Receptors Responsive to Protein Breakdown Products in G-Cells and D-Cells of Mouse, Swine and Human

Monitoring the luminal content in the stomach is of vital importance for adjusting the gastric activities, including the release of gastric hormones such as gastrin. Our previous studies have shown that in mice the gastrin-secreting G-cells express receptor types which are responsive to amino acids. Since the pig is considered as more suitable model for studying gastro-physiological aspects relevant for men, in this study we have analyzed the distribution of G-cells and D-cells in the gastric antrum of men, swine, and mouse and the expression of receptor types which may render these cells responsiveness to protein breakdown products. The results indicate that the number of G-cells per antral invagination was significantly higher in swine and human compared to mice and also the distribution pattern of G-cells differed between the species. The molecular phenotyping revealed that the receptors GPRC6A and CaSR were also expressed in G-cells and in a subpopulation of D-cells from swine and men. As an additional receptor type, the peptone-receptor GPR92, was found to be expressed in G-cells and a subpopulation of D-cells; this receptor type may be particular suitable for sensing protein breakdown products and thus be a key element to adjust the activity of G-cells and D-cells according to the progress of the digestive processes in the stomach. In search for elements of an intracellular signaling cascade it was found that G-cells express the G-protein subunit Gαq as well as the phospholipase C subtype PLCβ3; in contrast, D-cells expressed the subtype PLCβ2 and neither Gαq. These results indicate that there are significant species differences concerning the number and distribution pattern, but not concerning the molecular phenotype of the gastric endocrine cells. However, G-cells and D-cells significantly differ from each other regarding the repertoire of receptors and signaling elements

Band-like arrangement of taste-like sensory cells at the gastric groove: evidence for paracrine communication

The discovery of taste-related elements within the gastrointestinal tract has led to a growing interest in the mechanisms and physiological significance of chemosensory monitoring of chymus composition. Previous work suggests that brush cells located in the "gastric groove," which parallels the "limiting ridge," a structure in rodents that divides the fundus from the corpus, are candidate sensory cells. A novel sectioning technique revealed that these cells are arranged in a palisade-like manner forming a band which borders the whole length of the corpus epithelium. Using transgenic PLC beta 2 promoter-GFP mice and specific antibodies, we have demonstrated that most of these cells express gustducin, PLC beta 2, and TRPM5; typical signaling proteins of gustatory sensory "type II" cells. These molecular features strongly suggest that the cells may be capable of sensing nutrient or non-nutrient constituents of the ingested food. Since there is no evidence that brush cells are endocrine cells, attempts were made to explore how such putative chemosensory cells might transmit the information to "effector" cells. It was found that most of the cells express the neuronal nitric oxide synthase (NOS) suggesting some paracrine interaction with adjacent cells. Moreover, they also express choline acetyltransferase (ChAT) as well as the vesicular protein SNAP25, indicating the potential for cholinergic transmission, possibly with subjacent enteric nerve fibers

Putative interaction of brush cells with bicarbonate secreting cells in the proximal corpus mucosa

The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the "gastric groove," the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII), the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na+/H+ exchanger (NHE1). However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracnne interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1) and the neighboring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution

Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance: Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract

Short-term high fat feeding induces inflammatory responses of tuft cells and mucosal barrier cells in the murine stomach

Feeding mice with a high fat diet (HFD) induces inflammation and results in changes of gene expression and cellular composition in various tissues throughout the body, including the gastrointestinal tract. In the stomach, tuft cells expressing the receptor GPR120 are capable of sensing saturated long chain fatty acids (LCFAs) and thus may be involved in initiating mechanisms of mucosal inflammation. In this study, we assessed which cell types may additionally be affected by high fat feeding and which candidate molecular mediators might contribute to mucosaprotective immune responses. A high fat dietary intervention for 3 weeks caused an expansion of tuft cells that was accompanied by a higher frequency of mucosal mast cells and surface mucous cells which are a known source of the insult-associated cytokine interleukin 33 (IL-33). Our data demonstrate that both brush and mucosal mast cells comprise the enzyme ALOX5 and its activating protein FLAP and thus have the capacity for synthesizing leukotriene (LT). In HFD mice, several tuft cells showed a perinuclear colocalization of ALOX5 with FLAP which is indicative of an active LT synthesis. Monitoring changes in the expression of genes encoding elements of LT synthesis and signaling revealed that transcript levels of the leukotriene C4 synthase, LTC4S, catalyzing the first step in the biosynthesis of cysteinyl (cys) LTs, and the cysLT receptors, cysLTR2 and cysLTR3, were upregulated in mice on HFD. These mice also showed an increased expression level of IL-33 receptors, the membranebound ST2L and soluble isoform sST2, as well as the mast cell-specific protease MCPT1. Based on these findings it is conceivable that upon sensing saturated LCFAs tuft cells may elicit inflammatory responses which result in the production of cysLTs and activation of surface mucous cells as well as mucosal mast cells regulating gastric mucosal function and integrity

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice

Determination of apoptotic cells in testicular sections of wild-type and Tas1r1/mCherry knock-in mice.

<p>[<b>A</b>] Paraffin sections of Bouin-fixed wild-type and Tas1r1-deficient testes were used in a fluorescent TUNEL assay and counterstained with DAPI to visualize nuclei and thus cellular compartmentalization. The two photomicrographs for each genotype document representative staining patterns of TUNEL positive cells of 5 male littermates per genotype. Note that in wild-type animals [<i>+/+</i>] as well as in Tas1r1-deficient mice [<i>−/−</i>], spatial localization of TUNEL-reactive cells (red) showed the usual accumulation within the basal cell layer of the testicular tubules. Moreover, apoptotic cells for each genotype did not show obvious differences in their morphology (higher magnifications presented in the inserts in the two upper panels). Micrographs are composed by an overlay of the two fluorescent channels (TUNEL, [red]; DAPI, [blue]); apoptotic TUNEL-positive cells are highlighted by insets. [<b>B</b>] Quantitative analysis of apoptotic cells in testes of wild-type, heterozygous and Tas1r1 null animals. Numbers of TUNEL-positive cells of the three genotypes are presented as apoptotic cells per visual field. Note that Tas1r1-deficient mice ([−/−]) show a significantly increased rate of apoptosis compared to wild-type ([<i>+/+</i>]) and heterozygous ([+/−]) animals. Data presented are mean values ± SEM; statistical analysis was done using a paired Student's t-test comparing apoptotic rates of corresponding littermates (*: p≤0.05; **: p<0.01). Testes of littermate animals (n = 5) of each genotype were analyzed, and sections were taken from two different regions. 3–4 tissue sections of each testicular domain were quantified for TUNEL positive germ cells counting 3–4 randomly chosen microscopic fields containing 25–30 seminiferous tubules each.</p

Tas1r1 deletion results in increased spontaneous acrosome reaction and elevated cytosolic Ca²⁺ and cAMP levels.

<p>[<b>A</b>] Incidence of spontaneous loss of the acrosomal vesicle in sperm from Tas1r1 knock-out mice compared to control wild-type sperm. To quantify spontaneous acrosome reaction of uncapacitated and fully capacitated sperm, epididymal spermatozoa of wild-type and Tas1r1 null mutant mice with identical genetic background were either directly assessed for acrosomal secretion rates or incubated for 90 min in capacitation medium (HS/BSA/NaHCO₃). Data shown are mean values ± SEM of 15 independent experiments of different mouse sperm preparations. Obtained data were subjected to a Student's t-test for determination of significant differences (*: p≤0.05) between pairs of both genotypes. [<b>B</b>] Comparison of [Ca²⁺]_i, of wild-type and Tas1r1-deficient spermatozoa. To determine basal [Ca²⁺]_i in the head region of wild-type ([<i>+/+</i>], grey rhombs and squares) and Tas1r1-deficient ([<i>−/−</i>], black rhombs and squares) spermatozoa, epididymal sperm cells were either directly loaded with Fura-2AM ([<i>uncapacitated</i>], rhombs on the left side), or capacitated for 60 min prior Fura-2 loading ([<i>capacitated</i>], squares on the right side). Subsequently, Fura-2 fluorescence at 510 nm was measured at excitation wavelengths of 340 and 380 nm using a microscope based imaging system (TillPhotonics, Graefelfing, Germany). Fura-2 ratios (F340/F380) were determined for at least 14 cells per sperm preparation (total number of measured sperm cells: uncapacitated: 151 [<i>+/+</i>], 136 [<i>−/−</i>]); capacitated sperm: 168 [<i>+/+</i>], 181 [<i>−/−</i>]). [Ca²⁺]_i was calculated using the mean Fura-2 ratio of each animal (<i>F340/F380</i>) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032354#pone.0032354-Grynkiewicz1" target="_blank">[84]</a>. Only spermatozoa that showed [Ca²⁺]_i, increases upon stimulation with the calcium ionophore ionomycin were considered. Shown are vertical scatter plots of Fura-2 ratios of isolated spermatozoa of 5 animals for each genotype (littermates and animals with matched genetic background); the mean Fura-2 ratio is indicated by a bar. Mean values ± SEM of calculated [Ca²⁺]_i, for each genotype are given in numbers in the lower part of the graph.Statistical analyses were done using a paired Student's t-test (**: p<0.01). [<b>C</b>] Vertical scatter plot of basal cAMP concentration in uncapacitated spermatozoa. Shown are basal cAMP concentrations of epididymal sperm isolated in HS buffer. Littermate animals and animals with identical genetic background were prepared and assayed in parallel. cAMP values of corresponding animal pairs are connected by a line. Note that in 13 of 15 analyzed animal pairs, cAMP concentrations were higher in Tas1r1 -deficient [<i>−/−</i>] mice than in wild-type [<i>+/+</i>] animals. [<b>D–E</b>] cAMP concentrations in Tas1r1-deficient sperm compared to sperm of wild-type animals. Epididymal sperm of wild-type [<i>+/+</i>] and Tas1r1-deficient [<i>−/−</i>] mice were either isolated in HS (for 15 min) [<i>uncapacitated</i>] or in capacitation buffer (HS/BSA/NaHCO₃ for 60 min; [<i>capacitated</i>]), and subsequently treated for 5 min at 37°C with buffer alone [<b>D</b>] (uncapacitated: n = 15; capacitated: n = 11) or with 0.5 mM IMBX [<b>E</b>] (uncapacitated: n = 13; capacitated: n = 9). After shock-freezing the cells in liquid nitrogen, cAMP was extracted with PCA (7%), and quantified using a commercially available EIA kit. Data are mean values ± SEM. Sperm of littermate animals and animals with identical genetic background and age were assayed in parallel and compared using a paired student's T-Test (*: p≤0.05; **: p<0.01).</p

Genotype distribution of offspring from heterozygous Tas1r1 mating pairs.

<p>Breeding was carried out on a heterozygote-heterozygote base and the numbers of pubs of each genotype were determined [<i>number of pubs; observed</i>]. The percentage of each genotype from the total number of pubs is given in parentheses. The expected Mendelian distribution ratios [<i>number of pubs; expected</i>] and the p-value of the chi square test are given on the right. Note that for a total of 60 litters with 427 offspring of 15 heterozygous Tas1r1 breeding pairs, no significant deviation from the distribution predicted from Mendel's law was observed applying the chi square test (p≤0.05).</p