BQ788 reveals glial ETB receptor modulation of neuronal cholinergic and nitrergic pathways to inhibit intestinal motility: Linked to postoperative ileus.

BACKGROUND AND PURPOSE: ET-1 signalling modulates intestinal motility and inflammation, but the role of ET-1/ETB receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ETB signalling regulates neural-motor pathways of intestinal motility and inflammation. EXPERIMENTAL APPROACH: We studied ETB signalling using: ETB drugs (ET-1, SaTX, BQ788), activity-dependent stimulation of neurons (high K+ -depolarization, EFS), gliotoxins, Tg (Ednrb-EGFP)EP59Gsat/Mmucd mice, cell-specific mRNA in Sox10CreERT2 ;Rpl22-HAflx or ChATCre ;Rpl22-HAflx mice, Sox10CreERT2 ::GCaMP5g-tdT, Wnt1Cre2 ::GCaMP5g-tdT mice, muscle tension recordings, fluid-induced peristalsis, ET-1 expression, qPCR, western blots, 3-D LSM-immunofluorescence co-labelling studies in LMMP-CM and a postoperative ileus (POI) model of intestinal inflammation. KEY RESULTS: In the muscularis externa ETB receptor is expressed exclusively in glia. ET-1 is expressed in RiboTag (ChAT)-neurons, isolated ganglia and intra-ganglionic varicose-nerve fibres co-labelled with peripherin or SP. ET-1 release provides activity-dependent glial ETB receptor modulation of Ca2+ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca2+ responses and excitatory cholinergic contractions, sensitive to L-NAME. Gliotoxins disrupt SaTX-induced glial-Ca2+ waves and prevent BQ788 amplification of contractions. The ETB receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ETB up-regulation, SaTX-hypersensitivity and glial amplification of ETB signalling. In vivo BQ788 (i.p., 1 mg·kg-1 ) attenuates intestinal inflammation in POI. CONCLUSION AND IMPLICATIONS: Enteric glial ET-1/ETB signalling provides dual modulation of neural-motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ETB receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. METHODS: Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. RESULTS: Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. CONCLUSION AND INFERENCES: The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

Kokumi Taste Active Peptides Modulate Salt and Umami Taste.

Kokumi taste substances exemplified by γ-glutamyl peptides and Maillard Peptides modulate salt and umami tastes. However, the underlying mechanism for their action has not been delineated. Here, we investigated the effects of a kokumi taste active and inactive peptide fraction (500-10,000 Da) isolated from mature (FIIm) and immature (FIIim) Ganjang, a typical Korean soy sauce, on salt and umami taste responses in humans and rodents. Only FIIm (0.1-1.0%) produced a biphasic effect in rat chorda tympani (CT) taste nerve responses to lingual stimulation with 100 mM NaCl + 5 µM benzamil, a specific epithelial Na+ channel blocker. Both elevated temperature (42 °C) and FIIm produced synergistic effects on the NaCl + benzamil CT response. At 0.5% FIIm produced the maximum increase in rat CT response to NaCl + benzamil, and enhanced salt taste intensity in human subjects. At 2.5% FIIm enhanced rat CT response to glutamate that was equivalent to the enhancement observed with 1 mM IMP. In human subjects, 0.3% FIIm produced enhancement of umami taste. These results suggest that FIIm modulates amiloride-insensitive salt taste and umami taste at different concentration ranges in rats and humans.

Endoplasmic Reticulum Stress in Subepithelial Myofibroblasts Increases the TGF-ß1 Activity That Regulates Fibrosis in Crohn's Disease.

BACKGROUND: Endoplasmic reticulum (ER) stress is an essential response of epithelial and immune cells to inflammation in Crohn's disease. The presence and mechanisms that might regulate the ER stress response in subepithelial myofibroblasts (SEMFs) and its role in the development of fibrosis in patients with Crohn's disease have not been examined. METHODS: Subepithelial myofibroblasts were isolated from the affected ileum and normal ileum of patients with each Montreal phenotype of Crohn's disease and from normal ileum in non-Crohn's subjects. Binding of GRP78 to latent TGF-ß1 and its subcellular trafficking was examined using proximity ligation-hybridization assay (PLA). The effects of XBP1 and ATF6 on TGF-ß1 expression were measured using DNA-ChIP and luciferase reporter assay. Endoplasmic reticulum stress components, TGF-ß1, and collagen levels were analyzed in SEMF transfected with siRNA-mediated knockdown of DNMT1 and GRP78 or with DNMT1 inhibitor 5-Azacytidine or with overexpression of miR-199a-5p. RESULTS: In SEMF of strictured ileum from patients with B2 Crohn's disease, expression of ER stress sensors increased significantly. Tunicamycin elicited time-dependent increase in GRP78 protein levels, direct interaction with latent TGF-ß1, and activated TGF-ß1 signaling. The TGFB1 DNA-binding activity of ATF-6α and XBP1 were significantly increased and elicited increased TGFB1 transcription in SEMF-isolated from affected ileum. The levels of ER stress components, TGF-ß1, and collagen expression in SEMF were significantly decreased following knockdown of DNMT1 or GRP78 by 5-Azacytidine treatment or overexpression of miR-199a-5p. CONCLUSIONS: Endoplasmic reticulum stress is present in SEMF of patients susceptible to fibrostenotic Crohn's disease and can contribute to development of fibrosis. Targeting ER stress may represent a novel therapeutic target to prevent fibrosis in patients with fibrostenotic Crohn's disease.

Expression and function of umami receptors T1R1/T1R3 in gastric smooth muscle.

BACKGROUND: l-amino acids, such as monosodium glutamate (MSG), activate the umami receptor T1R1/T1R3. We previously showed increased peristalsis in response to activation of T1R1/T1R3 by MSG in mouse colon. However, the expression and function of these receptors in the different regions of the stomach are not clear. METHODS: Mouse gastric smooth muscle cells (SMCs) were isolated and cultured in Dulbecco's Modified Eagle Medium. Expression of T1R1 and T1R3 was measured by RT-PCR and Western blot. The effect of MSG with and without inosine monophosphate (IMP, an allosteric activator of T1R1/T1R3) on acetylcholine (ACh)-induced contraction was measured in muscle strips and isolated SMCs by scanning micrometry. The effect of MSG with or without IMP on activation of G proteins and ACh-induced Ca2+ release was measured in SMCs. KEY RESULTS: Monosodium glutamate inhibited ACh-induced contractions in muscle strips from both antrum and fundus and the effect of MSG was augmented by IMP; the effects were concentration-dependent and not affected by the nitric oxide synthase inhibitor, L-NNA, or tetrodotoxin suggesting a direct effect on SMCs. In isolated gastric SMCs, T1R1 and T1R3 transcripts and protein were identified. Addition of MSG with or without IMP inhibited ACh-induced Ca2+ release and muscle contraction; the effect on contraction was blocked by pertussis toxin suggesting activation of Gαi proteins. MSG in the presence of IMP selectively activated Gαi2 . CONCLUSIONS AND INFERENCES: Umami receptors (T1R1/T1R3) are present on SMCs of the stomach, and activation of these receptors induces muscle relaxation by decreasing [Ca2+ ]i via Gαi2 .

Expression patterns of L-amino acid receptors in the murine STC-1 enteroendocrine cell line.

Regulation of gut function depends on the detection and response to luminal contents. Luminal L-amino acids (L-AA) are detected by several receptors including metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4), calcium-sensing receptor (CaSR), GPRC family C group 6 subtype A receptor (GPRC6A) and umami taste receptor heterodimer T1R1/T1R3. Here, we show that murine mucosal homogenates and STC-1 cells, a murine enteroendocrine cell line, express mRNA for all L-AA receptors. Immunohistochemical analysis demonstrated the presence of all L-AA receptors on STC-1 with CaSR being most commonly expressed and T1R1 least expressed (35% versus 15% of cells); mGluRs and GPRC6a were intermediate (~ 20% of cells). Regarding coexpression of L-AA receptors, the mGluRs and T1R1 were similarly coexpressed with CaSR (10-12% of cells) whereas GPRC6a was coexpressed least (7% of cells). mGluR1 was coexpressed with GPRC6a in 11% of cells whereas coexpression between other receptors was less (2-8% of cells). CaSR and mGluR1 were coexpressed with glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) in 20-25% of cells whereas T1R1 and GPRC6a were coexpressed with GLP-1 and PYY less (8-12% of cells). Only mGluR4 showed differential coexpression with GLP-1 (13%) and PYY (21%). L-Phenylalanine (10 mM) caused a 3-fold increase in GLP-1 release, which was strongly inhibited by siRNA to CaSR indicating functional coupling of CaSR to GLP-1 release. The results suggest that not all STC-1 cells express (and coexpress) L-AA receptors to the same extent and that the pattern of response likely depends on the pattern of expression of L-AA receptors.

Identification of expression and function of the glucagon-like peptide-1 receptor in colonic smooth muscle.

The insulinotropic effects of the incretin hormone, glucagon-like peptide-1 (GLP-1) are mediated via GLP-1 receptors (GLP-1R) present on pancreatic ß cells. GLP-1 causes a decrease in the motility of stomach and intestine which involves both central and peripheral nervous systems. The expression and function of GLP-1R in gastrointestinal smooth muscle, however, are not clear. Muscle strips and isolated muscle cells were prepared from mouse colon and the effect of GLP-1(7-36) amide on acetylcholine (ACh)-induced contraction was measured. Muscle cells in culture were used to identify the expression of GLP-1R and the signaling pathways activated by GLP-1(7-36) amide. GLP-1R was expressed in the mucosal and non-mucosal tissue preparations derived from colon, and in smooth muscle cell cultures devoid of other cells such as enteric neurons. In colonic muscle strips, the addition of GLP-1(7-36) amide caused dose-dependent inhibition of acetylcholine-induced contractions. The effect of GLP-1(7-36) amide was partly inhibited by the neuronal blocker tetrodotoxin and nitric oxide (NO) synthase inhibitor l-NNA suggesting both NO-dependent neural and NO-independent direct effects on smooth muscle. In isolated colonic smooth muscle cells, GLP-1(7-36) amide caused an increase in Gαs activity, cAMP levels, and PKA activity, and inhibited ACh-induced contraction. The effect of GLP-1(7-36) amide on Gαs activity and cAMP levels was blocked by NF449, an inhibitor of Gαs, and the effect of GLP-1(7-36) amide on contraction was blocked by NF449 and myristoylated PKI, an inhibitor of PKA. We conclude that colonic smooth muscle cells express GLP-1R, and GLP-1(7-36) amide inhibits acetylcholine-induced contraction via GLP-1R coupled to the Gαs/cAMP/PKA pathway.

Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase γ and integrin-linked kinase in gastric smooth muscle.

BACKGROUND: Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. METHODS: Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/ßPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio. RESULTS: Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211). CONCLUSION: In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.

Branched Short-Chain Fatty Acid Isovaleric Acid Causes Colonic Smooth Muscle Relaxation via cAMP/PKA Pathway.

BACKGROUND: Isovaleric acid (IVA) is a 5-carbon branched-chain fatty acid present in fermented foods and produced in the colon by bacterial fermentation of leucine. We previously reported that the shorter, straight-chain fatty acids acetate, propionate and butyrate differentially affect colonic motility; however, the effect of branched-chain fatty acids on gut smooth muscle and motility is unknown. AIMS: To determine the effect of IVA on contractility of colonic smooth muscle. METHODS: Murine colonic segments were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with acetylcholine (ACh), and the effects of IVA on ACh-induced contraction were measured in the absence and presence of tetrodotoxin (TTx) or inhibitors of nitric oxide synthase [L-N-nitroarginine (L-NNA)] or adenylate cyclase (SQ22536). The effect of IVA on ACh-induced contraction was also measured in isolated muscle cells in the presence or absence of SQ22536 or protein kinase A (PKA) inhibitor (H-89). Direct activation of PKA was measured in isolated muscle cells. RESULTS: In colonic segments, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion; the IVA response was not affected by TTx or L-NNA but inhibited by SQ22536. Similarly, in isolated colonic muscle cells, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion and the effect blocked by SQ22536 and H-89. IVA also increased PKA activity in isolated smooth muscle cells. CONCLUSIONS: The branched-chain fatty acid IVA acts directly on colonic smooth muscle and causes muscle relaxation via the PKA pathway.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-ß/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-ß1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, ß (α and ß PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

Nicotinic acetylcholine receptor (CHRN) expression and function in cultured human adult fungiform (HBO) taste cells.

In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca2+]i. Nicotine effects on [Ca2+]i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca2+]i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.

Nicotinic acetylcholine receptors (nAChRs) are expressed in Trpm5 positive taste receptor cells (TRCs).

Nicotine evokes chorda tympani (CT) taste nerve responses and an aversive behavior in Trpm5 knockout (KO) mice. The agonists and antagonists of nicotinic acetylcholine receptors (nAChRs) modulate neural and behavioral responses to nicotine in wildtype (WT) mice, Trpm5 KO mice and rats. This indicates that nicotine evokes bitter taste by activating a Trpm5-dependent pathway and a Trpm5-independent but nAChR-dependent pathway. Rat CT responses to ethanol are also partially inhibited by nAChR blockers, mecamylamine and dihydro-ß-erythroidine. This indicates that a component of the bitter taste of ethanol is also nAChR-dependent. However, at present the expression and localization of nAChR subunits has not been investigated in detail in taste receptor cells (TRCs). To this end, in situ hybridization, immunohistochemistry and q-RT-PCR techniques were utilized to localize nAChR subunits in fungiform and circumvallate TRCs in WT mice, Trpm5-GFP transgenic mice, nAChR KO mice, and rats. The expression of mRNAs for α7, ß2 and ß4 nAChR subunits was observed in a subset of rat and WT mouse circumvallate and fungiform TRCs. Specific α3, α4, α7, ß2, and ß4 antibodies localized to a subset of WT mouse circumvallate and fungiform TRCs. In Trpm5-GFP mice α3, α4, α7, and ß4 antibody binding was observed in a subset of Trpm5-positive circumvallate TRCs. Giving nicotine (100 µg/ml) in drinking water to WT mice for 3 weeks differentially increased the expression of α3, α4, α5, α6, α7, ß2 and ß4 mRNAs in circumvallate TRCs to varying degrees. Giving ethanol (5%) in drinking water to WT mice induced an increase in the expression of α5 and ß4 mRNAs in circumvallate TRCs with a significant decrease in the expression of α3, α6 and ß2 mRNAs. We conclude that nAChR subunits are expressed in Trpm5-positive TRCs and their expression levels are differentially altered by chronic oral exposure to nicotine and ethanol.

Matrix Metalloprotease-1 and Elastase Are Novel Uterotonic Agents Acting Through Protease-Activated Receptor 1.

Matrix metalloproteinase-1 (MMP-1) and neutrophil elastase are proteolytic enzymes involved in tissue remodeling, but a role for them as uterotonic agents has not been considered. However, both these proteases activate protease-activated receptor 1 (PAR-1) that mediates thrombin-induced contractions. Matrix metalloproteinase-1 and elastase are products of neutrophils that infiltrate intrauterine tissues at the time of labor, so we tested the hypothesis that these proteases might be novel uterotonic agents acting via PAR-1. Decidual tissue was collected from fetal membranes of term not-in-labor (TNL), term labor (TL), and preterm labor (PTL) women and analyzed for gene and protein expression of MMP-1 and neutrophil elastase. Contractile effects of MMP-1 and elastase were tested with uterine strips of day 19 and 20 gestation rats. Expression of MMP-1 and neutrophil elastase was increased in TL and PTL as compared to TNL. Expression of both the pro- and active enzymes of MMP-1 increased progressively from TNL to TL to PTL. Tumor necrosis factor-α, a neutrophil product, increased MMP-1 in decidual and myometrial cells. Both MMP-1 and elastase stimulated strong contractions of myometrial strips, which were prevented by inhibition of PAR-1 and inhibition of inositol trisphosphate receptor or calcium channel blockade. Indomethacin did not prevent protease-induced contractions. These data suggest that MMP-1 and neutrophil elastase may be important but heretofore unrecognized players in stimulating uterine contractions at the time of labor, and they may explain why indomethacin delays, but does not prevent, PTL because indomethacin inhibits the prostaglandin component but not the protease component of labor.

Diabetes-induced oxidative stress mediates upregulation of RhoA/Rho kinase pathway and hypercontractility of gastric smooth muscle.

The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.

Augmentation of cGMP/PKG pathway and colonic motility by hydrogen sulfide.

Hydrogen sulfide (H2S), like nitric oxide (NO), causes smooth muscle relaxation, but unlike NO, does not stimulate soluble guanylyl cyclase (sGC) activity and generate cyclic guanosine 5'-monophosphate (cGMP). The aim of this study was to investigate the interplay between NO and H2S in colonic smooth muscle. In colonic smooth muscle from rabbit, mouse, and human, l-cysteine, substrate of cystathionine-γ-lyase (CSE), or NaHS, an H2S donor, inhibited phosphodiesterase 5 (PDE5) activity and augmented the increase in cGMP levels, IP3 receptor phosphorylation at Ser1756 (measured as a proxy for PKG activation), and muscle relaxation in response to NO donor S-nitrosoglutathione (GSNO), suggesting augmentation of cGMP/PKG pathway by H2S. The inhibitory effect of l-cysteine, but not NaHS, on PDE5 activity was blocked in cells transfected with CSE siRNA or treated with CSE inhibitor d,l-propargylglycine (dl-PPG), suggesting activation of CSE and generation of H2S in response to l-cysteine. H2S levels were increased in response to l-cysteine, and the effect of l-cysteine was augmented by GSNO in a cGMP-dependent protein kinase-sensitive manner, suggesting augmentation of CSE/H2S by cGMP/PKG pathway. As a result, GSNO-induced relaxation was inhibited by dl-PPG. In flat-sheet preparation of colon, l-cysteine augmented calcitonin gene-related peptide release in response to mucosal stimulation, and in intact segments, l-cysteine increased the velocity of pellet propulsion. These results demonstrate that in colonic smooth muscle, there is a novel interplay between NO and H2S. NO generates H2S via cGMP/PKG pathway, and H2S, in turn, inhibits PDE5 activity and augments NO-induced cGMP levels. In the intact colon, H2S promotes colonic transit.NEW & NOTEWORTHY Hydrogen sulfide (H2S) and nitric oxide (NO) are important regulators of gastrointestinal motility. The studies herein provide the cross talk between NO and H2S signaling to mediate smooth muscle relaxation and colonic transit. H2S inhibits phosphodiesterase 5 activity to augment cGMP levels in response to NO, which, in turn, via cGMP/PKG pathway, generates H2S. These studies suggest that interventions targeted at restoring NO and H2S homeostasis within the smooth muscle may provide novel therapeutic approaches to mitigate motility disorders.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and ß nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-ß-erythroidine, a α4ß2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and ß4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Enhanced Sensitivity of α3ß4 Nicotinic Receptors in Enteric Neurons after Long-Term Morphine: Implication for Opioid-Induced Constipation.

Opioid-induced constipation is a major side effect that persists with long-term opioid use. Previous studies demonstrated that nicotine-induced contractions are enhanced after long-term morphine exposure in guinea pig ileum. In the present study, we examined whether the increased sensitivity to nicotine could be observed in single enteric neurons after long-term morphine exposure, determined the subunits in mouse enteric neurons, and examined the effect of nicotine in reversing opioid-induced constipation. Nicotine (0.03-1 mM) dose-dependently induced inward currents from a holding potential of -60 mV in isolated single enteric neurons from the mouse ileum. The amplitude of the currents, but not the potency to nicotine, was significantly increased in neurons receiving long-term (16-24 h) but not short-term (10 min) exposure to morphine. Quantitative mRNA analysis showed that nicotinic acetylcholine receptor (nAChR) subunit expression in the mouse ileum was α3 ≥ ß2 > ß4 > α5 > α4 > ß3 > α6. Nicotine-induced currents were obtained in neurons from α7, ß2, α5, and α6 knockout mice. The currents were, however, inhibited by mecamylamine (10 µM) and the α3ß4 blocker α-conotoxin AuIB (3 µM), suggesting that nicotine-induced currents were mediated by the α3ß4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3ß4 nAChR, enhanced fecal pellet expulsion in a dose-dependent manner in mice that received long-term, but not short-term, morphine treatment. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after long-term morphine exposure, and activation of the α3ß4 subtype of nAChR reverses chronic, but not acute, morphine-induced constipation.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis.

The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic ß cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases ß cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic ß cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and ß cells by switching from glucagon to GLP-1 to restore ß cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus.

Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury.

Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Met(fl/fl); Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1-1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Met(fl/fl); Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Met(fl/fl); Wnt1Cre+ mice. Finally, Met(fl/fl); Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT: The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation.