opiates its poor ability to internalize µ opioid receptors (µORs). However, chronic treatment with morphine produces adaptational changes at the dynamin level, which enhance the efficiency of acute morphine stimulation to promote µOR internalization in enteric neurons. This study tested the effect of chronic treatment with fentanyl, a µOR-internalizing agonist, on ligand-induced endocytosis and the expression of the intracellular trafficking proteins, dynamin and ß-arrestin, in enteric neurons using organotypic cultures of the guinea pig ileum. In enteric neurons from guinea pigs chronically treated with fentanyl, µOR immunoreactivity was predominantly at the cell surface after acute exposure to morphine with a low level of µOR translocation, slightly higher than in neurons from naïve animals. This internalization was not due to morphine's direct effect, because it was also observed in neurons exposed to medium alone. By contrast, D-Ala2-N-Me-Phe4-Gly-ol5-enkephalin (DAMGO), a potent µOR-internalizing agonist, induced pronounced and rapid µOR endocytosis in enteric neurons from animals chronically treated with fentanyl or from naïve animals. Chronic fentanyl treatment did not alter dynamin or ß-arrestin expression. These findings indicate that prolonged activation of µORs with an internalizing agonist such as fentanyl does not enhance the ability of acute morphine to trigger µOR endocytosis or induce changes in intracellular trafficking proteins, as observed with prolonged activation of µORs with a poorly internalizing agonist such as morphine. Cellular adaptations induced by chronic opiate treatment might be ligand dependent and vary with the agonist efficiency to induce receptor internalization.

Enteroendocrine profile of α-transducin immunoreactive cells in the gastrointestinal tract of the European sea bass (Dicentrarchus labrax).

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G(αtran)) and α-gustducin (G(αgust)). This study was aimed at characterising the cells expressing G(αtran) immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G(αtran) immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G(αtran) immunoreactive cells in the stomach contained G(αgust) immunoreactivity. Gastric G(αtran) immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G(αtran) immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G(αtran) and G(αgust) antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract.

Microbiota-Dependent Upregulation of Bitter Taste Receptor Subtypes in the Mouse Large Intestine in High-Fat Diet-Induced Obesity.

Bitter taste receptors (Tas2rs in mice) detect bitterness, a warning signal for toxins and poisons, and are expressed in enteroendocrine cells. We tested the hypothesis that Tas2r138 and Tas2r116 mRNAs are modulated by microbiota alterations induced by a long-term high-fat diet (HFD) and antibiotics (ABX) (ampicillin and neomycin) administered in drinking water. Cecum and colon specimens and luminal contents were collected from C57BL/6 female and male mice for qRT-PCR and microbial luminal 16S sequencing. HFD with/without ABX significantly increased body weight and fat mass at 4, 6, and 8 weeks. Tas2r138 and Tas2r116 mRNAs were significantly increased in mice fed HFD for 8 weeks vs. normal diet, and this increase was prevented by ABX. There was a distinct microbiota separation in each experimental group and significant changes in the composition and diversity of microbiome in mice fed a HFD with/without ABX. Tas2r mRNA expression in HFD was associated with several genera, particularly with Akkermansia, a Gram-negative mucus-resident bacterium. These studies indicate that luminal bacterial composition is affected by sex, diet, and ABX and support a microbial dependent upregulation of Tas2rs in HFD-induced obesity, suggesting an adaptive host response to specific diet-induced dysbiosis.

Morphine induces µ opioid receptor endocytosis in guinea pig enteric neurons following prolonged receptor activation.

BACKGROUND & AIMS: The µ opioid receptor (µOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of µOR affects morphine's ability to induce receptor endocytosis in enteric neurons. METHODS: We compared the effects of morphine, a poor µOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent µOR-internalizing agonist, on µOR trafficking in enteric neurons and on the expression of dynamin and ß-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. RESULTS: Morphine (100 µmol/L) strongly induced endocytosis of µOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 µmol/L) strongly induced internalization of µOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced µOR endocytosis resulted from direct interactions between the ligand and the µOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced µOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect ß-arrestin immunoreactivity. CONCLUSIONS: Chronic activation of µORs increases the ability of morphine to induce µOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.

Protective role of µ opioid receptor activation in intestinal inflammation induced by mesenteric ischemia/reperfusion in mice.

Intestinal ischemia is a clinical emergency with high morbidity and mortality. We investigated whether activation of µ opioid receptor (µOR) protects from the inflammation induced by intestinal ischemia and reperfusion (I/R) in mice. Ischemia was induced by occlusion of the superior mesenteric artery (45 min), followed by reperfusion (5 hr). Sham-operated (SO) and normal (N) mice served as controls. Each group received subcutaneously 1) saline solution, 2) the µOR selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 0.01 mg kg(-1) ), 3) DAMGO and the selective µOR antagonist [H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] (CTAP; 0.1 mg kg(-1) ), or 4) CTAP alone. I/R induced intestinal inflammation as indicated by histological damage and the significant increase in myeloperoxidase (MPO) activity, an index of tissue neutrophil accumulation. Tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) mRNA levels were also increased in I/R mice compared with SO. DAMGO significantly reduced tissue damage, MPO activity, and TNF-α mRNA levels in I/R, and these effects were reversed by CTAP. By contrast, DAMGO did not modify IL-10 mRNA levels or gastrointestinal transit. DAMGO's effects are receptor mediated and likely are due to activation of peripheral µORs, because it does not readily cross the blood-brain barrier. These findings suggest that activation of peripheral µOR protects from the inflammatory response induced by I/R through a pathway involving the proinflammatory cytokine TNF-α. Reduction of acute inflammation might prevent I/R complications, including motility impairment, which develop at a later stage of reperfusion and likely are due to inflammatory cell infiltrates.

Enteric Neuromyopathies: Highlights on Genetic Mechanisms Underlying Chronic Intestinal Pseudo-Obstruction.

Severe gut motility disorders are characterized by the ineffective propulsion of intestinal contents. As a result, the patients develop disabling/distressful symptoms, such as nausea and vomiting along with altered bowel habits up to radiologically demonstrable intestinal sub-obstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility. This syndrome occurs due to changes altering the morpho-functional integrity of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), the interstitial cells of Cajal (ICC) (mesenchymopathy), and smooth muscle cells (myopathy). In the last years, several genes have been identified in different subsets of CIPO patients. The focus of this review is to cover the most recent update on enteric dysmotility related to CIPO, highlighting (a) forms with predominant underlying neuropathy, (b) forms with predominant myopathy, and (c) mitochondrial disorders with a clear gut dysfunction as part of their clinical phenotype. We will provide a thorough description of the genes that have been proven through recent evidence to cause neuro-(ICC)-myopathies leading to abnormal gut contractility patterns in CIPO. The discovery of susceptibility genes for this severe condition may pave the way for developing target therapies for enteric neuro-(ICC)-myopathies underlying CIPO and other forms of gut dysmotility.

Pharmacological characterization of naloxegol: In vitro and in vivo studies.

Opioid-induced constipation is the most prevalent adverse effect of opioid drugs. Peripherally acting mu opioid receptor antagonists (PAMORAs), including naloxegol, are indicated for the treatment of opioid-induced constipation. The aim of this study was the in vitro and in vivo pharmacological characterization of naloxegol in comparison with naloxone. In vitro experiments were performed to measure calcium mobilization in cells coexpressing opioid receptors and chimeric G proteins and mu receptor interaction with G protein and ß-arrestin 2 using bioluminescence resonance energy transfer. In vivo experiments were performed in mice to measure pain threshold using the tail withdrawal assay and colonic transit using the bead expulsion assay. In vitro, naloxegol behaved as a selective and competitive mu receptor antagonist similarly to naloxone, being 3-10-fold less potent. In vivo, naloxone was effective in blocking fentanyl actions when given subcutaneously (sc), but not per os (po). In contrast, naloxegol elicited very similar effects with sc or po administration counteracting in a dose dependent manner the constipating effects of fentanyl without interfering with the fentanyl mediated analgesia. Thus, a useful PAMORA action could be obtained with naloxegol both after po and sc administration.

Novel understanding on genetic mechanisms of enteric neuropathies leading to severe gut dysmotility.

The enteric nervous system (ENS) is the third division of the autonomic autonomic nervous system and the largest collection of neurons outside the central nervous system (CNS). The ENS has been referred to as "the brain in the gut" or "the second brain of the human body" because of its highly integrated neural circuits controlling a vast repertoire of gut functions, including absorption/secretion, splanchnic blood vessels, some immunological aspects, intestinal epithelial barrier, and gastrointestinal (GI) motility. The latter function is the result of the ENS fine-tuning over smooth musculature, along with the contribution of other key cells, such as enteric glia (astrocyte like cells supporting and contributing to neuronal activity), interstitial cells of Cajal (the pacemaker cells of the GI tract involved in neuromuscular transmission), and enteroendocrine cells (releasing bioactive substances, which affect gut physiology). Any noxa insult perturbing the ENS complexity may determine a neuropathy with variable degree of neuro-muscular dysfunction. In this review, we aim to cover the most recent update on genetic mechanisms leading to enteric neuropathies ranging from Hirschsprung's disease (characterized by lack of any enteric neurons in the gut wall) up to more generalized form of dysmotility such as chronic intestinal pseudo-obstruction (CIPO) with a significant reduction of enteric neurons. In this line, we will discuss the role of the RAD21 mutation, which we have demonstrated in a family whose affected members exhibited severe gut dysmotility. Other genes contributing to gut motility abnormalities will also be presented. In conclusion, the knowledge on the molecular mechanisms involved in enteric neuropathy may unveil strategies to better manage patients with neurogenic gut dysmotility and pave the way to targeted therapies.

NIH Workshop Report: sensory nutrition and disease.

In November 2019, the NIH held the "Sensory Nutrition and Disease" workshop to challenge multidisciplinary researchers working at the interface of sensory science, food science, psychology, neuroscience, nutrition, and health sciences to explore how chemosensation influences dietary choice and health. This report summarizes deliberations of the workshop, as well as follow-up discussion in the wake of the current pandemic. Three topics were addressed: A) the need to optimize human chemosensory testing and assessment, B) the plasticity of chemosensory systems, and C) the interplay of chemosensory signals, cognitive signals, dietary intake, and metabolism. Several ways to advance sensory nutrition research emerged from the workshop: 1) refining methods to measure chemosensation in large cohort studies and validating measures that reflect perception of complex chemosensations relevant to dietary choice; 2) characterizing interindividual differences in chemosensory function and how they affect ingestive behaviors, health, and disease risk; 3) defining circuit-level organization and function that link and interact with gustatory, olfactory, homeostatic, visceral, and cognitive systems; and 4) discovering new ligands for chemosensory receptors (e.g., those produced by the microbiome) and cataloging cell types expressing these receptors. Several of these priorities were made more urgent by the current pandemic because infection with sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease of 2019 has direct short- and perhaps long-term effects on flavor perception. There is increasing evidence of functional interactions between the chemosensory and nutritional sciences. Better characterization of this interface is expected to yield insights to promote health, mitigate disease risk, and guide nutrition policy.

Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2.

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and alpha-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)). We sought to identify the mechanism by which amino acids induce Ca(2+) signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na(+)-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca(2+) signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca(2+) signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca(2+) signaling in STC-1 cells was suppressed by removing Na(+) from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca(2+)](i); 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca(2+) signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca(2+)](i) was associated with membrane depolarization and mediated by Ca(2+) influx, since it depended on extracellular Ca(2+); 6) the increase in [Ca(2+)](i) in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1-10 muM) or nitrendipine (1 muM), which block L-type voltage-sensitive Ca(2+) channels. We hypothesize that the inward current of Na(+) associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca(2+) channels that mediate Ca(2+) influx, thereby leading to an increase in the [Ca(2+)](i) in enteroendocrine STC-1 cells.