TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the specificity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deficient mice. The phytonutrients increased currents in the duodenum with the relative potencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 μM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 μM), and were greatly diminished in Trpa1-/- duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.

Recent studies reveal complex patterns of hormone coexpression within enteroendocrine cells (EECs), contrary to the traditional view that gut hormones are expressed individually in EECs. Moreover, different hormones have been found in separate subcellular vesicles. However, detailed analysis of relative expression of multiple hormones has not been made. Subcellular studies have been confined to peptide hormones, and have not included the indolamine 5-hydroxytryptamine (5-HT) or the neuroendocrine protein chromogranin A (CgA). In the present work, coexpression of 5-HT, CgA, secretin, cholecystokinin (CCK), ghrelin, and glucagonlike peptide (GLP)-1 in mouse duodenum was quantified at a cellular and subcellular level by semiautomated cell counting and quantitative vesicle measurements. We investigated whether relative numbers of cells with colocalized hormones analyzed at a cell level matched the numbers revealed by examination of individual storage vesicles within cells. CgA and 5-HT were frequently expressed in EECs that contained combinations of GLP-1, ghrelin, secretin, and CCK. Separate subcellular stores of 5-HT, CgA, secretin, CCK, ghrelin, and GLP-1 were identified. In some cases, high-resolution analysis revealed small numbers of immunoreactive vesicles in cells dominated by a different hormone. Thus the observed incidence of cells with colocalized hormones is greater when analyzed at a subcellular, compared with a cellular, level. Subcellular analysis also showed that relative numbers of vesicles differ considerably between cells. Thus separate packaging of hormones that are colocalized is a general feature of EECs, and EECs exhibit substantial heterogeneity, including the colocalization of hormones that were formerly thought to be in cells of different lineages.

Recent studies reveal substantial species and regional differences in enteroendocrine cell (EEC) populations, including differences in patterns of hormone coexpression, which limit extrapolation between animal models and human. In this study, jejunal samples, with no histologically identifiable pathology, from patients undergoing Whipple's procedure were investigated for the presence of gastrointestinal hormones using double- and triple-labelling immunohistochemistry and high-resolution confocal microscopy. Ten hormones (5-HT, CCK, secretin, proglucagon-derived peptides, PYY, GIP, somatostatin, neurotensin, ghrelin and motilin) were localised in EEC of the human jejunum. If only single staining is considered, the most numerous EEC were those containing 5-HT, CCK, ghrelin, GIP, motilin, secretin and proglucagon-derived peptides. All hormones had some degree of colocalisation with other hormones. This included a population of EEC in which GIP, CCK and proglucagon-derived peptides are costored, and four 5-HT cell populations, 5-HT/GIP, 5-HT/ghrelin, 5-HT/PYY, and 5-HT/secretin cell groups, and a high degree of overlap between motilin and ghrelin. The presence of 5-HT in many secretin cells is consistent across species, whereas lack of 5-HT and CCK colocalisation distinguishes human from mouse. It seems likely that the different subclasses of 5-HT cells subserve different roles. At a subcellular level, we examined the vesicular localisation of secretin and 5-HT, and found these to be separately stored. We conclude that hormone-containing cells in the human jejunum do not comply with a one-cell, one-hormone classification and that colocalisations of hormones are likely to define subtypes of EEC that have different roles.

Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine (5-HT) to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify fourteen EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.

We use a monoclonal antibody against the C-terminal of oxyntomodulin (OXM) to investigate enteroendocrine cells (EEC) in mouse, rat, human and pig. This antibody has cross-reactivity with the OXM precursor, glicentin (Gli) but does not recognise glucagon. The antibody stained EEC in the jejunum and colon of each species. We compared OXM/Gli immunoreactivity with that revealed by antibodies against structurally related peptides, GLP-1 and glucagon and against GIP and PYY that are predicted to be in some EEC that express OXM/Gli. We used super-resolution to locate immunoreactive vesicles. In the pancreas, OXM/Gli was in glucagon cells but was located in separate storage vesicles to glucagon. In jejunal EEC, OXM/Gli and GIP were in many of the same cells but often in separate vesicles, whereas PYY and OXM/Gli were commonly colocalised in the same storage vesicles of colonic EEC. When binding of anti-GLP-1 to the structurally related GIP was removed by absorption with GIP peptide, GLP-1 and OXM/Gli immunoreactivities were contained in the same population of EEC in the intestine. We conclude that anti-OXM/Gli is a more reliable marker than anti-GLP-1 for EEC expressing preproglucagon products. Storage vesicles that were immunoreactive for OXM/Gli were almost always immunoreactive for GLP-1. OXM concentrations, measured by ELISA, were highest in the distal ileum and colon. Lesser concentrations were found in more proximal parts of small intestine and pancreas. Very little was in the stomach. In EEC containing GIP and OXM/Gli, these hormones are packaged in different secretory vesicles. Separate packaging also occurred for OXM and glucagon, whereas OXM/Gli and PYY and OXM/Gli and GLP-1 were commonly contained together in secretory vesicles.

The highly innervated cornea is susceptible to nerve loss secondary to systemic diseases such as diabetes and metabolic disturbances caused by high-fat diet. In this study, we characterize the effect of high-fat diet on the mouse corneal neuroimmune phenotype, including changes to corneal nerve density and resident immune cells, alongside the clinical assessment of corneal thickness and endothelial cell density.

Although the pig is an accepted model species for human digestive physiology, no previous study of the pig gastric mucosa and gastric enteroendocrine cells has investigated the parallels between pig and human. In this study, we have investigated markers for each of the classes of gastric endocrine cells, gastrin, ghrelin, somatostatin, 5-hydroxytryptamine, histidine decarboxylase, and PYY cells in pig stomach. The lining of the proximal stomach consisted of a collar of stratified squamous epithelium surrounded by gastric cardiac glands in the fundus. This differs considerably from human that has only a narrow band of cardiac glands at its entrance, surrounded by a fundic mucosa consisting of oxyntic glands. However, the linings of the corpus and antrum are similar in pig and human. Likewise, the endocrine cell types are similar and similarly distributed in the two species. As in human, gastrin cells were almost exclusively in the antrum, ghrelin cells were most abundant in the oxyntic mucosa and PYY cells were rare. In the pig, 70% of enterochromaffin-like (ECL) cells in the antrum and 95% in the fundus contained 5-hydroxytryptamine (5-HT), higher proportions than in human. Unlike the enteroendocrine of the small intestine, most gastric enteroendocrine cells (EEC) did not contain colocalised hormones. This is similar to human and other species. We conclude that the pig stomach has substantial similarity to human, except that the pig has a protective lining at its entrance that may reflect the difference between a pig diet with hard abrasive components and the soft foods consumed by humans.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

5-HT containing enteroendocrine cells (EEC), the most abundant type of EEC in the gut, regulate many functions including motility, secretion and inflammatory responses. We examined the morphologies of 5-HT cells from stomach to rectum, patterns of hormone co-expression in the stomach and colon, and the relationship of 5-HT cells with nerve fibres. We also reviewed some of the relevant literature. The morphologies of 5-HT cells were distinct, depending on their location in the gut. A noticeable feature of some 5-HT cells in the antrum and colon was their long basal processes, which resembled processes of neurons, whereas 5-HT cells in the small intestinal mucosa lacked basal processes. In the stomach, numerous 5-HT cells, including cells with basal processes, were identified as enterochromaffin-like cells by their expression of histidine decarboxylase. In the colon, we observed a small number of 5-HT cells that were in close contact with, but distinct from, oxyntomodulin (OXM) and PYY immunoreactive EEC. We did not find specific relationships between nerve fibres and the processes of colonic 5-HT cells. We conclude that five major features, i.e., gut region, morphology, hormone content, receptor repertoire and cell lineage, can be used to define 5-HT cells.