David R. Linden

Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea‐pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 ± 2 mV compared to 20 ± 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 ± 1 compared to 1.6 ± 0.2); they had a smaller afterhyperpolarisation (9 ± 2 mV s compared to 20 ± 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+‐sensitive rectification of the current–voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis‐induced augmentation of the hyperpolarisation‐activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.

Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells

Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5‐hydroxytryptamine (5‐HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ‐free (GF) or humanized (HM; ex‐GF colonized with human gut microbiota). 5‐HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5‐HT synthesis; P < 0.01] and chromogranin A (neuroendocrine secretion; P < 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5‐HT4, or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein (P < 0.05) and 5‐HT concentrations (GF, 17 ± 3 ng/mg; HM, 25 ± 2 ng/mg; and CR, 35 ± 3 ng/mg; P < 0.05). Enterochromaffin (EC) cell numbers (cells producing 5‐HT) were unchanged. Short‐chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5‐HT production and homeostasis.—Reigstad, C. S., Salmonson, C. E., Rainey, III, J. F., Szurszewski, J. H., Linden, D. R., Sonnenburg, J. L., Farrugia, G., Kashyap, P. C. Gut microbes promote colonic serotonin production through an effect of short‐chain fatty acids on enterochromaffin cells. FASEB J. 29, 1395‐1403 (2015). www.fasebj.org

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

Abstract  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.

CD206-Positive M2 Macrophages That Express Heme Oxygenase-1 Protect Against Diabetic Gastroparesis in Mice

BACKGROUND & AIMS Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues

The gaseous molecule hydrogen sulfide (H2S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H2S. This production occurs through the activity of two enzymes, cystathionine‐γ‐lyase and cystathionine‐β‐synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H2S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.

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

Abstract  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.

Effect of Age on the Enteric Nervous System of the Human Colon

Abstract  The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age‐related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33–99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu‐positive neurons declined with age (slope = −1.3 neurons/mm/10 years, P = 0.03). The number of ChAT‐positive neurons also declined with age (slope = −1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS‐positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = −0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS‐positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits

Inflammation of the gastrointestinal (GI) tract has pronounced effects on GI function. Many of the functions of the GI tract are subject to neural regulation by the enteric nervous system (ENS) and its extrinsic connections. Therefore, it is possible that inflammatory effects on the ENS contribute to altered function during GI inflammation. The reflex circuitry of the ENS is comprised of sensory transducers in the mucosa (enteroendocrine cells), afferent neurons, interneurons and motor neurons. This review focuses on recent data that describe inflammation-induced changes to the ENS and mucosal enteroendocrine cells. Studies of tissues from patients with inflammatory bowel disease (IBD) and from animal models of IBD have demonstrated marked changes in mucosal enteroendocrine cell signaling. These changes, which have been studied most intensely in 5-HT-containing enterochromaffin cells, involve changes in the number of cells, their signaling molecule content or their means of signal termination. Morphological evidence of enteric neuropathy during inflammation has been obtained from human samples and animal models of IBD. The neuropathy can reduce the number of enteric neurons in the inflamed region and is often accompanied by a change in the neurochemical coding of enteric neurons, both in the inflamed region and at distant sites. Electrophysiological recordings have been made from enteric neurons in inflamed regions of the colon of animal models of IBD. These studies have consistently found that inflammation increases excitability of intrinsic primary afferent neurons and alters synaptic transmission to interneurons and motor neurons. These data set the stage for a comprehensive examination of the role of altered neuronal and enteroendocrine cell signaling in symptom generation during GI inflammation.

Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.

We have previously demonstrated that trinitrobenzene sulphonic acid (TNBS)‐induced colitis in guinea pig is associated with hyperexcitability of myenteric AH neurones, enhanced synaptic activity in the myenteric plexus, increased serotonin (5‐HT) availability in the mucosa, and decreased propulsive motor activity. The current study tested the hypothesis that the activation of cyclooxygenase (COX) contributes to these alterations in bowel functions. DFU inhibition of COX‐2, but not SC‐560 inhibition of COX‐1, restored to normal levels the electrical properties of myenteric AH neurones, the proportion of S neurones exhibiting slow EPSPs, and the rate of propulsive motor activity. Neither inhibitor was effective in altering the level of inflammation, the increased availability of mucosal 5‐HT, or the enhanced fast EPSPs in myenteric AH and S neurones. COX‐2 expression is enhanced in the myenteric plexus and cells within the smooth muscle layers during colitis, possibly reflecting the site at which COX‐2 inhibition acts to allow recovery of motor function. In support of this concept, COX‐1, but not COX‐2, inhibition was effective in restoring normal mucosal prostaglandin levels. These results indicate that the various changes that occur in the motor neural pathways of the distal colon in TNBS‐induced colitis do not involve a single neuroimmune mechanism. COX‐2 activation is a critical step in the enhanced excitability of AH neurones as well as diminished propulsive motility in TNBS colitis, whereas other yet to be resolved pathways, that do not involve COX‐1 or COX‐2 activation, lead to altered 5‐HT content in the mucosa and an augmentation of fast EPSPs.

Endogenous Production of H2S in the Gastrointestinal Tract: Still in Search of a Physiologic Function

Hydrogen sulfide (H(2)S) has long been associated with the gastrointestinal tract, especially the bacteria-derived H(2)S present in flatus. Along with evidence from other organ systems, the finding that gastrointestinal tissues are capable of endogenous production of H(2)S has led to the hypothesis that H(2)S is an endogenous gaseous signaling molecule. In this review, the criteria of gasotransmitters are reexamined, and evidence from the literature regarding H(2)S as a gaseous signaling molecule is discussed. H(2)S is produced enzymatically by gastrointestinal tissues, but evidence is lacking on whether H(2)S production is regulated. H(2)S causes well-defined physiologic effects in gastrointestinal tissues, but evidence for a receptor for H(2)S is lacking. H(2)S is inactivated through enzymatic oxidation, but evidence is lacking on whether manipulating H(2)S oxidation alters endogenous cell signaling. Remaining questions regarding the role of H(2)S as a gaseous signaling molecule in the gastrointestinal tract suggest that H(2)S currently remains a molecule in search of a physiologic function.