Candidate chemosensory cells in the stomach mucosa of young postnatal mice during the phases of dietary changes

The complex physiology of the gastrointestinal (GI) tract must permanently be adjusted according to the composition of ingested food, which requires continuous monitoring by appropriate sensory systems. Sensing the dietary constituents is thought to be mediated by chemosensory cells residing in the mucosa of the GI tract. We have examined the appearance and differentiation of candidate chemosensory cells at distinct postnatal stages and visualized cells that express gustducin or TRPM5. Two critical stages have been considered: the suckling period when the neonates are nourished exclusively on milk and the weaning period when the diet gradually changes to solid food. At early postnatal stages, only a few gustducin- or TRPM5-expressing cells have been found; they display an immature morphology. At the time of weaning, numerous gustducin- or TRPM5-positive cells are present in the gastric mucosa and are isomorphic to adult candidate chemosensory cells. The typical accumulation of gustducin and TRPM5 cells at the border between the forestomach and corpus region and the characteristic tissue fold or “limiting ridge” have not been observed at early postnatal stages but are complete at the time of weaning. The appearance of candidate chemosensory cells at the strategic position occurs within the last few days before weaning but after the formation of the limiting ridge. Thus, both the topographic arrangement of the cells and the limiting ridge seem to be important features for the processing of solid food in the mouse stomach.     

T1R3 is expressed in brush cells and ghrelin-producing cells of murine stomach

Various digestive and enteroendocrine signaling processes are constantly being adapted to the chemical composition and quantity of the chyme contained in the diverse compartments of the gastrointestinal tract. The chemosensory monitoring that underlies the adaptive capacity of the gut is thought to be performed by so-called brush cells that share morphological and molecular features with gustatory sensory cells. A substantial population of brush cells is localized in the gastric mucosa. However, no chemosensory receptors have been found to be expressed in these cells so far, challenging the concept that they serve a chemosensory function. The canonical chemoreceptors for the detection of macronutrients are taste receptors belonging to the T1R family; these have been identified in several tissues in addition to the gustatory system including the small intestine. We demonstrate the expression of the T1R subtype T1R3, which is essential for the detection of both sugars and amino acids in the gustatory system, in two distinct cell populations of the gastric mucosa. One population corresponds to open-type brush cells, emphasizing the notion that they are a chemosensory cell type; T1R3 immunoreactivity in these cells is restricted to the apical cell pole, which might provide the basis for the detection of luminal macronutrient compounds. The second gastric T1R3-positive population consists of closed-type endocrine cells that produce ghrelin. This finding suggests that ghrelin-releasing cells, which lack access to the stomach lumen, might receive chemosensory input from macronutrients in the circulation via T1R3.     

Fatty acid detection during food consumption and digestion: Associations with ingestive behavior and obesity

The inability of humans to adequately regulate fat consumption is a salient contributor to the development of obesity. The macronutrients, fat, protein and carbohydrate, within foods are detected at various stages of consumption, during which their digestive products, fatty acids, amino acids and sugars, interact with chemosensory cells within the oral epithelium (taste receptor cells) and gastrointestinal (GI) tract (enteroendocrine cells). This chemoreception initiates functional responses, including taste perception, peptide secretion and alterations in GI motility, that play an important role in liking of food, appetite regulation and satiety. This review will summarize the available evidence relating to the oral and GI regulation of fat intake and how chemoreception at both locations is associated with digestive behavior, satiety and weight regulation.     

Nutrient sensing receptors in gastric endocrine cells

Sensing protein breakdown products in the luminal content is of particular importance for the regulation of digestive activities in the stomach which are mainly governed by gastric hormones. The molecular basis for tuning the release of hormones according to the protein content is still elusive. In this study we have analysed the murine stomach for candidate nutrient receptors. As a promising candidate we have concentrated on the broadly tuned amino acid receptor GPRC6A. Expression of GPRC6A could be demonstrated in different regions of the murine stomach; especially in the gastric antrum. Using immunohistochemical approaches, a large cell population of GPRC6A-positive cells was visualized in the basal half of the antral gastric mucosa. Molecular phenotyping of GPRC6A-immunoreactive cells revealed that most of them contained the peptide hormone gastrin. A small population turned out to be immunoreactive for somatostatin. In search for additional amino acid receptors in antral gastric mucosa, we obtained evidence for expression of the gustatory amino acid receptor subunit T1R3 and the calcium-sensing receptor CaSR. Many CaSR-cells were found in the gastric antrum and most of them also contained gastrin; very similar to GPRC6A-cells. In contrast, T1R3 was found only in a small population of gastrin-negative cells. The finding that GPRC6A-and CaSR-receptors are both expressed in many if not all gastrin cells strongly suggests that both receptor types are co-expressed in the same cells, where they could form heterodimers providing a unique response spectrum of these cells.     

Gastric tuft cells express DCLK1 and are expanded in hyperplasia

Epithelial tuft cells are named after their characteristic microtubule bundles located at the cell apex where these are exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical “tuft” is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, and newborn and postnatal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum, but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g.,TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia, and metaplasia.     

Taste-signaling proteins are coexpressed in solitary intestinal epithelial cells

The taste system, made up of taste receptor cells clustered in taste buds at the surface of the tongue and the soft palate, plays a key role in the decision to ingest or reject food and thereby is essential in protecting organisms against harmful toxins and in selecting the most appropriate nutrients. To determine if a similar chemosensory system exists in the gastrointestinal tract, we used immunohistochemistry and real-time polymerase chain reaction (PCR) to investigate which taste-signaling molecules are expressed in the intestinal mucosa. The PCR data showed that T1r1, T1r2, T1r3, alpha-gustducin, phospholipase Cbeta2 (PLCbeta2), and Trpm5 are expressed in the stomach, small intestine, and colon of mice and humans, with the exception of T1r2, which was not detected in the mouse and human stomach or in the mouse colon. Using transgenic mice expressing enhanced green fluorescent protein under the control of the Trpm5 promoter, we found colocalization of Trpm5 and alpha-gustducin in tufted cells at the surface epithelium of the colon, but these cells did not express T1r3 or PLCbeta2. In the duodenal glands, 43%, 33%, and 38% of Trpm5-expressing cells also express PLCbeta2, T1r3, or alpha-gustducin, respectively. The duodenal gland cells that coexpress PLCbeta2 and Trpm5 morphologically resemble enteroendocrine cells. We found a large degree of colocalization of Trpm5, alpha-gustducin, T1r1, and T1r3 in tufted cells of the duodenal villi, but these cells rarely expressed PLCbeta2. The data suggest that these duodenal cells are possibly involved in sensing amino acids.     

Gastrin-releasing peptide: neuronal distribution and spatial relation to endocrine cells in the human upper gut

By using immunocytochemical techniques, we have studied the distribution of gastrin releasing peptide (GRP)-containing neurons as well as the spatial relationship between these neurons and the endocrine cells in the human stomach and duodenum. Moderate numbers of immunoreactive fibers were distributed in the smooth muscle and submucosa of the stomach; they were more rare in the duodenal wall. Numerous GRP-containing nerve fibers were found in the oxyntic mucosa, the antral mucosa harboured only few GRP immunoreactive nerve fibers. The mucosa of the proximal duodenum was found to be virtually devoid of such fibers. Only occasionally did we observe signs of a direct contact between GRP-containing nerve fibers and gastrin and somatostatin cells in the antral mucosa. In the oxyntic mucosa GRP-containing nerve fibers sometimes seemed to contact endocrine cells, including somatostatin cells as well as individual parietal cells. In conclusion, although GRP-containing nerve fibers were quite numerous in the wall of the human upper gastro-intestinal (GI)-tract, we observed a lack of intimate spatial relationship between these fibers and endocrine cells in the antral mucosa, suggesting additive mechanisms to a direct innervation of gastrin cells and somatostatin cells by GRP nerve fibers explaining the physiological effects on hormonal release.     

Taste receptors in the gastrointestinal tract. I. Bitter taste receptors and alpha-gustducin in the mammalian gut

Molecular sensing by gastrointestinal (GI) cells plays a critical role in the control of multiple fundamental functions in digestion and also initiates hormonal and/or neural pathways leading to the regulation of caloric intake, pancreatic insulin secretion, and metabolism. Molecular sensing in the GI tract is also responsible for the detection of ingested harmful drugs and toxins, thereby initiating responses critical for survival. The initial recognition events and mechanism(s) involved remain incompletely understood. The notion to be discussed in this article is that there are important similarities between the chemosensory machinery elucidated in specialized neuroepithelial taste receptor cells of the lingual epithelium and the molecular transducers localized recently in enteroendocrine open GI cells that sense the chemical composition of the luminal contents of the gut.     

Gastrointestinal chemosensation: chemosensory cells in the alimentary tract

Sensing potentially beneficial or harmful constituents in the luminal content by specialized cells in the gastrointestinal mucosa is an essential prerequisite for governing digestive processes, initiating protective responses and regulating food intake. Until recently, it was poorly understood how the gastrointestinal tract senses and responds to nutrients and non-nutrients in the diet; however, the enormous progress in unraveling the molecular machinery underlying the responsiveness of gustatory cells in the lingual taste buds to these compounds has been an important starting point for studying intestinal chemosensation. Currently, the field of nutrient sensing in the gastrointestinal tract is evolving rapidly and is benefiting from the deorphanization of previously unliganded G-protein-coupled receptors which respond to important nutrients, such as protein degradation products and free fatty acids as well as from the FACS-assisted isolation of distinct cell populations. This review focuses on mechanisms and principles underlying the chemosensory responsiveness of the alimentary tract. It describes the cell types which might potentially contribute to chemosensation within the gut: cells that can operate as specialized sensors and transducers for luminal factors and which communicate information from the gut lumen by releasing paracrine or endocrine acting messenger molecules. Furthermore, it addresses the current knowledge regarding the expression and localization of molecular elements that may be part of the chemosensory machinery which render some of the mucosal cells responsive to constituents of the luminal content, concentrating on candidate receptors and transporters for sensing nutrients.     

Comparative analysis of gastrin-immunoreactive and argyrophil cells in bony fish larvae

A comparative study was made of enteroendocrine cells in the larvae of bony fishes (Teleostei): carp (Cyprinus carpio L.), big head (Aristichyts nobilis Richardson), silver carp (Hypophthalmichthys molitrix Valenciennes), and atlantic salmon (salmo salar L.). Argyrophil cells demonstrated by the method after Grimelius were found in all examined fish species. They are located in the pyloric appendices, stomach and intestinal mucosa. Gastrin-immunoreactive cells demonstrated by the immunocytochemical method were located in the pyloric part of the stomach in all examined fishes, except for the atlantic salmon in which they were not identified. The first argyrophil cells were identified at the age of about 10 days, while gastrin-immunoreactive cells proved to appear 5-7 days later.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Morphological and histochemical characterisation of the mucosa of the digestive tract in matrinxã Brycon amazonicus (Teleostei: Characiformes)

This study describes anatomical, histological and histochemical features of the digestive tract mucosal layer of the matrinxã Brycon amazonicus, an omnivorous freshwater fish endemic from the Amazon basin. This species presents short thick oesophagus with longitudinal folds, that allow the passage of large food items. The mucosa is lined with a stratified secretory epithelium rich in goblet cells that secrete neutral and acid mucins. The two mucin types provide different viscosity in anterior and posterior oesophagus related to the protective and lubricant functions, respectively. The stomach is a highly distensible Y-shaped saccular organ. Here, it is proposed that this anatomical shape plays an essential role in food storage when food availability is abundant. The stomach mucosa is composed of epithelial cells with intense neutral mucin secretion to protects against gastric juice. The intestine is slightly coiled and presents internally a complex pattern of transversal folds that increases the absorption surface and the retention time of food. Goblet cells in the intestine secrete acid and neutral mucins that lubricate the epithelium and aid in the digestive processes. In the rectum, an increase in goblet cells population occurs that may be related to better lubrication.     

Immunohistochemical characterization of brush cells in the rat larynx

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.     

Satiety: the roles of peptides from the stomach and the intestine

Rats were surgically prepared to allow perfusions of anatomically limited portions of the gastrointestinal (GI) surface during test meals. The results demonstrated that at least one potent satiety signal was generated when ingested food accumulated in the stomach and did not enter the small intestine. This gastric satiety signal did not require the vagus nerve for its operation. In addition, at least one other potent satiety signal was generated when food perfused the small intestine. This intestinal satiety signal did not require gastric distension for its operation. We tested a variety of GI peptides to determine whether any met the criteria imposed by this evidence for regionally specific satiety signals. Bombesin (BBS), a peptide present in high concentration in the stomach, was a potent and behaviorally specific inhibitor of food intake. Its satiating effect was not altered by subdiaphragmatic vagotomy. Cholecystokinin (CCK), a peptide hormone that is released from the small intestine by food, was also a potent and behaviorally specific inhibitor of food intake; its satiating effect did not require gastric distension for its expression, but its satiating effect was markedly reduced or abolished by subdiaphragmatic vagotomy. Thus, BBS and CCK may mediate at least part of the satiating effect of food acting in the stomach and in the small intestine, respectively.     

The retinoblastoma protein, RB, is required for gastrointestinal endocrine cells to exit the cell cycle, but not for hormone expression

Important functions of the RB family proteins include inhibition of cell cycle progression and regulation of terminal differentiation. We have examined the role of RB and the related protein, p107, in regulating cell cycle activity and differentiation of gastrointestinal endocrine cells, a relatively quiescent cell population, by conditionally disrupting the RB gene in neurogenin3 (Ngn3)-expressing cells in both p107^+/+ and p107^−/− mice. Endocrine cells in the small intestine, colon, pancreas, and stomach were present in normal numbers in RB and RB-p107 mutants except for an increase in serotonin cells and decrease in ghrelin cells in the antral stomach. Deletion of RB resulted in a dramatic increase in proliferating serotonin cells in the antral stomach and intestine, whereas other enteroendocrine cell types exhibited much lower cell cycle activity or remained quiescent. The related p107 protein appears dispensable for enteroendocrine differentiation and does not functionally compensate for the loss of RB. Our results suggest that RB is required for enteroendocrine cells, particularly serotonin cells, to undergo cell cycle arrest as they terminally differentiate. RB has relatively subtle effects on enteroendocrine cell differentiation and is not required for the expression of the normal repertoire of hormones in the gastrointestinal tract.     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

Transdifferentiation of mucous neck cells into chief cells in fundic gastric glands shown by GNA lectin histochemistry

The epithelium of the gastric mucosa and its glands in the corpus of rat stomach contains mucous surface cells (MSCs), parietal cells, mucous neck cells (MNCs), zymogenic or chief cells (ZCs), several types of enteroendocrine cells, and intermediate cells with characteristics between MNCs and ZCs also called transitional or prezymogenic cells (pre-ZCs).The aim of our work was to analyze the expression of Mannose (Man) in the rat gastric glands by means of Galanthus nivalis lectin (GNA) histochemistry to identify the differences between MNC, pre-ZCs and ZCs and to establish the relationships between these cells. Most of the cytoplasm of MNCs was negative for GNA histochemistry. Intensity of GNA labeling in the gastric gland showed a graduation from pre-ZCs (weak labeling) to ZCs (moderate labeling). Labeling of ZCs was stronger at the perinuclear and apical cytoplasm.In the last years, strong evidence has been reported supporting that ZCs differentiate from MNCs. Our work also supports the origin of ZCs from MNCs, because the GNA labeling graduation might be due to oligosaccharides which are not expressed in MNCs, start to express in pre-ZCs and are more abundant in ZCs, indicating that differentiation from MNCs to ZCs is a process in which glycans with Man moieties are synthesized.