BACKGROUND AND PURPOSE Hydrogen sulphide (H2S) 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 H2S involves SQR.
EXPERIMENTAL APPROACH H2S 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, H235S was catabolized to 35S‐thiosulphate and 35S‐sulphate, and stigmatellin reduced both the consumption of H235S and formation of 35S‐thiosulphate. Stigmatellin also enhanced H2S release by the colonic muscularis externa. In the brain, catabolism of H235S to 35S‐thiosulphate and 35S‐sulphate, which was stigmatellin‐insensitive, partially accounted for H235S consumption, while the remainder was captured as unidentified 35S 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 H2S 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 H2S signal termination occurs partially through protein sequestration and partially through catabolism not involving SQR. As H2S has beneficial effects in animal models of human disease, we suggest that selective inhibition of SQR is an attractive target for pharmaceutical development.
Background: Interstitial cells of Cajal (ICC) are required for normal intestinal motility. ICC are found throughout the human colon and are decreased in the sigmoid colon of patients with slow ...transit constipation. Aims: The aims of this study were to determine the normal distribution of ICC within the human colon and to determine if ICC are decreased throughout the colon in slow transit constipation. Patients: The caecum, ascending, transverse, and sigmoid colons from six patients with slow transit constipation and colonic tissue from patients with resected colon cancer were used for this study. Methods: ICC cells were identified with a polyclonal antibody to c-Kit, serial 0.5 μm sections were obtained by confocal microscopy, and three dimensional software was employed to reconstruct the entire thickness of the colonic muscularis propria and submucosa. Results: ICC were located within both the longitudinal and circular muscle layers. Two networks of ICC were identified, one in the myenteric plexus region and another, less defined network, in the submucosal border. Caecum, ascending colon, transverse colon, and sigmoid colon displayed similar ICC volumes. ICC volume was significantly lower in the slow transit constipation patients across all colonic regions. Conclusions: The data suggest that ICC distribution is relatively uniform throughout the human colon and that decreased ICC volume is pan-colonic in idiopathic slow transit constipation.
Background & Aims: Gastrointestinal complications of long-standing diabetes include nausea, vomiting, abdominal pain, diarrhea, and constipation.The pathophysiology underlying these symptoms is ...poorly understood.Recent evidence suggests an important role for interstitial cells of Cajal in controlling gastrointestinal motility.The aim of this study was to determine changes in interstitial cells of Cajal and enteric innervation in a patient with insulin-dependent diabetes.Methods: A full thickness jejunal biopsy was obtained from a 38-year-old insulin-dependent diabetic with evidence for diabetic gastroenteropathy.Immunohistochemistry, confocal microscopy, and 3-dimensional reconstruction techniques were used to quantify changes in the volume of interstitial cells of Cajal and enteric innervation.Results: Interstitial cells of Cajal were markedly decreased throughout the entire thickness of the jejunum.A decrease in neuronal nitric oxide synthase, vasoactive intestinal peptide, PACAP, and tyrosine hydroxylase immunopositive nerve fibers was observed in circular muscle layer while substance P immunoreactivity was increased.Conclusions: The data suggest that long-standing diabetes is associated with a decrease in interstitial cells of Cajal volume and a decrease in inhibitory innervation, associated with an increase in excitatory innervation.The changes in interstitial cells of Cajal volume and enteric nerves may underlie the pathophysiology of gastrointestinal complications associated with diabetes and suggest future therapeutic targets.
GASTROENTEROLOGY 2001;121:427-434
Key points
A CO‐dependent transwall gradient of resting membrane potential exists across the circular muscle layer in the gastrointestinal tract; this gradient regulates the strength of phasic muscle ...contraction during electrical slow wave activity.
H2S, CO and NO are endogenously generated in the muscle layers of the gut wall.
Inhibiting the H2S producing enzyme CSE with the CSE inhibitor PAG had no significant effect on transwall resting membrane potential gradient in control preparations but significantly shifted the entire gradient in the depolarizing direction in preparations pretreated with the NO synthase (NOS) inhibitor l‐NNA.
PAG significantly shifted the gradient in the depolarizing direction in mouse preparations lacking neuronal NOS (nNOS‐KO) and the gradient was significantly shifted in the depolarizing direction in mouse preparations lacking both nNOS and CSE (CSE‐KO–nNOS‐KO).
NO production was significantly higher in CSE‐KO mouse preparations compared to wild‐type mouse preparations and the amplitude of NO‐mediated slow inhibitory junction potentials (S‐IJPs) was significantly higher in CSE‐KO mouse preparations compared to the amplitude of S‐IJPs in wild‐type mouse preparations.
Nearly all submucosal neurons and myenteric neurons were CSE positive and 11% of submucosal neurons and 50% of myenteric neurons were nNOS positive.
Our novel findings suggested that endogenous H2S is a stealth hyperpolarizing factor on smooth muscle cells and that endogenous H2S inhibits NO production from nNOS.
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.
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 13C‐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.
High temporal resolution gastric emptying breath tests can measure accelerated or delayed gastric emptying of liquids and solids and resolve 1‐phase vs two‐phase gastric emptying. These analyses discovered that two‐phase gastric emptying is more prevalent that previously described, is strain‐dependent and may involve intestinal feedback, yet contribute little to the overall rate of gastric emptying.
The factors underlying the survival and maintenance of interstitial cells of Cajal (ICC) are not well understood. Loss of ICC is often associated with loss of neuronal nitric oxide synthase (nNOS) in ...humans, suggesting a possible link. The aim of this study was to determine the effect of neuronal NO on ICC in the mouse gastric body. The volumes of ICC were determined in nNOS−/− and control mice in the gastric body and in organotypic cultures using immunohistochemistry, laser scanning confocal microscopy and three‐dimensional reconstruction. ICC numbers were determined in primary cell cultures after treatment with an NO donor or an NOS inhibitor. The volumes of myenteric c‐Kit‐immunoreactive networks of ICC from nNOS−/− mice were significantly reduced compared with control mice. No significant differences in the volumes of c‐Kit‐positive ICC were observed in the longitudinal muscle layers. ICC volumes were either decreased or unaltered in the circular muscle layer after normalization for the volume of circular smooth muscle. The number of ICC was increased after incubation with S‐nitroso‐N‐acetylpenicillamine and decreased by N(G)‐nitro‐l‐arginine. Neuronally derived NO modulates ICC numbers and network volume in the mouse gastric body. NO appears to be a survival factor for ICC.
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.
A strong, positive correlation was identified between colonic expression of serotonin transporter Slc6a4 and serotonin receptor Htr4 across different colonization states and sexes. These data suggest that expression of Slc6a4 and Htr4 involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut.
Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 ...(HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes (nNOSΔ /Δand HO2Δ /Δ) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOSΔ /Δand HO2Δ /Δmice are depolarized, with apparent additive effects in the double knockouts (HO2Δ /Δ/nNOSΔ /Δ). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2Δ /Δpreparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.
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.--Sha, L., Farrugia, G., Linden, D. R., Szurszewski, J. H. The transwall gradient across the mouse colonic circular muscle layer is carbon monoxide dependent.
Interstitial cells of Cajal (ICC) are specialized mesenchyme‐derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility ...disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c‐Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase‐3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double‐labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 ± 0.2% of ICC were positive for activated caspase‐3 and in the circular muscle layer 2.1 ± 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.
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