Enteric Neurotransmission Research Articles

The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.

Glucagon-like Peptide-2 Depresses Ileal Contractility in Preparations from Mice through Opposite Modulatory Effects on Nitrergic and Cholinergic Neurotransmission.

Glucagon-like peptide-2 (GLP-2) has been reported to influence gastrointestinal motor responses, exerting a modulatory role on enteric neurotransmission. To our knowledge, no data on GLP-2 effects on the motility of the isolated ileum are available; therefore, we investigated whether GLP-2 affects the contractile activity of mouse ileal preparations and the neurotransmitters engaged. Ileal preparations showed tetrodotoxin (TTX)- and atropine-insensitive spontaneous contractile activity, which was unaffected by the nitric oxide synthesis inhibitor, L-NNA. GLP-2 depressed the spontaneous contractility, an effect that was abolished by TTX or L-NNA and not influenced by atropine. Electrical field stimulation induced TTX- and atropine-sensitive contractile responses, which were reduced in amplitude by GLP-2 even in the presence of L-NNA. Immunohistochemical results showed a significant increase in nNOS-positive fibers in the ileal muscle wall and a significant decrease in ChAT-positive myenteric neurons in GLP-2-exposed preparations. The present results offer the first evidence that GLP-2 acts on ileal preparations. The hormone appears to depress ileal contractility through a dual opposite modulatory effect on inhibitory nitrergic and excitatory cholinergic neurotransmission. From a physiological point of view, it could be hypothesized that GLP-2 inhibitory actions on ileal contractility can increase transit time, facilitating nutrient absorption.

Novel insights into mechanisms of inhibition of colonic motility by loperamide.

It is well known that opiates slow gastrointestinal (GI) transit, via suppression of enteric cholinergic neurotransmission throughout the GI tract, particularly the large intestine where constipation is commonly induced. It is not clear whether there is uniform suppression of enteric neurotransmission and colonic motility across the full length of the colon. Here, we investigated whether regional changes in colonic motility occur using the peripherally-restricted mu opioid agonist, loperamide to inhibit colonic motor complexes (CMCs) in isolated mouse colon. High-resolution video imaging was performed to monitor colonic wall diameter on isolated whole mouse colon. Regional changes in the effects of loperamide on the pattern generator underlying cyclical CMCs and their propagation across the full length of large intestine were determined. The sensitivity of CMCs to loperamide across the length of colon varied significantly. Although there was a dose-dependent inhibition of CMCs with increasing concentrations of loperamide (10 nM - 1 μM), a major observation was that in the mid and distal colon, CMCs were abolished at low doses of loperamide (100 nM), while in the proximal colon, CMCs persisted at the same low concentration, albeit at a significantly slower frequency. Propagation velocity of CMCs was significantly reduced by 46%. The inhibitory effects of loperamide on CMCs were reversed by naloxone (1 μM). Naloxone alone did not change ongoing CMC characteristics. The results show pronounced differences in the inhibitory action of loperamide across the length of large intestine. The most potent effect of loperamide to retard colonic transit occurred between the proximal colon and mid/distal regions of colon. One of the possibilities as to why this occurs is because the greatest density of mu opioid receptors are located on interneurons responsible for neuro-neuronal transmission underlying CMCs propagation between the proximal and mid/distal colon. The absence of effect of naloxone alone on CMC characteristics suggest that the mu opioid receptor has little ongoing constitutive activity under our recording conditions.

Region-specific remodeling of the enteric nervous system and enteroendocrine cells in the colon of spinal cord injury patients

Patients with spinal cord injury (SCI) suffer from major bowel dysfunction, whose exact pathophysiology, particularly the involvement of the enteric nervous system or epithelial dysfunction is poorly understood. Herein, we aimed to characterize the mucosal biopsies of the right and left colon in SCI patients vs controls (CT): (1) remodeling of key enteric neurotransmitters, (2) remodeling of enteroendocrine cells, and (3) mucosal inflammation compared to those in controls. In SCI, mucosal ACh concentration was lower in the right colon as compared to CT, but no change was observed in the left colon, and AChE expression was lower in both the right and left colons than in CT. While the VIP concentration was similar in the right and left colons, VIP mRNA expression was increased in the right colon and decreased in the left colon, in SCI patients as compared to CT. Interestingly, 5-HT concentration was reduced in the left colon but not in the right colon in SCI patients. Moreover, in SCI patients, as compared to CT, SERT mRNA expression was selectively increased in the left colon while TPH1 mRNA expression was increased in the right and left colons. Although mucosal TNFα and IL-1β mRNA expression did not significantly differ between SCI and CT groups, we identified a significant positive correlation between TNFα and IL-1β mRNA expression and left colon transit time in the SCI group. In conclusion, region-specific changes occur in the enteric neurotransmitter, serotonergic, and inflammatory pathways in the colon of SCI patients. The significant correlations between these pathways and clinical parameters in the left colon further set a scientific basis for designing therapeutic targets to improve colonic motor dysfunction in patients.Biobank information: Spinal cord injury patients: PHRC ConstiCAPE—clinical trial NCT02566746. Controls: Anosain—clinical trial NCT03054415 and biobank of the “Institut des Maladies de l’Appareil Digestif (IMAD)” registered under number DC-2008-402.

Pasteurized Akkermansia muciniphila improves glucose metabolism is linked with increased hypothalamic nitric oxide release

Background and objectivePasteurized Akkermansia muciniphila cells have shown anti-diabetic effects in rodents and human. Although, its primary site of action consists in maintaining the gut barrier function, there are no study exploring if A. muciniphila controls glycemia via a gut to brain axis. Targeting the gut motility represents an alternative pathway to treat hyperglycemia. Here, we tested the impact of pasteurized A. muciniphila on gut motility, gut-brain axis and glucose metabolism. MethodsWe used mice fed a 45% high-fat (HFD) treated or not with pasteurized A. muciniphila MucT during 12 weeks. We measured the effects of the treatment on body weight gain, glucose metabolism (insulin, glycemia, glucose tolerance), gut contraction and enteric neurotransmitter release, and hypothalamic nitric oxide (NO) release. ResultsWe show that pasteurized A. muciniphila exerts positive effects on different metabolic parameters such as body weight, fat mass, insulin, glycemia and glucose tolerance. This could be explained by the ability of pasteurized A. muciniphila supplementation to decrease duodenal contraction and to increase hypothalamic NO release in HFD mice. ConclusionWe demonstrate a novel mode of action of pasteurized A. muciniphila explaining its beneficial impact on the control of glycemia in a preclinical model of type 2 diabetes via gut-brain axis signaling.

Acetylsalicylic Acid Supplementation Affects the Neurochemical Phenotyping of Porcine Duodenal Neurons.

Aspirin (ASA) is a popular nonsteroidal anti-inflammatory drug (NSAID), which exerts its therapeutic properties through the inhibition of cyclooxygenase (COX) isoform 2 (COX-2), while the inhibition of COX-1 by ASA results in the formation of gastrointestinal side effects. Due to the fact that the enteric nervous system (ENS) is involved in the regulation of digestive functions both in physiological and pathological states, the aim of this study was to determine the influence of ASA on the neurochemical profile of enteric neurons in the porcine duodenum. Our research, conducted using the double immunofluorescence technique, proved an increase in the expression of selected enteric neurotransmitters in the duodenum as a result of ASA treatment. The mechanisms of the visualized changes are not entirely clear but are probably related to the enteric adaptation to inflammatory conditions resulting from aspirin supplementation. A detailed understanding of the role of the ENS in the development of drug-induced inflammation will contribute to the establishment of new strategies for the treatment of NSAID-induced lesions.

Interstitial Cells of Cajal and Enteric Nervous System in Gastrointestinal and Neurological Pathology, Relation to Oxidative Stress

The enteric nervous system (ENS) is organized into two plexuses-submucosal and myenteric-which regulate smooth muscle contraction, secretion, and blood flow along the gastrointestinal tract under the influence of the rest of the autonomic nervous system (ANS). Interstitial cells of Cajal (ICCs) are mainly located in the submucosa between the two muscle layers and at the intramuscular level. They communicate with neurons of the enteric nerve plexuses and smooth muscle fibers and generate slow waves that contribute to the control of gastrointestinal motility. They are also involved in enteric neurotransmission and exhibit mechanoreceptor activity. A close relationship appears to exist between oxidative stress and gastrointestinal diseases, in which ICCs can play a prominent role. Thus, gastrointestinal motility disorders in patients with neurological diseases may have a common ENS and central nervous system (CNS) nexus. In fact, the deleterious effects of free radicals could affect the fine interactions between ICCs and the ENS, as well as between the ENS and the CNS. In this review, we discuss possible disturbances in enteric neurotransmission and ICC function that may cause anomalous motility in the gut.

Cinnamic acid regulates the intestinal microbiome and short-chain fatty acids to treat slow transit constipation.

Slow transit constipation (STC) is a disorder with delayed colonic transit. Cinnamic acid (CA) is an organic acid in natural plants, such as Radix Scrophulariae (Xuan Shen), with low toxicity and biological activities to modulate the intestinal microbiome. To explore the potential effects of CA on the intestinal microbiome and the primary endogenous metabolites-short-chain fatty acids (SCFAs) and evaluate the therapeutic effects of CA in STC. Loperamide was applied to induce STC in mice. The treatment effects of CA on STC mice were assessed from the 24 h defecations, fecal moisture and intestinal transit rate. The enteric neurotransmitters: 5-hydroxytryptamine (5-HT) and vasoactive intestinal peptide (VIP) were determined by the enzyme-linked immunosorbent assay. Hematoxylin-eosin and Alcian blue and Periodic acid Schiff staining were used to evaluate intestinal mucosa's histopathological performance and secretory function. 16S rDNA was employed to analyze the composition and abundance of the intestinal microbiome. The SCFAs in stool samples were quantitatively detected by gas chromatography-mass spectrometry. CA ameliorated the symptoms of STC and treated STC effectively. CA ameliorated the infiltration of neutrophils and lymphocytes, increased the number of goblet cells and acidic mucus secretion of the mucosa. In addition, CA significantly increased the concentration of 5-HT and reduced VIP. CA significantly improved the diversity and abundance of the beneficial microbiome. Furthermore, the production of SCFAs [including acetic acid (AA), butyric acid (BA), propionic acid (PA) and valeric acid (VA)] was significantly promoted by CA. The changed abundance of Firmicutes, Akkermansia, Lachnoclostridium, Monoglobus, UCG.005, Paenalcaligenes, Psychrobacter and Acinetobacter were involved in the production of AA, BA, PA and VA. CA could treat STC effectively by ameliorating the composition and abundance of the intestinal microbiome to regulate the production of SCFAs.

SARS-CoV-2 Induces Epithelial-Enteric Neuronal Crosstalk Stimulating VIP Release.

Diarrhea is present in up to 30-50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.

Group I Metabotropic Glutamate Receptors Modulate Motility and Enteric Neural Activity in the Mouse Colon.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of Grm1 (mGlu1) and Grm5 (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.

Soluble dietary fiber and cellulose from Saccharina japonica by-product ameliorate Loperamide-induced constipation via modulating enteric neurotransmitters, short-chain fatty acids and gut microbiota

The effects of soluble dietary fiber (SDF) and cellulose (IDF) from Saccharina japonica by-product and their differences in improving constipation were further clarified in the present study. We demonstrated that SDF was mainly made up of d-mannuronic acid and d-mannose while IDF consisted of d-glucose , which is different from other reported dietary fibers of terrestrial plants. In this research, both SDF and IDF improved fecal-related indicators, gastrointestinal transit rate and histological morphology in Lop-induced mice. Moreover, they could increase the level of antioxidant enzymes (SOD and GSH-Px), restore the expression of enteric neurotransmitters, and maintain the function of ZO-1, JAM-1 as well as Occludin. Interestingly, SDF and IDF had a significant up-regulated effect on the proportion of Muribaculacea, Prevotellaceaen and Lachnospiraceae, which are critical to preserving intestinal immune homeostasis. Besides, they promoted the biosynthesis of short-chain fatty acids (SCFAs). The overall index showed that SDF is more effective for constipation due to its better water retention capacity. Thus, they can be used as a safe dietary supplement for the treatment of chronic or occasional constipation in humans.

Calcium transients in intramuscular interstitial cells of Cajal of the murine gastric fundus and their regulation by neuroeffector transmission.

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca2+ and post-junctional electrical responses to neural stimulation. ICC-IM were highly active, generating intracellular Ca2+ -transients that occurred stochastically, from multiple independent sites in single ICC-IM. Ca2+ -transients were not entrained in single ICC-IM or between neighbouring ICC-IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca2+ -transients in ICC-IM. This inhibitory response was often preceded by a summation of Ca2+ -transients that led to a global rise in Ca2+ . Individual ICC-IM responded to nerve stimulation by a global rise in Ca2+ followed by inhibition of Ca2+ -transients. The inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in intracellular Ca2+ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video-imaging revealed that the rise in Ca2+ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca2+ -transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. KEY POINTS: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC-IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs. Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC-IM were examined. ICC-IM were highly active, generating stochastic intracellular Ca2+ -transients. Stimulation of enteric motor nerves abolished Ca2+ -transients in ICC-IM. This inhibitory response was preceded by a global rise in intracellular Ca2+ . Individual ICC-IM responded to nerve stimulation with a rise in Ca2+ followed by inhibition of Ca2+ -transients. Inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in Ca2+ was inhibited by the muscarinic antagonist atropine. Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca2+ and inhibition of Ca2+ -transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials. The data presented support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

LimosiLactobacillus pentosus Isolated from Mustard Relieves Drug-induced Constipation in Mice Fed a High-fat Diet by Modulating Enteric Neurotransmitter Function.

Functional constipation is one of the most common gastrointestinal disorders. Oxidative stress can aggravate organ dysfunction. Enteric neurotransmitters have significant effects on the regulation of the enteric nervous system and intestinal muscle contraction. Oxidative stress and reduced gastrointestinal motility are considered to be one of the main causes of constipation. This study aimed to investigate whether LimosiLactobacillus pentosus CQZC02 alleviated loperamide hydrochloride (Lop)-induced constipation in mice under high-fat diet (HFD) conditions and to elucidate the underlying mechanism, focusing on enteric neurotransmitters. Four-week-old female BALB/c mice were randomly divided into five groups: normal group (Nor), constipation model group (H-Lop), L. pentosus CQZC02 low-dose group (H-Lop + ZC02L), L. pentosus CQZC02 high-dose group (H-Lop + ZC02H), and LimosiLactobacillus bulgaricus control group (H-Lop + LB). The fecal weight, water content, and total gastrointestinal transit time were measured to determine whether the mice were constipated. Small bowel and colon tissue damage was assessed by hematoxylin and eosin staining, while the degree of damage was determined by double-blind scoring. The levels of serum oxidative stress markers malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase and neurotransmitters motilin, gastrin, substance P, endothelin, somatostatin, and vasoactive intestinal peptide were measured. The gene expression levels of endothelial nitric oxide synthase, inducible nitric oxide synthase, neuronal nitric oxide synthase, nuclear factor kappa-B, and cyclooxygenase-2 in small intestine tissue were calculated. The constipation symptoms of mice in H-Lop group were manifested by a variety of physiological indicators. In addition, compared with the H-Lop group, H-Lop + ZC02H could effectively relieve the symptoms of constipation in mice. In symptom characterization, the mice in the H-Lop + ZC02H group lost weight and increased feces and water content. In functional experiments, gastrointestinal motility was enhanced; the inflammation score of intestinal tissue was decreased, and gene expression levels were modulated; serum oxidative factor levels were modulated, and oxidative stress levels were decreased.

Small intestine neuromuscular dysfunction in a mouse model of dextran sulfate sodium-induced ileitis: Involvement of dopaminergic neurotransmission

AimsAnomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and preclinical models of IBD. Thus, we aimed to evaluate the impact of dextran sodium sulfate (DSS)-induced ileitis on enteric dopaminergic pathways. Materials and methodsMale C57/Bl6 mice (10 ± 2 weeks old) received 2% DSS in drinking water for 5 days and were then switched to regular drinking water for 3 days. To measure ileitis severity inflammatory cytokines (IL-1β, TNFα, IL-6) levels were assessed. Changes in ileal muscle tension were isometrically recorded following: 1) cumulative addition of dopamine on basal tone (0.1–1000 μM); ii) 4-Hz electric field stimulation (EFS) in the presence of 30 μM dopamine with/without 10 μM SCH-23390 (dopamine D1 receptor (D1R) antagonist) or 10 μM sulpiride (D2R antagonist). Immunofluorescence distribution of the neuronal HuC/D protein, glial S100β marker, D1R, and dopamine transporter (DAT) were determined in longitudinal-muscle-myenteric plexus whole-mounts (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. Key findingsDSS caused an inflammatory process in the small intestine associated to dysmotility and altered barrier permeability, as suggested by decreased fecal output and enhanced stool water content. DSS treatment caused a significant increase of DAT and D1R myenteric immunoreactivity as well as of D1R and D2R mRNA levels, accompanied by a significant reduction of dopamine-mediated relaxation, involving primarily D1-like receptors. SignificanceMouse ileitis affects enteric dopaminergic neurotransmission mainly involving D1R-mediated responses. These findings provide novel information on the participation of dopaminergic pathways in IBD-mediated neuromuscular dysfunction.

P014 Impact of experimental ileitis and Toll-Like Receptor 4 signaling on enteric inhibitory neurotransmission

Abstract Background Alterations in Toll-like receptor 4 (TLR4) expression and changes in enteric neurotransmissions influence the onset and severity of inflammatory bowel disease (IBD). We aimed to assess the interaction between the enteric inhibitory pathways and TLR4 signaling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced small intestine inflammation. Methods Male TLR4-/- mice (9±2 weeks old) were intrarectally treated with 2.5% DNBS. Pro-inflammatory cytokines (IL-6 and TNF-α) were measured in ileal samples. Changes in ileal muscle tension were recorded following exposure to: i) 10-Hz electric field stimulation in non-adrenergic-non-cholinergic (NANC) conditions (1 μM guanethidine + 1 μM atropine) with or without 10 µM 1400W (a inducible nitric oxide synthase (iNOS) inhibitor), or 0.1 µM Nω-nitro-L-arginine methyl ester (L-NAME; a pan-NOS inhibitor); ii) 30 μM dopamine or 30 μM SKF38393 (a dopamine 1 receptor (D1R) agonist) or bromocriptine (a D2R agonist). Immunoreactivity of neuronal (HuC/D) and glial (S100β) markers as well as nitrergic chemical coding (i.e. nNOS and iNOS) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. Results No changes in IL-6 and TNF-α ileal mRNA transcripts were found after DNBS administration. The development of ileitis did not affect the NANC-inhibitory response, that was partially reduced in presence of 1400W or L-NAME, suggesting that TLR4 signaling influences NO-mediated inhibitory responses. DNBS-treated mice showed a significant increase in dopamine-induced relaxation (+31%) and in D1R- and D2R-mediated inhibitory responses (by +57% and +75%, respectively). A significant reduction of the total number of HuC/D+ neurons (-25%), with a proportional increase in nNOS+ neurons (+11%) was found in LMMPs of DNBS mice, associated with a significant reduction of S100β immunofluorescence (-12%) and no changes in iNOS immunoreactivity, highlighting a potential involvement of TLR4 signaling in the development of neuroplasticity and reactive gliosis. Conclusion The present data suggest that the severity of small intestine ileitis and neuronal dysfunction depend on the interplay occurring between TLR4 signaling and enteric inhibitory pathways. This evidence further strengthens the importance of characterizing neuroimmune interactions to identify potential molecular mechanism(s) representing putative pharmacological targets for the development of efficacious IBD therapies.

Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy.

Simple SummaryDopamine is involved in a variety of physiological functions in the gastrointestinal tract (GI). Using a Drosophila model, we investigated the effects of dopamine administration on intestinal physiology and gut motility to gain new insights into what could be a potential future promise candidate for GI pharmacology. We investigated whether giving a dopamine-supplemented food medium to adult flies modified the gut contents, defecation rate, and excreta nature. The effects of dopamine on adult gut spontaneous contraction and motility were also studied. We discovered significant gender differences in the effect of dopamine. Drosophila dopamine D1-like receptors (Dop1R1 and Dop1R2) were also displayed by immunohistochemistry to be expressed in all smooth muscles in both larval and adult flies. Furthermore, we showed for the first time that dopamine drives phospholipase C Beta (PLC-β) translocation from the cytoplasm to the plasma membrane in enterocytes. Overall, the data provided new insights into the epidemiology and clinical aspects of neurodegenerative diseases associated with dopamine deficiency, as well as what may be a potential future prospect for GI pharmacotherapy.Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, we used Drosophila as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males’ gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies’ gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. Our findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency.

Neurotransmitters responsible for purinergic motor neurotransmission and regulation of GI motility.

Classical concepts of peripheral neurotransmission were insufficient to explain enteric inhibitory neurotransmission. Geoffrey Burnstock and colleagues developed the idea that ATP or a related purine satisfies the criteria for a neurotransmitter and serves as an enteric inhibitory neurotransmitter in GI muscles. Cloning of purinergic receptors and development of specific drugs and transgenic mice have shown that enteric inhibitory responses depend upon P2Y1 receptors in post-junctional cells. The post-junctional cells that transduce purinergic neurotransmitters in the GI tract are PDGFRα+ cells and not smooth muscle cells (SMCs). PDGFRα+ cells express P2Y1 receptors, are activated by enteric inhibitory nerve stimulation and generate Ca2+ oscillations, express small-conductance Ca2+-activated K+ channels (SK3), and generate outward currents when exposed to P2Y1 agonists. These properties are consistent with post-junctional purinergic responses, and similar responses and effectors are not functional in SMCs. Refinements in methodologies to measure purines in tissue superfusates, such as high-performance liquid chromatography (HPLC) coupled with etheno-derivatization of purines and fluorescence detection, revealed that multiple purines are released during stimulation of intrinsic nerves. β-NAD+ and other purines, better satisfy criteria for the purinergic neurotransmitter than ATP. HPLC has also allowed better detection of purine metabolites, and coupled with isolation of specific types of post-junctional cells, has provided new concepts about deactivation of purine neurotransmitters. In spite of steady progress, many unknowns about purinergic neurotransmission remain and require additional investigation to understand this important regulatory mechanism in GI motility.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Interaction between the Renin–Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

Activation of KCNQ (KV7) K+ channels in enteric neurons inhibits epithelial Cl- secretion in mouse distal colon.

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).