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Background & Aims Celiac disease is characterized by HLA-DQ2/8-restricted responses of CD4+ T cells to cereal gluten proteins. A diagnosis of celiac disease based on serologic and histologic evidence requires patients to be on gluten-containing diets. The growing number of individuals adhering to a gluten-free diet (GFD) without exclusion of celiac disease complicates its detection. HLA-DQ–gluten tetramers can be used to detect gluten-specific T cells in blood of patients with celiac disease, even if they are on a GFD. We investigated whether an HLA-DQ–gluten tetramer-based assay accurately identifies patients with celiac disease. Methods We produced HLA-DQ–gluten tetramers and added them to peripheral blood mononuclear cells isolated from 143 HLA-DQ2.5 + subjects (62 subjects with celiac disease on a GFD, 19 subjects without celiac disease on a GFD [due to self-reported gluten sensitivity], 10 subjects with celiac disease on a gluten-containing diet, and 52 presumed healthy individuals [controls]). T cells that bound HLA-DQ–gluten tetramers were quantified by flow cytometry. Laboratory tests and flow cytometry gating analyses were performed by researchers blinded to sample type, except for samples from subjects with celiac disease on a gluten-containing diet. Test precision analyses were performed using samples from 10 subjects. Results For the HLA-DQ–gluten tetramer-based assay, we combined flow-cytometry variables in a multiple regression model that identified individuals with celiac disease on a GFD with an area under the receiver operating characteristic curve value of 0.96 (95% confidence interval [CI] 0.89–1.00) vs subjects without celiac disease on a GFD. The assay detected individuals with celiac disease on a gluten-containing diet vs controls with an area under the receiver operating characteristic curve value of 0.95 (95% CI 0.90–1.00). Optimized cutoff values identified subjects with celiac disease on a GFD with 97% sensitivity (95% CI 0.92–1.00) and 95% specificity (95% CI 0.84–1.00) vs subjects without celiac disease on a GFD. The values identified subjects with celiac disease on a gluten-containing diet with 100% sensitivity (95% CI 1.00–1.00]) and 90% specificity (95% CI 0.83–0.98) vs controls. In an analysis of 4 controls with positive results from the HLA-DQ–gluten tetramer test, 2 had unrecognized celiac disease and the remaining 2 had T cells that proliferated in response to gluten antigen in vitro. Conclusions An HLA-DQ–gluten tetramer-based assays that detects gluten-reactive T cells identifies patients with and without celiac disease with a high level of accuracy, regardless of whether the individuals are on a GFD. This test would allow individuals with suspected celiac disease to avoid gluten challenge and duodenal biopsy, but requires validation in a larger study. Clinicaltrials.gov no: NCT02442219.

Background & Aims Non-celiac gluten sensitivity is characterized by symptom improvement after gluten withdrawal in absence of celiac disease. The mechanisms of non-celiac gluten sensitivity are unclear, and there are no biomarkers for this disorder. Foods with gluten often contain fructans, a type of fermentable oligo-, di-, monosaccharides and polyols. We aimed to investigate the effect of gluten and fructans separately in individuals with self-reported gluten sensitivity. Methods We performed a double-blind crossover challenge of 59 individuals on a self-instituted gluten-free diet, for whom celiac disease had been excluded. The study was performed at Oslo University Hospital in Norway from October 2014 through May 2016. Participants were randomly assigned to groups placed on diets containing gluten (5.7 g), fructans (2.1 g), or placebo, concealed in muesli bars, for 7 days. Following a minimum 7-day washout period (until the symptoms induced by the previous challenge were resolved), participants crossed over into a different group, until they completed all 3 challenges (gluten, fructan, and placebo). Symptoms were measured by Gastrointestinal Symptom Rating Scale Irritable Bowel Syndrome (GSRS-IBS) version. A linear mixed model for analysis was used. Results Overall GSRS-IBS scores differed significantly during gluten, fructan, and placebo challenges; mean values were 33.1 ± 13.3, 38.6 ± 12.3, and 34.3 ± 13.9, respectively ( P = .04). Mean scores for GSRS-IBS bloating were 9.3 ± 3.5, 11.6 ± 3.5, and 10.1 ± 3.7, respectively, during the gluten, fructan, and placebo challenges ( P = .004). The overall GSRS-IBS score for participants consuming fructans was significantly higher than for participants consuming gluten ( P = .049), as was the GSRS bloating score ( P = .003). Thirteen participants had the highest overall GSRS-IBS score after consuming gluten, 24 had the highest score after consuming fructan, and 22 had the highest score after consuming placebo. There was no difference in GSRS-IBS scores between gluten and placebo groups. Conclusions In a randomized, double-blind, placebo-controlled crossover study of individuals with self-reported non-celiac gluten sensitivity, we found fructans to induce symptoms, measured by the GSRS-IBS. Clinicaltrials.gov no: NCT02464150.

The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects.Primary cultures of human colonic circular smooth muscle cells and muscle strips were used.The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha.The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

Transcription factor nuclear factor kappa B (NF-kappa B) plays a critical role in transcriptional changes in several diseases, including inflammation. The aim of this study was to investigate whether NF-kappa B is activated by inflammation and oxidative stress in colonic circular smooth muscle cells and whether that leads to suppression of their contractility.The experiments were performed on freshly dissociated single cells using electrophoretic mobility shift assay, Western immunoblotting, and immunofluorescence imaging.The NF-kappa B DNA binding was approximately 6-fold greater in cells from the inflamed colon vs. those from the normal colon. Supershift assay indicated that the antibodies to p65, p50, and c-Rel, but not that to p52, shifted the NF-kappa B band. Western immunoblotting and immunofluorescence imaging also demonstrated the presence of p65, p50, and c-Rel proteins in the cytoplasm and their translocation to the nucleus by H(2)O(2)-induced oxidative stress. H(2)O(2) treatment degraded I kappa B(beta), but not I kappa B(alpha), to translocate NF-kappa B to the nucleus. Hydrogen peroxide concentration and time dependently activated NF-kappa B DNA binding and suppressed cell contraction to acetylcholine. NF-kappa B inhibitors significantly inhibited these effects. Inhibition of NF-kappa B prior to and during inflammation in intact dogs also reversed the suppression of contractility.Transcription factor NF-kappa B is activated in colonic circular muscle cells by inflammation and oxidative stress. This activation of NF-kappa B mediates the suppression of cell contractility.

Background & Aims: Arachidonic acid (AA) is a critical second messenger in several cell types. We examined whether cholinergic AA acts as a second messenger in contraction of colonic circular muscle cells and if this role is altered by inflammation. Methods: The experiments were performed on single dispersed cells. AA release was measured by high-performance liquid chromatography. Escherichia coli membranes labeled with 3 H-AA were used as a substrate for determining phospholipase A 2 (PLA 2 ) activity, and Western immunoblotting for protein expression. Results: Acetylcholine and the PLA 2 activator melittin induced cell contractions and AA release. Both effects were inhibited by the PLA 2 inhibitor ONO-RS-082. Cytosolic and membrane PLA 2 activities increased in response to acetylcholine. These were blocked by ONO-RS-082 and cytosolic PLA 2 100-kilodalton antibody, but not by dithiothreitol, a secretory PLA 2 inhibitor. Acetylcholine- and melittin-stimulated release of AA and their contractile response were attenuated in inflamed cells. Immunoblotting indicated that the protein expression of cPLA 2 was suppressed during inflammation. Conclusions: AA acts as a second messenger in muscarinic receptor–activated contractions of colonic circular muscle cells. cPLA 2 is the primary enzyme that releases AA in these cells; its expression as well as activation are significantly attenuated by inflammation. The attenuated release of AA may partly account for the inhibition of colonic circular muscle tone and phasic contractions observed during inflammation. GASTROENTEROLOGY 2000;119:62-70

Background & Aims: The characteristics of colonic circular smooth muscle slow waves are altered during inflammation. The aim of this study was to examine whether inflammation modulates the open-state probability of Ca 2+ -activated K + (K Ca ) channels in these cells to contribute to these alterations. Methods: The experiments were performed on freshly dissociated single smooth muscle cells from the canine colon using standard patch clamp methods. Inflammation was induced by mucosal exposure to ethanol and acetic acid. Results: Inflammation decreased the open-state probability of large-conductance K Ca (BK) channels in the cell-attached and excised inside-out configurations. The voltage sensitivity of the channels was also reduced during inflammation. Inflammation had no significant effect on the large, medium, and small conductances or the unitary current levels of channel openings. However, it decreased the maximum number of simultaneous channel openings. The channels were Ca 2+ -dependent and were blocked by tetraethylammonium and charybdotoxin in normal and inflamed cells. Conclusions: Inflammation decreases the open-state probability of BK channels. This may partially reverse the decrease in duration and amplitude of slow waves and depolarization of membrane potential seen in inflammation. GASTROENTEROLOGY 1999;116:884-892

Background & Aims Gastric hypersensitivity (GHS) contributes to epigastric pain in patients with functional dyspepsia (FD); the etiology and cellular mechanisms of this dysfunction remain unknown. We investigated whether inflammatory insult to the colons of neonatal rats induced GHS in adult life. Methods We used cellular, molecular, and in vivo approaches to investigate the mechanisms of GHS in adult rats subjected to neonatal colonic insult by intraluminal administration of trinitrobenzene sulfonic acid; controls received saline. Six to 8 weeks later, rats were evaluated for GHS and tissue was collected for molecular experiments. Results Inflammatory insult to the colon on post-natal day 10 caused an aberrant increase of corticosterone on post-natal day 15 and induced GHS in adult life. We called these FD-like rats . Inhibition of glucocorticoid receptors after neonatal insult blocked the induction of GHS in adult rats. The aberrant increase of plasma corticosterone in neonates increased the plasma concentration of norepinephrine, nerve growth factor in the gastric fundus muscularis externae, brain-derived neurotrophic factor in the thoracic dorsal root ganglia and spinal cord, and down-regulated K v 1.1 messenger RNA in thoracic dorsal root ganglia without affecting the expression of K v 1.4, Na v 1.8, TrpA1, TrpV1, or P2X3 in FD-like rats. Inhibition of glucocorticoid receptors during neonatal insult or the inhibition of adrenergic receptors, nerve growth factor, or brain-derived neurotrophic factor in FD-like rats suppressed GHS. The intrathecal administration of small interfering RNAs against K v 1.1 increased GHS in naive rats. Conclusions Inflammatory insult to the colons of rat pups leads to GHS in adult life. GHS is caused by altered expression of genes encoding neurotrophins and ion channels, and altered activity of the sympathetic nervous system.

The hypothesis that gastric antrum controls the phasic contractions of gallbladder cyclic motor activity in the fasting state was tested. Gallbladder, gastric, and small bowel motor and myoelectric activity was recorded by strain gauge transducers and bipolar electrodes. Gallbladder pressure was measured manometrically by a surgically implanted intraluminal catheter. After control recordings for 4 to 6 weeks, antrectomy and gastroduodenostomy were performed. Six weeks later, bilateral truncal vagotomy was performed in each dog. Recordings were made after each surgical procedure. In the control state, the gallbladder exhibited cyclic motor activity consisting of phasic contractions at a frequency of 0.75 ± 0.02/min superimposed on an increase in baseline pressure. Antrectomy and gastroduodenostomy completely abolished the phasic contractions of gallbladder cyclic motor activity and significantly decreased the incidence of the cyclic increase in baseline pressure. Subsequent vagotomy had no additional effect on gallbladder cyclic motor activity. In intact dogs, the gallbladder filled from 0% to 80% and emptied from 80% to 100% of the duodenal migrating motor complex cycle, which was considered to begin at the start of phase I activity. Antrectomy significantly altered this pattern; after antrectomy, the gallbladder filled from 0% to 10% and from 90% to 100% and emptied during the remainder of the duodenal migrating motor complex cycle. Subsequent vagotomy had no additional effect on periodic gallbladder filling and emptying. It is concluded that major changes occur in gallbladder cyclic motor activity and its periodic filling and emptying pattern in the fasting state after antrectomy and vagotomy. It is hypothesized that in the absence of cyclic phasic contractions after antrectomy, periodic stirring and agitation of gallbladder bile and its mixing with fresh hepatic bile may not occur in the fasting state. The absence of this phenomenon may lead to supersaturation of bile near the mucosal surface and increase the propensity for precipitation of salts and formation of gallstones.

The effects of an antibacterially effective IV dose of erythromycin on gastrointestinal motor activity were investigated in eight normal healthy human volunteers in the fasted state and the fed state. Motor activity was recorded by a multilumen manometric tube. Data were analyzed visually and by a computer method. Blood samples were obtained for erythromycin and motilin assays. In the gastric antrum, erythromycin significantly increased the total duration, amplitude, and area under contractions from 0 to 60 minutes and frequency of contractions from 0 to 30 minutes from the start of its infusion in the fasted state. A similar response in the fed state occurred mostly from 0 to 30 minutes after the start of erythromycin infusion. By contrast, erythromycin inhibited the frequency and decreased the duration of small intestinal contractions in the fed state but had no effect in the fasted state. The gastric motor response was related to the plasma concentration of erythromycin, but not to plasma motilin. Erythromycin significantly shortened the duration of migrating motor complex disruption by a meal. Erythromycin also induced symptoms of upper abdominal pain, bloating, and nausea. Abdominal pain was related to strong antral contractions in both fasted and fed states; bloating occurred only in the fed state. Nausea occurred in both fasted and fed states, but it was not related to any specific pattern of motor activity. It is concluded that the strong antral contractions induced by erythromycin may accelerate the rate of gastric emptying, but they may also be responsible for causing the sensations of upper abdominal pain and bloating. The motor response to erythromycin is less during the fed than during the fasted state. The strong antral contractions induced by erythromycin are not mediated by the release of motilin.

The effect of Trichinella spiralis infection on small intestinal transit and motor activity in the fed state during the intestinal phase of infection was studied. Contractions were recorded by strain gauge transducers, and mean transit time was measured by marker dilution technique. The mean amplitude and area of individual phasic contractions decreased, but no change occurred in their mean duration during trichinosis. The total amplitude and area of phasic contractions also decreased; this was caused by a decrease in the frequency of contractions as well as a decrease in the mean parameters. The reduction in the total duration was entirely caused by the decrease in frequency. The reduction in the total parameters of all contractions was the result of a reduction in the same parameters for both propagating and nonpropagating contractions. However, the decrease in the parameters of propagating contractions was much greater. Also, there was a decrease in the distance of propagation of phasic contractions. The transit time as a result of phasic contractions increased during T. spiralis infection. Additionally, T. spiralis infection induced giant migrating contractions in the fed state that were never observed during control. Chyme was propelled very rapidly and effectively by giant migrating contractions. The findings of the present study suggest that during diarrhea induced by T. spiralis infection, the phasic contractions may act to decrease transit and, hence, allow more contact time for absorption of water and nutrients. However, this response may be counter-balanced by giant migrating contractions that rapidly propel chyme into the colon and compound the diarrhea associated with T. spiralis infection.

G A A b st ra ct s medicine with ghrelin enhancing properties, is used to treat gastroparesis (Takahashi T et al. World J Surg 2009). Aim: To assess the brain sites activated by abdominal surgery in mice and whether rikkunshito modified this pattern and the inhibited food intake.Methods: RKT (0.5 g/kg/day) or vehicle (distilled water, 4 ml/kg) was given daily by gavage for 7 days and on day 8 at 6 h and 1 h before the surgery. Male mice (C57BL/6, 23-26 g) fasted for 6 h received laparotomy and cecal palpation under isoflurane anesthesia 2 h before the dark phase. Controls remained in home cages. For Fos immunohistochemistry in brain sections, mice were euthanized 2 h post-surgery. For food intake monitoring, mice were maintained in an automated feeding monitoring system (BioDAQ, Research Diet). Results: In vehicle-treated mice, there was moderate Fos immunoreactivity (ir) in the paraventricular nucleus (PVN), arcuate nucleus (Arc), and preoptic, lateral and dorsomedial areas of the hypothalamus, medial bed nucleus of stria terminalis and Edinger-westphal nucleus (E-W), which could be resulted from gavage and 6-h fasting. Abdominal surgery induced robust Fos-ir in the lateral septum, supraoptic nuclei (SON), PVN, Arc, lateral parabrachial nucleus, locus ceruleus, ventrolateral medulla and nucleus tractus solitarius (NTS); and moderate increase in Fos-ir in the prelimbic, insular and cingulate cortex, nucleus of accumbens, subfornical organ, E-W, periaqueductal area (PAG), Barrington's nucleus, A5 and area postrema. Compared to vehicle, RKT treatment in control mice induced significant more numbers of Fos-ir neurons of the SON (1.9±0.8 vs 6.2±1.0 cells/section) and E-W (22.7±1.1 vs 31.3±2.6). In mice received surgery, RKT induced a significant increase in Fos-ir in the lateral PAG (68.3±4.8 vs 49.5±5.4) and decrease in the NTS (82.4±4.1 vs 96.1±8.0). Abdominal surgery reduced food intake significantly at 6, 12 and 24 h by 83%, 94% and 94%, respectively compared to control. RKT resulted in a small, but significant increase in food intake at 6 and 12 h after surgery compared to vehicle plus surgery (0.31±0.04 vs 0.11±0.04, and 0.62±0.11 vs 0.37±0.01 g). Conclusion: Brain circuits of mice responsive to abdominal surgery include visceral regulatory centers, forebrain and midbrain areas involved in pain, and integrative nuclei related to stress. The partially restored food intake by RKT after abdominal surgery may reply on reducing the inhibitory inputs from the NTS related to visceral afferents and enhancing the descending pain inhibitory pathway via the PAG. Supported by Tsumura &Co., and NIH DDK-41303

Background & Aims: Vasoactive intestinal polypeptide (VIP) relaxes smooth muscle by generation of cAMP and activation of protein kinase A (PKA). However, PKA activation also phosphorylates the transcription factor CREB. The aim of this study was to investigate whether the phosphorylation of CREB induces gene expression of the pore-forming α1C subunit of Cav1.2 channels (L-type calcium channels), whose promoter has 2 binding sites for CREB. Methods: The experiments were performed on primary cultures of human colonic circular smooth muscle cells and freshly obtained human and rat colonic circular muscle strips. Results: The incubation of human colonic circular smooth muscle cells or muscle strips with VIP for 24 hours enhanced the expression of α1C protein and mRNA as well as the contractile response to acetylcholine and KCl. On the contrary, incubation of the muscle strips with VIP antagonist for 24 hours suppressed cell contractility. The incubation of the cells with VIP caused sustained generation of cAMP for 24 hours, but PKA activation and CREB phosphorylation were transient. The inhibition of PKA by H-89 or silencing of CREB gene with targeted RNAi blocked the transcription of α1C. Progressive 5′ deletions of hα1C1b promoter and site-directed mutations of the 2 CREB binding cis-elements indicated that most of α1C transcription was mediated by the 5′ cAMP response element. Conclusions: The excitation–transcription coupling stimulated by VIP induces expression of the Cav1.2 channels. The influx of calcium through these channels is a critical step in excitation–contraction coupling in smooth muscle cells.

Nitric oxide is a major inhibitory neurotransmitter in the enteric nervous system. Loss or dysfunction of nitrinergic neurons is associated with serious disruptions of motility, intractable symptoms, and long-term suffering. The aim of this study was to evaluate the effect of intrapyloric transplantation of neural stem cells (NSCs) on gastric emptying and pyloric function in nNOS-/- mice, a well-established genetic model of gastroparesis.NSCs were isolated from embryonic mice transgenically engineered to express green fluorescent protein and transplanted into the pylorus of nNOS-/- mice. Grafted cells were visualized in pyloric sections and further characterized by immunofluorescence staining. One week posttransplantation, gastric emptying to a non-nutrient meal was measured using the phenol red method and pyloric function was assessed by measuring the relaxation of pyloric strips in an organ bath in response to electrical field stimulation (EFS) under nonadrenergic, noncholinergic conditions.One week following implantation, grafted NSCs differentiated into neurons and expressed neuronal nitric oxide synthase. Gastric emptying was significantly increased in mice that received NSCs as compared with vehicle-injected controls (49.67% vs 35.09%; P.01 by Student t test). EFS-induced relaxation of pyloric strips was also significantly increased (P.01 by 2-way analysis of variance). The nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester and the neuronal blocker tetrodotoxin blocked the EFS-induced relaxation, indicating that the observed effect is NO mediated and neuronally derived.Our results support the potential of NSC transplantation as a viable therapeutic option for neuroenteric disorders.

Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha.Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips.TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFalpha. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C zeta (PKCzeta) inhibition before or 3 hours after stimulation with TNFalpha also blocked the expression of ICAM-1. TNFalpha treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect.The expression of ICAM-1 on HCCSMCs in response to TNFalpha is regulated by transcription factors Sp1 and NF-kappaB binding independently to the -192/-172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFalpha.

Background & Aims: Protein kinase C (PKC) is a key signaling molecule in excitation-contraction coupling in several types of smooth muscle cells. We investigated whether the attenuated contraction in inflamed colon cells is caused by alterations in the expression, distribution, and activation of specific PKC isozymes. Methods: Kinase assays, immunofluorescence imaging, and Western immunoblotting were performed on single circular smooth muscle cells obtained from the normal dog colon as well as from colon with experimental colitis induced by mucosal exposure to ethanol and acetic acid, to determine the distribution, expression, and activation of PKC isozymes. Results: Classical (α, β, and γ), novel (δ and ϵ), and the atypical PKC (ι, λ, and ζ) isozymes were detected in colonic circular muscle cells. The expression of PKC α, β, and ϵ isozymes was down-regulated, whereas that of PKC ι and λ isozymes was up-regulated; other isozymes were not affected by inflammation. Acetylcholine (ACh) treatment translocated only the PKC α, β, and ϵ isozymes from the cytosol to the membrane in normal cells; this translocation was absent in inflamed colon cells. Immunofluorescence imaging confirmed the translocation of PKC α from the cytosol to the membrane in response to ACh in normal cells. PKC inhibitors, chelerythrine, and myristoylated peptides to α, β, and ϵ isozymes inhibited the contractile response to ACh in normal, but not in inflamed, cells. PKC ι and λ did not participate in the contractile response to ACh. Conclusions: ACh-induced contraction is mediated by PKC α, β, and ϵ isozymes in normal colonic circular muscle cells. Contractile dysfunction in inflamed colon cells is, in part, caused by decreased expression and impaired activation of specific PKC isozymes. GASTROENTEROLOGY 2002;122:483-494

G A A b st ra ct s neurons. In contrast to control rats, UK14304 decreased the amplitude of IPSC in 12 of 17 rats that underwent homotypic stress (2hrs restrain, 20min forced swim, 40min cold @4°C and 2hrs water avoidance) for 5 days in a concentration-dependent manner (91±1%, 78±1% and 53±9% of baseline amplitude in response to perfusion with 0.1, 1 and 3μM, respectively; P

Background & Aims: Circular smooth muscle phasic contractions and tone are suppressed during colonic inflammation, but the contributing factors are poorly understood. This study investigated if the expression level of voltage-gated long-lasting (L-type) Ca 2+ channel protein and functional Ca 2+ channel current are down-regulated in the circular muscle cells of the inflamed canine colon. Methods: L-type Ca 2+ channel expression was compared between normal and inflamed smooth muscle cells by Western immunoblots using an antibody directed against the pore-forming α1C-subunit, and patch-clamp methods were used to evaluate Ca 2+ channel current density. Results: The expression of the L-type Ca 2+ channel protein was significantly reduced in inflamed compared with normal circular smooth muscle cell membranes, and this finding was associated with suppressed levels of Ca 2+ channel current in patch-clamped cells. The L-type Ca 2+ channel current in normal and inflamed cells increased proportionately in response to Bay K 8644, but the maximal current density was still lower in the inflamed cells. Acetylcholine increased the L-type Ca 2+ channel current in normal but not in inflamed cells. Conclusions: The expression level of L-type Ca 2+ channels is down-regulated in the circular smooth muscle cell membranes of the inflamed colon, which may result in reduced Ca 2+ influx. The functional and pharmacologic properties of the channels seem normal. Although some Ca 2+ channels are still present in the inflamed cells, acetylcholine does not activate these channels, which may be caused by additional upstream defects in the receptor signaling cascade. The down-regulation of L-type Ca 2+ channel expression may suppress circular smooth muscle contractions in the inflamed colon and contribute to the abnormalities in motility and digestion observed during inflammatory disorders. GASTROENTEROLOGY 2001;120:480-489

BACKGROUND & AIMS: Spontaneous in vivo phasic contractions are suppressed during ileal inflammation. The aim of this study was to test the hypothesis that modulation of the contractile response to muscarinic receptor activation contributes to this inhibition. METHODS: Muscarine, methacholine, and arecaidine propargyl ester were infused close-intra-arterially in the normal and inflamed ileum of conscious dogs. Concurrent studies were performed on in vitro muscle strips. RESULTS: Prior close-intra-arterial infusions of tetrodotoxin or hexamethonium had no significant effect on the contractile response to any agonist, indicating that they acted primarily on smooth muscle muscarinic receptors. The contractile response to each agonist was suppressed significantly during inflammation. The median inhibitory dose of 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) was 57-244-fold smaller than that of pirenzepine, methoctramine, and tropicamide to inhibit the contractile response to muscarine. Inflammation induced a significant leftward shift in the inhibitory dose-response curve to methoctramine but not to 4-DAMP, pirenzepine, or tropicamide. The data from in vitro muscle strips were similar to those in conscious dogs. CONCLUSIONS: Inflammation suppresses the phasic contractile response to muscarinic receptor activation in circular smooth muscle cells. The spontaneous and muscarinic agonist-stimulated in vivo phasic contractions of ileal circular smooth muscle cells are mediated primarily by M3 receptors. (Gastroenterology 1997 Mar;112(3):864-74)