A cluster of gustducin-expressing cells in the mouse stomach associated with two distinct populations of enteroendocrine cells

In the gastrointestinal (GI) tract, a variety of digestive processes are continually adapted to the changing composition of ingested foods, which requires a precise chemosensory monitoring of luminal contents. Gustducin-expressing brush cells scattered throughout the GI mucosa are considered candidate sensory cells for accomplishing this task. In this study, we have investigated a large cluster of gustducin-positive cells which is located exactly at the boundary between the fundic and the oxyntic mucosa of the mouse stomach, at the so-called “limiting ridge”. In close association with the candidate chemosensory cluster, we found two populations of enteroendocrine cells: one population containing the satiety regulating hormone ghrelin, the other population comprising serotonin-secreting enterochromaffin cells. The particular arrangement of gustducin-expressing cells and enteroendocrine cells at the limiting ridge suggests a direct interplay between these cell types with immediate implications, not only for digestive processes in the stomach, but also for parameters controlling the satiety status.     

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.     

Ontogeny of apelin and its receptor in the rodent gastrointestinal tract

Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin–APJ system in the regulation of smooth muscle, epithelial and goblet cell function in the GI tract.     

Role of CCK1 and Y2 receptors in activation of hindbrain neurons induced by intragastric administration of bitter taste receptor ligands

G-protein-coupled receptors signaling bitter taste (T2Rs) in the oral gustatory system and the alpha-subunit of the taste-specific G-protein gustducin are expressed in the gastrointestinal (GI) tract. alpha-Subunit of the taste-specific G-protein gustducin colocalizes with markers of enteroendocrine cells in human and mouse GI mucosa, including peptide YY. Activation of T2Rs increases cholecystokinin (CCK) release from the enteroendocrine cell line, STC-1. The aim of this study was to determine whether T2R agonists in the GI tract activate neurons in the nucleus of the solitary tract (NTS) and whether this activation is mediated by CCK and peptide YY acting at CCK(1) and Y(2) receptors. Immunocytochemistry for the protooncogene c-Fos protein, a marker for neuronal activation, was used to determine activation of neurons in the midregion of the NTS, the region where vagal afferents from the GI tract terminate. Intragastric administration of the T2R agonist denatonium benzoate (DB), or phenylthiocarbamide (PTC), or a combination of T2R agonists significantly increased the number of Fos-positive neurons in the mid-NTS; subdiaphragmatic vagotomy abolished the NTS response to the mixture of T2R agonists. Deletion of CCK(1) receptor gene or blockade of CCK(1) receptors with devazepide abolishes the activation of NTS neurons in response to DB, but had no effect on the response to PTC. Administration of the Y(2) receptor antagonist BIIE0246 blocks the activation of NTS neurons to DB, but not PTC. These findings suggest that activation of neurons in the NTS following administration of T2R agonists to the GI tract involves CCK(1) and Y(2) receptors located on vagal afferent terminals in the gut wall. T2Rs may regulate GI function via release of regulatory peptides and activation of the vagal reflex pathway.     

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.     

Perinatal olfactory learning in the domestic dog

The ability of individuals to learn about chemosensory stimuli in the prenatal, or immediate postnatal, period may be advantageous in acquiring information about "safe" foods after weaning. In this study, we examined the influence of perinatal exposure to aniseed via the mother's diet on a two-choice food test in the domestic dog. Pups were tested at 10 weeks of age following "prenatal" exposure to aniseed (the last 20 days of gestation), "postnatal" exposure to aniseed (the first 20 days after birth), "perinatal" exposure to aniseed (pre- and postnatal exposure combined), or no exposure to aniseed prenatally or postnatally (control). Perinatal exposure resulted in a significantly greater preference for the aniseed food than the other types of exposure. At 10 weeks, there was no evidence for the retention of any prenatal learning of the aniseed. It is suggested that exposure to a chemosensory stimulus across the perinatal period results in a greater effect than simply the sum of pre- and postnatal exposure due to priming of the chemosensory system via prenatal chemosensory experience. Such a system may confer survival advantages by promoting the acquisition of information about safe foods.     

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.     

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

Background

Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.

Methods

We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).

Results

Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.

Conclusions

Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.     

Expression of taste signal transduction molecules in the caecum of common marmosets

The extraoral presence of taste signal transduction proteins has recently been reported in rodents and humans. Here, we report for the first time the presence of these signal transduction proteins in the caecum of a non-human primate, the common marmoset. Quantitative RT-PCR data on the gene expression of taste signal transduction molecules (gustducin and TRPM5) in common marmosets suggested high expression in the caecum, which was not observed in other non-human primates. Immunohistochemical analysis confirmed the specific presence of gustducin and taste receptors in marmoset caecal cells. These results may relate to the specific feeding behaviour of marmosets, which consume plant exudates, primarily gums.     

Role of gastrin in the development of gastric mucosa,ECL cells and A-like cells in newborn and young rats

Histamine-producing ECL cells and ghrelin-producing A-like cells are endocrine/paracrine cell populations in the acid-producing part of the rat stomach. While the A-like cells operate independently of gastrin, the ECL cells respond to gastrin with mobilization of histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. Gastrin is often assumed to be the driving force behind the postnatal development of the gastric mucosa in general and the ECL cells in particular. We tested this assumption by examining the oxyntic mucosa (with ECL cells and A-like cells) in developing rats under the influence of YF476, a cholecystokinin-2 (CCK(2)) receptor antagonist. The drug was administered by weekly subcutaneous injections starting at birth. The body weight gain was not affected. Weaning occurred at days 15-22 in both YF476-treated and age-matched control rats. Circulating gastrin was low at birth and reached adult levels 2 weeks after birth. During and after weaning (but not before), YF476 greatly raised the serum gastrin concentration (because of abolished acid feedback inhibition of gastrin release). The weight of the stomach was unaffected by YF476 during the first 2-3 weeks after birth. From 4 to 5 weeks of age, the weight and thickness of the gastric mucosa were lower in YF476-treated rats than in controls. Pancreastatin-immunoreactive cells (i.e. all endocrine cells in the stomach) and ghrelin-immunoreactive cells (A-like cells) were few at birth and increased gradually in number until 6-8 weeks of age (control rats). At first, YF476 did not affect the development of the pancreastatin-immunoreactive cells, but a few weeks after weaning, the cells were fewer in the YF476 rats. The ECL-cell parameters (oxyntic mucosal histamine and pancreastatin concentrations, the histidine decarboxylase (HDC) activity, the HDC mRNA levels and serum pancreastatin concentration) increased slowly until weaning in both YF476-treated and control rats. From then on, there was a further increase in the ECL-cell parameters in control rats but not in YF476 rats. The postnatal development of the ghrelin cells (i.e. the A-like cells) and of the A-like cell parameters (the oxyntic mucosal ghrelin concentration and the serum ghrelin concentrations) was not affected by YF476 at any point.We conclude that gastrin affects neither the oxyntic mucosa nor the endocrine cells before weaning. After weaning, CCK(2) receptor blockade is associated with a somewhat impaired development of the oxyntic mucosa and the ECL cells. While gastrin stimulation is of crucial importance for the onset of acid secretion during weaning and for the activation of ECL-cell histamine formation and secretion, the mucosal and ECL-cell growth at this stage is only partly gastrin-dependent. In contrast, the development of the A-like cells is independent of gastrin at all stages.     

Potential Role of Transient Receptor Potential Channel M5 in Sensing Putative Pheromones in Mouse Olfactory Sensory Neurons

Based on pharmacological studies of chemosensory transduction in transient receptor potential channel M5 (TRPM5) knockout mice it was hypothesized that this channel is involved in transduction for a subset of putative pheromones in mouse olfactory sensory neurons (OSNs). Yet, in the same study an electroolfactogram (EOG) in the mouse olfactory epithelium showed no significant difference in the responses to pheromones (and odors) between wild type and TRPM5 knockout mice. Here we show that the number of OSNs expressing TRPM5 is increased by unilateral naris occlusion. Importantly, EOG experiments show that mice lacking TRPM5 show a decreased response in the occluded epithelia to putative pheromones as opposed to wild type mice that show no change upon unilateral naris occlusion. This evidence indicates that under decreased olfactory sensory input TRPM5 plays a role in mediating putative pheromone transduction. Furthermore, we demonstrate that cyclic nucleotide gated channel A2 knockout (CNGA2-KO) mice that show substantially decreased or absent responses to odors and pheromones also have elevated levels of TRPM5 compared to wild type mice. Taken together, our evidence suggests that TRPM5 plays a role in mediating transduction for putative pheromones under conditions of reduced chemosensory input.     

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Regulation of the mucosal gastrin receptor

The gastrin receptor in rat oxyntic gland mucosa is highly regulated. This regulation has so far only been found to be directed at the total numbers of receptors present. The affinity of the receptor is not altered significantly by agents which affect receptor numbers. Homospecific regulation occurs in that gastrin upregulates its receptor over long periods of time. Upregulation, however, appears to be preceded by a brief period of downregulation. During development corticosterone triggers the synthesis, or at least the appearance of gastrin receptors. Receptor development is maintained by solid food and presumably the gastrin it releases, but the change in diet which occurs at weaning does not in itself induce development. In female rats, estrogens prevent the increase of gastrin receptor levels to those found in males. These results with the gastrin receptor emphasize the importance of studying receptor concentrations as well as hormone levels to the total understanding of an endocrine response. They also suggest that the receptors of the other gastrointestinal hormones are likely to be regulated and that this regulation will probably be important in understanding some of the diseases of 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     

Spermidine-induced glycoprotein fucosylation in immature rat intestine.

In rat small intestine, during postnatal development, the glycoprotein fucosylation is markedly increased at weaning. At the same time, a rise in the intestinal spermidine level was observed, partly due to the increase in the spermidine content of solid food given to animals at this period as compared to the spermidine content of milk. In order to mimic the spermidine increase observed in weanling rat intestines, we had treated suckling rats with spermidine by oral ingestion to study its role as maturation factor of the small intestine. In spermidine-treated suckling rats, the spermidine and N-acetyl-spermidine contents were highly increased. Spermidine treatment induced the rise in alpha-1,2-fucosyltransferase activity and the precocious appearance in the brush-border membrane of some alpha-1,2-fucoproteins in weaned rats. Such results indicate that spermidine could be a maturation factor implicated in the appearance of alpha-1,2-fucoproteins naturally observed at weaning time.     

Localization, physiological significance and possible clinical implication of gastrointestinal melatonin.

The gastrointestinal tract (GIT) is a major source of extrapineal melatonin. In some animals, tissue concentrations of melatonin in the GIT surpass blood levels by 10-100 times and the digestive tract contributes significantly to melatonin concentrations in the peripheral blood, particularly during the day. Some melatonin found in the GIT may originate from the pineal gland, as the organs of the digestive system contain binding sites, which in some species exhibit circadian variation. Unlike the production of pineal melatonin, which is under the photoperiodic control, release of GI melatonin seems to be related to periodicity of food intake. Melatonin and melatonin binding sites were localized in all GI tissues of mammalian and avian embryos. Postnatally, melatonin was localized in the GIT of newborn mice and rats. Phylogenetically, melatonin and melatonin binding sites were detected in GIT of numerous mammals, birds and lower vertebrates. Melatonin is probably produced in the serotonin-rich enterochromaffin cells (EC) of the GI mucosa and can be released into the portal vein postprandially. In addition, melatonin can act as an autocrine or a paracrine hormone affecting the function of GI epithelium, lymphatic tissues of the immune system and the smooth muscles of the digestive tube. Finally, melatonin may act as a luminal hormone, synchronizing the sequential digestive processes. Higher peripheral and tissue levels of melatonin were observed not only after food intake but also after a long-term food deprivation. Such melatonin release may have a direct effect on the various GI tissues but may also act indirectly via the CNS; such action might be mediated by sympathetic or parasympathetic nerves. Melatonin can protect GI mucosa from ulceration by its antioxidant action, stimulation of the immune system and by fostering microcirculation and epithelial regeneration. Melatonin may reduce the secretion of pepsin and the hydrochloric acid and influence the activity of the myoelectric complexes of the gut via its action in the CNS. Tissue or blood levels of melatonin may serve as a marker of GI lesions or tumors. Clinically, melatonin has a potential for a prevention or treatment of colorectal cancer, ulcerative colitis, irritable bowel syndrome, children colic and diarrhea.     

Difference between the effect of acute and chronic surgical vagotomy on the cytoprotective action of atropine against indomethacin-induced mucosal lesions on the gastrointestinal tract in rats

The cytoprotective effect of a small dose of atropine was proved against the indomethacin (IND)-caused gastrointestinal (GI) mucosal damage. This protective effect of atropine disappeared in the acute phase of surgical vagotomy (ASV) on the vagally-innervated parts of GI tract. The aims of our observations were: 1) to examine the effect of chronic surgical vagotomy (CSV) on the cytoprotective action of atropine in the GI tract; and 2) to compare the effects of ASV and CSV on the GI cytoprotection caused by atropine against IND-induced mucosal damage and vascular permeability in rats. The IND was given s.c. 24 h prior to the killing of the animals in a dose of 20 mg x kg(-1). Bilateral surgical vagotomy or sham operation were carried out 24 h (ASV) or 14 d (CSV) before IND-application. Atropine was given i.p. every 5 h after IND-treatment in a dose of 0.1 mg x kg(-1). The number of macroscopical mucosal ulcerations was noted and its severity was calculated by semiquantitative scale in the stomach, small intestine and three equal parts of colon. Vascular permeability was measured by Evans-blue leakage into the mucosal tissue. It has been found that: 1) Tte small dose of atropine significantly decreased the IND-induced mucosal damage and vascular permeability on the stomach, small intestine and the vascular permeability on the proximal colon; 2) the small dose of atropine did not cause any changes in the appearance of IND-induced mucosal lesions and in Evans blue concentration in the mucosa after ASV, but it significantly decreased the IND-caused mucosal damage and Evans blue concentration in the mucosa of stomach, small intestine and proximal colon after CSV; 3) the IND-induced mucosal damage and vascular permeability treated with atropine (given in cytoprotective dose) were significantly smaller after CSV than that after ASV on the stomach, small intestine and proximal colon. It has been concluded that the intact vagal nerve has an essential role in the appearance of cytoprotective mechanisms of atropine in GI tract.