Expression of RAD21 immunoreactivity in myenteric neurons of the human and mouse small intestine.

BACKGROUND: RAD21 is a double-strand-break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss-of-function mutation was found in cases of chronic intestinal pseudo-obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression in the enteric nervous system is lacking, thus we aimed to characterize RAD21 immunoreactivity (IR) in myenteric ganglia. METHODS: Double labeling immunofluorescence in mouse and human jejunum was used to determine colocalization of RAD21 with HuC/D, PGP9.5, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT), Kit, platelet-derived growth factor receptor-α (PDGFRα), and glial fibrillary acid protein (GFAP) IRs. RESULTS: A subset of PGP9.5- and HuC/D-IR neuronal cell bodies and nerve fibers in the myenteric plexus of human and mouse small intestine also displayed cytoplasmic RAD21-IR Cytoplasmic RAD21-IR was found in 43% of HuC/D-IR neurons in adult and neonatal mice but did not colocalize with nNOS. A subset of ChAT-positive neurons had cytoplasmic RAD21-IR Punctate RAD21-IR was restricted to the nucleus in most cell types consistent with labeling of the cohesin complex. Cytoplasmic RAD21-IR was not detected in interstitial cells of Cajal, fibroblast-like cells or glia. Subsets of neurons in primary culture exhibited cytoplasmic RAD21-IR Suppression of RAD21 expression by shRNA knockdown abolished RAD21-IR in cultured neurons. CONCLUSIONS: Our data showing cytoplasmic RAD21 expression in enteric neurons provide a basis toward understanding how mutations of this gene may contribute to altered neuronal function/survival thus leading to gut-motor abnormalities.

High temporal resolution gastric emptying breath tests in mice.

BACKGROUND: Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. METHODS: We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13 C]-octanoic acid and off axis- integrated cavity output spectroscopy (OA-ICOS). Stretched gamma variate and 2-component stretched gamma variate models fit measured breath excretion data. KEY RESULTS: These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin-induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two-phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two-phase gastric emptying curves. A greater proportion of two-phase gastric emptying was induced in Balb/c mice with the administration of PYY (8-80 nmol) 60 min following meal ingestion. CONCLUSIONS AND INFERENCES: Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two-phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.

Correlated gene expression encoding serotonin (5-HT) receptor 4 and 5-HT transporter in proximal colonic segments of mice across different colonization states and sexes.

The production and handling of serotonin (5-HT) is an important determinant of colonic motility and has been reported to be altered in gastrointestinal (GI) disorders such as irritable bowel syndrome (IBS). Recent studies suggest that the intestinal microbiota and sex of the host can influence expression of genes involved in 5-HT biosynthesis and signaling. While expression of genes in serotonergic pathways has been shown to be variable, it remains unclear whether genes within this pathway are coregulated. As a first step in that direction, we investigated potential correlations in relative mRNA expression of serotonergic genes, in the proximal colon isolated from male and female mice in different states of microbial association: germ-free (GF), humanized (ex-germ-free colonized with human gut microbiota, HM), and conventionally raised (CR) mice. Among the 10 pairwise comparisons conducted between five serotonergic transcripts, Tph1, Chga, Maoa, Slc6a4, and Htr4, we found a strong, positive correlation between colonic expression of Slc6a4 and Htr4 across different colonization states and sexes. We also identified a positive correlation between the expression of Tph1 and Chga; however, there were no correlations observed between any other tested pair of 5-HT-related transcripts. These data suggest that correlated expression of Slc6a4 and Htr4 likely involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut. Further work will need to be done to understand the pathways and cell types responsible for this correlated expression, given the important role of 5-HT in gastrointestinal physiology.

Walker 256 tumor-bearing rats demonstrate altered interstitial cells of Cajal. Effects on ICC in the Walker 256 tumor model.

BACKGROUND: Cachexia is a significant problem in patients with cancer. The effect of cancer on interstitial cells of Cajal (ICC) and neurons of the gastrointestinal tract have not been studied previously. Although supplementation with L-glutamine 2% may have beneficial effects in cancer-related cachexia, and be protective of ICC in models of oxidative stress such as diabetes, its effects on ICC in cancer have also not been studied. METHODS: Twenty-eight male Wistar rats were divided into four groups: control (C), control supplemented with L-glutamine (CG), Walker 256 tumor (WT), and Walker 256 tumor supplemented with L-glutamine (WTG). Rats were implanted with tumor cells or injected with saline in the right flank. After 14 days, the jejunal tissues were collected and processed for immunohistochemical techniques including whole mounts and cryosections and Western blot analysis. KEY RESULTS: Tumor-bearing rats demonstrate reduced numbers of Myenteric ICC and deep muscular plexus ICC and yet increased Ano1 protein expression and enhanced ICC networks. In addition, there is more nNOS protein expressed in tumor-bearing rats compared to controls. L-glutamine treatment had a variety of effects on ICC that may be related to the disease state and the interaction of ICC and nNOS neurons. Regardless, L-glutamine reduced the size of tumors and also tumor-induced cachexia that was not due to altered food intake. CONCLUSIONS & INFERENCES: There are significant effects on ICC in the Walker 256 tumor model. Although supplementation with L-glutamine has differential and complex effects of ICC, it reduces tumor size and tumor-associated cachexia, which supports its beneficial therapeutic role in cancer.

Changes in nitrergic and tachykininergic pathways in rat proximal colon in response to chronic treatment with otilonium bromide.

BACKGROUND: Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. METHODS: Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. KEY RESULTS: Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. CONCLUSIONS & INFERENCES: Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Effects of gestation and lactation on forage intake, digestion, and passage rates of primiparous beef heifers and multiparous beef cows.

Angus-cross cows (n = 13; 8 pregnant, BW 610 ± 24 kg, and 5 nonpregnant, BW 571 ± 23 kg) and heifers (n = 13; 8 pregnant, BW 511 ± 40 kg, and 5 nonpregnant, BW 451 ± 60 kg) were individually fed chopped warm-season grass hay (5.5% CP, 67% NDF) for ad libitum intake and soybean meal (46% CP) at 450 g/d. Intake was measured daily, and DM digestibility, digesta passage rate, and plasma glucose and ß-hydroxybutyrate (BHBA) concentrations were measured every 14 d from 49 d prepartum to 49 d postpartum. Prepartum DMI (% of BW) increased over time for pregnant heifers through 2 wk prepartum before declining but did not change over time for pregnant cows. Dry matter digestibility decreased with advancing gestation (P < 0.001); pregnant animals had greater digestibility than nonpregnant cows and heifers (P = 0.02). Digestibility was not influenced by age (P = 0.99). Pregnant cows and heifers had faster digesta passage rates than their nonpregnant counterparts (P = 0.02). Pregnant animals had lower plasma glucose (P < 0.001). Plasma BHBA concentrations were greater in pregnant animals than in nonpregnant animals (P < 0.001) but were not influenced by age (P = 0.27) or time prepartum (P = 0.98). Postpartum DMI (% of BW) was greater for lactating heifers than other groups (age × lactation status; P = 0.05) and increased over time (P < 0.001). Diet digestibility increased with time postpartum (P < 0.001), and heifers had greater digestibility than cows from 3 to 7 wk postpartum but not at 1 wk postpartum (age × time; P = 0.02). Postpartum passage rate was not influenced by age or lactation status (P > 0.23). Lactating animals had lower plasma glucose and greater plasma BHBA concentrations postpartum than nonlactating animals (P < 0.001). Calves from mature cows grew faster than calves from heifers (age × time; P < 0.001). These data show that although primiparous beef heifers have similar DM digestibility, passage rates, and plasma glucose and BHBA concentrations, intake patterns differ between heifers and cows. Although DMI (% of BW) and digestibility did not differ between pregnant beef heifers and pregnant mature cows, the DMI (% of BW) was greater for lactating primiparous cows (heifers) than for lactating multiparous cows. Even with their postpartum increase in DMI, primiparous beef heifers were not able to consume adequate amounts of the warm-season forage to support their requirements for maintenance, growth, and lactation.

On the fiftieth anniversary. Postinfectious irritable bowel syndrome: mechanisms related to pathogens.

BACKGROUND: Gastrointestinal (GI) infections resulting from bacterial, viral, and parasitic pathogens predispose to postinfectious irritable bowel syndrome (PI-IBS) and other functional GI disorders. Existing literature supports the role of enterochromaffin cell hyperplasia, serotonin synthesis and reuptake, impaired barrier function, altered immune activation, and potentially mast cell activation in the pathophysiology of PI-IBS. PURPOSE: The objective of this review was to summarize from the literature the characteristics of the pathogens commonly implicated in PI-IBS, their acute enteritis phases, and the changes seen in the postinfectious phase that may contribute toward development of IBS. A limitation of our current understanding is that the postinfectious GI sequelae reported in prior studies followed epidemic diarrheal outbreaks often involving more than one pathogen, or the studies focused on highly selected, tertiary referral patients. Understanding the mechanisms, natural history, and optimized management of individuals suffering PI-IBS following the more typical sporadic infection requires larger studies of PI-IBS following GI infections encountered in community settings. These studies should include genetic, physiological, and molecular studies to provide more generalizable information that can ultimately be used to diagnose, manage, and potentially prevent the development of PI-IBS.

Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle.

A transwall gradient in resting membrane potential (RMP) exists across the circular muscle layer in the mouse colon. This gradient is dependent on endogenous generation of CO. H2S is also generated in muscle layers of the mouse colon. The effect of endogenously generated H2S on the transwall gradient is not known. The aim was to investigate the role of endogenous H2S. Our results showed that the CSE inhibitor dl-propargylglycine (PAG, 500 µm) had no effect on the transwall gradient. However, in preparations pretreated with the nitric oxide synthase inhibitor N-nitro-l-arginine (l-NNA, 200 µm) and in nNOS-knockout (KO) mouse preparations, PAG shifted the transwall gradient in the depolarizing direction. In CSE-KO-nNOS-KO mice, the gradient was shifted in the depolarizing direction. Endogenous generation of NO was significantly higher in muscle preparations of CSE-KO mice compared to wild-type (WT) mice. The amplitude of NO-mediated slow inhibitory junction potentials (S-IJPs) evoked by electric field stimulation was significantly higher in CSE-KO mouse preparations compared to the amplitude of S-IJPs in wild-type mouse preparations. CSE was present in all submucosal ganglion neurons and in almost all myenteric ganglion neurons. Eleven per cent of CSE positive neurons in the submucosal plexus and 50% of CSE positive neurons in the myenteric plexus also contained nNOS. Our results suggest that endogenously generated H2S acts as a stealth hyperpolarizing factor on smooth muscle cells to maintain the CO-dependent transwall gradient and inhibits NO production from nNOS.

Sulphide quinone reductase contributes to hydrogen sulphide metabolism in murine peripheral tissues but not in the CNS.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2) S) is gaining acceptance as a gaseous signal molecule. However, mechanisms regarding signal termination are not understood. We used stigmatellin and antimycin A, inhibitors of sulphide quinone reductase (SQR), to test the hypothesis that the catabolism of H(2) S involves SQR. EXPERIMENTAL APPROACH: H(2) S production and consumption were determined in living and intact mouse brain, liver and colonic muscularis externa using gas chromatography and HPLC. Expressions of SQR, ethylmalonic encephalopathy 1 (Ethe1) and thiosulphate transferase (TST; rhodanese) were determined by RT-PCR and immunohistochemistry. KEY RESULTS: In the colonic muscularis externa, H(2) (35) S was catabolized to [(35) S]-thiosulphate and [(35) S]-sulphate, and stigmatellin reduced both the consumption of H(2) (35) S and formation of [(35) S]-thiosulphate. Stigmatellin also enhanced H(2) S release by the colonic muscularis externa. In the brain, catabolism of H(2) (35) S to [(35) S]-thiosulphate and [(35) S]-sulphate, which was stigmatellin-insensitive, partially accounted for H(2) (35) S consumption, while the remainder was captured as unidentified (35) S that was probably bound to proteins. Levels of mRNA encoding SQR were higher in the colonic muscularis externa and the liver than in the brain. CONCLUSIONS AND IMPLICATIONS: These data support the concept that termination of endogenous H(2) S signalling in the colonic muscularis externa occurs via catabolism to thiosulphate and sulphate partially via a mechanism involving SQR. In the brain, it appears that H(2) S signal termination occurs partially through protein sequestration and partially through catabolism not involving SQR. As H(2) S has beneficial effects in animal models of human disease, we suggest that selective inhibition of SQR is an attractive target for pharmaceutical development.

Effects of mineral-supplement delivery system on frequency, duration, and timing of supplement use by beef cows grazing topographically rugged, native rangeland in the Kansas Flint Hills.

The effects of mineral-supplement delivery system on patterns of supplement use by grazing beef cows were measured in 2 studies. Study 1 was conducted on 4 pastures grazed by pregnant, mature beef cows (BW = 562 ± 38 kg) from February to May. Study 2 was conducted on 4 pastures grazed by lactating beef cows (BW = 579 ± 54 kg) and their calves from May to September. Treatments were mineral delivered in salt-based, granular form (salty) or mineral provided in a low-protein, cooked, molasses-based block (sweet); both were fed ad libitum. The salty supplement was supplied to cattle via a covered mineral feeder; the sweet supplement was supplied via an open-topped barrel. Both salty and sweet supplements were deployed in each pasture. No additional salt was supplied to cattle. Forage use in the vicinity of each supplement-deployment site and the frequency and duration of herd visits to each supplement-deployment site were measured during four 14-d periods during study 1 and seven 14-d periods during study 2. Supplements were moved to new locations within pastures at the beginning of each period. Consumption of the sweet supplement was greater than salty during each data-collection period in study 1; however, relative differences in consumption diminished over time (treatment × time, P = 0.03). In study 2, sweet consumption was greater than salty in periods 1, 6, and 7 but was not different from salty during periods 2, 3, 4, and 5 (treatment × time, P < 0.01). Increased consumption of the sweet supplement in study 1 translated to greater frequency of herd visits to supplement-deployment sites compared with the salty sites (2.82 vs. 2.47 herd visits/d; P = 0.02) and longer herd visits to supplement-deployment sites compared with the salty sites (125.7 vs. 54.9 min/herd visit; P < 0.01). The frequency of herd visits to mineral feeding sites in study 2 was similar (P > 0.10) between treatments for periods 1 through 6; however, herds visited the sweet sites more often than salty during period 7 (P < 0.01). Herd visits to the sweet sites were longer than those to the salty sites in study 2 (83.8 vs. 51.4 min/herd visit; P < 0.01). Forage disappearance within 100 m of supplement-deployment sites was not influenced (P ≥ 0.54) by treatment in either study. Results were interpreted to suggest that the sweet supplement influenced the location of grazing cattle more strongly than the salty supplement and may be more effective for luring cattle into specific areas of pasture during the winter, spring, and early fall but not during summer.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging.

BACKGROUND: Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. METHODS: Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy. KEY RESULTS: Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. CONCLUSIONS & INFERENCES: Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.

The transwall gradient across the mouse colonic circular muscle layer is carbon monoxide dependent.

Gastric and small intestinal circular smooth muscle layers have a transwall resting membrane potential (RMP) gradient that is dependent on release of carbon monoxide (CO) from interstitial cells of Cajal (ICCs). Our aim was to determine whether a RMP gradient exists in the mouse colon and whether the gradient is CO dependent. Microelectrodes were used to record RMPs from muscle cells at different depths of the circular muscle layer from wild-type and heme oxygenase-2-knockout (HO-2-KO) mice. A transwall RMP gradient was present in wild-type mice. The CO scavenger oxyhemoglobin (20 µM) and the heme oxygenase inhibitor chromium mesoporphyrin IX (CrMP, 5 µM) abolished the transwall gradient. The gradient was absent in HO-2-KO mice. Tetrodotoxin (1 µM) caused a significant depolarization in circular smooth muscle cells throughout the circular muscle layer and abolished the transwall gradient. Removal of the submucosal neurons abolished the gradient. The majority of submucosal neurons contained HO-2 immunoreactivity (HO-2-IR), while ICCs did not. These data show for the first time that a transwall gradient exists across the circular smooth muscle layer of the mouse colon, that the gradient is due to CO, and that the source of CO is the submucosal neurons.

Linaclotide - a secretagogue and antihyperalgesic agent - what next?

Ongoing clinical trials suggest that linaclotide, a first-in-class, 14-amino acid peptide guanylate cyclase-C (GC-C) receptor agonist and intestinal secretagogue is an effective treatment for chronic constipation. A study in this issue of the Journal suggests that linaclotide also has antihyperalgesic effects in three common rat models of inflammation- and stress-induced hypersensitivity (i.e., acute trinitrobenzene sulfonic acid colitis, water avoidance stress [WAS], and restraint-induced stress) but not in naïve animals. In mice, linaclotide at least partly reduces hyperalgesia via GC-C receptors. Dose-effect relationships of linaclotide were complicated and non-linear. This viewpoint discusses human clinical trials with linaclotide and the results of this study. Potential mechanisms and clinical significance of these findings are explored. Collectively, these data suggest that GC-C receptors exert other, as yet poorly understood, effects on gastrointestinal sensitivity in conditions associated with inflammation and/or stress-induced increased intestinal permeability. However, the data need to be confirmed in humans and in long-term animal models. Further studies are also necessary to elucidate the mechanisms as these effects cannot be explained by linaclotide's known effects on epithelial GC-C receptors.

Post-inflammatory colonic afferent sensitisation: different subtypes, different pathways and different time courses.

OBJECTIVE: Intestinal infection evokes hypersensitivity in a subgroup of patients with irritable bowel syndrome (IBS) long after healing of the initial injury. Trinitrobenzene sulfonic acid (TNBS)-induced colitis in rodents likewise results in delayed maintained hypersensitivity, regarded as a model of some aspects of IBS. The colon and rectum have a complex sensory innervation, comprising five classes of mechanosensitive afferents in the splanchnic and pelvic nerves. Their plasticity may hold the key to underlying mechanisms in IBS. Our aim was therefore to determine the contribution of each afferent class in each pathway towards post-inflammatory visceral hypersensitivity. DESIGN: TNBS was administered rectally and mice were studied after 7 (acute) or 28 (recovery) days. In vitro preparations of mouse colorectum with attached pelvic or splanchnic nerves were used to examine the mechanosensitivity of individual colonic afferents. RESULTS: Mild inflammation of the colon was evident acutely which was absent at the recovery stage. TNBS treatment did not alter proportions of the five afferent classes between treatment groups. In pelvic afferents little or no difference in response to mechanical stimuli was apparent in any class between control and acute mice. However, major increases in mechanosensitivity were recorded from serosal afferents in mice after recovery, while responses from other subtypes were unchanged. Both serosal and mesenteric splanchnic afferents were hypersensitive at both acute and recovery stages. CONCLUSIONS: Colonic afferents with high mechanosensory thresholds contribute to inflammatory hypersensitivity, but not those with low thresholds. Pelvic afferents become involved mainly following recovery from inflammation, whereas splanchnic afferents are implicated during both inflammation and recovery.

Novel promoter and alternate transcription start site of the human serotonin reuptake transporter in intestinal mucosa.

Selective serotonin-reuptake inhibitors are therapies for psychological and bowel disorders, but produce adverse effects in the non-targeted system. To determine whether human serotonin-selective reuptake transporter (SERT) transcripts in the intestine are different from the brain, rapid amplification of cDNA ends, primer extension and RT-PCR assays were used to evaluate SERT transcripts from each region. Potential SLC6A4 gene promoter constructs were evaluated with a secreted alkaline phosphatase reporter assay. A novel transcript of the human SLC6A4 gene was discovered that predominates in the intestine, and differs from previous transcripts in the 5'-untranslated region. The distinct transcriptional start site and alternate promoter suggest that gastrointestinal SERT can be differentially regulated from brain SERT, may explain why the polymorphism in the previously identified promoter is associated with affective disorders, but not associated with gastrointestinal dysfunction, and suggest the intriguing possibility of the development of site-specific therapeutics for SERT regulation in the treatment of multiple disorders.

Changes in colonic motility and the electrophysiological properties of myenteric neurons persist following recovery from trinitrobenzene sulfonic acid colitis in the guinea pig.

Persistent changes in gastrointestinal motility frequently accompany the resolution of colitis, through mechanisms that remain to be determined. Trinitrobenzene sulfonic acid (TNBS) colitis in the guinea pig decreases the rate of propulsive motility, causes hyperexcitability of AH neurons, and induces synaptic facilitation. The changes in motility and AH neurons are sensitive to cyclooxygenase-2 (COX-2) inhibition. The aim of this investigation was to determine if the motility and neurophysiological changes persist following recovery from colitis. Evaluations of inflammation, colonic motility and intracellular electrophysiology of myenteric neurons 8 weeks after TNBS administration were performed and compared to matched control conditions. Myeloperoxidase levels in the colons were comparable to control levels 56 days after TNBS treatment. At this time point, the rate of colonic motility was decreased relative to controls following treatment with TNBS alone or TNBS plus a COX-2 inhibitor. Furthermore, the electrical properties of AH neurons and fast synaptic potentials in S neurons were significantly different from controls and comparable to those detected during active inflammation. Collectively, these data suggest that altered myenteric neurophysiology initiated during active colitis persists long term, and provide a potential mechanism underlying altered gut function in individuals during remission from inflammatory bowel disease.

Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon.

This investigation was conducted to establish whether guinea-pig trinitrobenzene sulfonic acid (TNBS)-colitis was associated with a change in the number of neurones of the myenteric plexus, and, if so, whether select subpopulations of neurones were affected. Total neurones were quantified with human (Hu) antiserum, and subpopulations were evaluated with antisera directed against choline acetyltransferase, nitric oxide synthase, calretinin, neuronal nuclear protein or vasoactive intestinal peptide (VIP). Colitis was associated with a loss of 20% of the myenteric neurones, most of which occurred during the first 12 h past-TNBS administration. During this period, myenteric ganglia were infiltrated with neutrophils while lymphocytes appeared at a later time-point. The neuronal loss persisted at a 56-day time-point, when inflammation had resolved. The decrease in myenteric neurones was not associated with a decrease in any given subpopulation of neurones, but the proportion of VIP-immunoreactive neurones increased 6 days following TNBS administration and returned to the control range at the 56 days. These findings indicate that there is an indiscriminant loss of myenteric neurones that occurs during the onset of TNBS-colitis, and the loss of neurones may be associated with the appearance of neutrophils in the region.

Serotonin transporter function and expression are reduced in mice with TNBS-induced colitis.

Regulated release of serotonin (5-HT) from enterochromaffin (EC) cells activates neural reflexes that are involved in gut motility, secretion, vascular perfusion and sensation. The 5-HT-selective reuptake transporter (SERT) terminates serotonergic signalling in the intestinal mucosa. The aim of this investigation was to determine whether mucosal 5-HT content, release, and/or reuptake are altered in a murine model of immune cell-mediated colitis. Experiments were conducted 6 days after colitis was induced by 2,4,6-trinitrobenzene sulfonic acid, a time point when macroscopic and histological damage scores indicated significant inflammation. During inflammation, SERT transcript levels and immunoreactivity were reduced, and the uptake of [3H] 5-HT was impaired. Increases in mucosal 5-HT content and the number of 5-HT-immunoreactive mast cells in the lamina propria were also detected in the inflamed region, whereas EC cell numbers did not change. Mucosal 5-HT released under basal and stimulated conditions was unchanged in animals with colitis. These data suggest that murine colitis alters 5-HT signalling by increasing 5-HT availability through decreased 5-HT uptake by mucosal epithelial cells. These findings support the concept that altered 5-HT signalling could be a contributing factor in altered gut function and sensitivity in inflammatory bowel disease.

Agonists of proteinase-activated receptor 2 excite guinea pig ileal myenteric neurons.

The effects of proteinase-activated receptor 2 (PAR2) agonists on the electrical properties of intact guinea pig ileal myenteric neurons were measured with intracellular microelectrodes. Approximately 52% of AH neurons and 41% of S neurons responded to pressure ejection of SLIGRL-NH(2) or trypsin with a prolonged depolarization that was often accompanied by increased excitability. When added to the bathing solution, trypsin caused a concentration-dependent depolarization of responding neurons with an estimated EC(50) value of 87 nM. Collectively, these novel observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.

Time-dependent changes in neurokinin(3) receptors and tachykinins during adjuvant-induced peripheral inflammation in the rat.

Although considerable evidence exists that spinal neurokinin(1) receptors are involved in central sensitization of nociception, recent evidence from knockout studies indicates that other neurokinin receptors in the spinal cord may mediate a portion of the hyperalgesia caused by substance P and neurokinin A. The present study determined whether the second most abundant class of neurokinin receptors, neurokinin(3) receptors, are regulated during persistent peripheral inflammation. Inflammation in the hind paw of the rat was induced by intraplantar injection of complete Freund's adjuvant. Receptor autoradiography revealed specific binding of [125I]-MePhe(7)-NKB, a selective ligand for neurokinin(3) receptors, in the superficial dorsal horn of the spinal cord. Specific binding of [125I]-MePhe(7)-NKB in the medial dorsal horn was reduced bilaterally two days after unilateral injection of complete Freund's adjuvant. Binding returned to basal levels four days after injection of complete Freund's adjuvant. Neurokinin(3) receptor messenger RNA levels doubled in the dorsal spinal cord at 12h and remained elevated for at least four days. The change in neurokinin(3) receptor binding and messenger RNA during adjuvant-induced inflammation may be a consequence of activation of the receptor. Spinal levels of potential endogenous ligands for spinal neurokinin(3) receptors were measured by radioimmunohistochemistry. Immunoreactive substance P but not neurokinin B peptide 2, a marker for neurokinin B, was reduced bilaterally during adjuvant-induced inflammation.Collectively, these data indicate that spinal neurokinin(3) receptors may play a role in spinal neurotransmission of injured rats and require consideration of other tachykinins as physiologically relevant ligands to spinal neurokinin(3) receptors.