Patricia Widmayer

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 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.

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.

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.

Altered expression of gustatory-signaling elements in gastric tissue of morbidly obese patients

Objective:Sensing of nutrients in the stomach is of crucial importance for the regulation of ingestive behavior especially in the context of metabolic dysfunctions such as obesity. Cells in the gastric mucosa with taste-signaling elements are considered as candidates for sensing the composition of ingested food and consequently modulate gastrointestinal processes. To assess whether obesity might have an impact on gastric chemosensory cells, gastric tissue samples from morbidly obese patients and normal-weight subjects were compared using a reverse transcriptase (RT)-PCR, qPCR and immunohistochemical approach.Results:Analysis of biopsy tissue samples from human stomach revealed that transcripts for the taste-signaling elements, including the receptor T1R3 involved in the reception of amino acids and carbohydrates, the fatty acid receptor GPR120, the G protein gustducin, the effector enzyme PLCβ2 and the ion channel TRPM5 are present in the human gastric mucosa and led to the visualization of candidate chemosensory cells in the stomach expressing gustatory marker molecules. RT-PCR and qPCR analyses indicated striking differences in the expression profiles of specimens from obese subjects compared with controls. For GPR120, gustducin, PLCβ2 and TRPM5 the expression levels were increased, whereas for T1R3 the level decreased. Using TRPM5 as an example, we found that the higher expression level was associated with a higher number of TRPM5 cells in gastric tissue samples from obese patients. This remarkable change was accompanied by an increased number of ghrelin-positive cells.Conclusions:Our findings argue for a relationship between the amount of food intake and/or the energy status and the number of candidate chemosensory cells in the gastric mucosa.

Expression of the G-protein α-subunit gustducin in mammalian spermatozoa

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit α-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether α-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-α-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the α-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether α-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for α-gustducin. Although a possible function for α-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein α-subunit may process intracellular signals controlling sperm motility.

Expression of a pheromone receptor in ovipositor sensilla of the female moth (Heliothis virescens).

Female moths release pheromones that influence various behavioral and physiological processes. The highly specific responses elicited by pheromones are mediated via specific chemosensory proteins, pheromone binding proteins and chemoreceptors, operating in the antennal sensory neurons. In Heliothis virescens, the response to the major pheromone component (Z)‐11‐hexadecenal (Z11‐16:Al) is mediated by the pheromone binding protein PBP2 and the receptor type HR13. PCR experiments revealed that transcripts for relevant chemosensory molecules are also present in the abdomen suggesting an additional role. In the female, mRNA for HR13 as well as for the related PBP2 was found in the ovipositor tip and in an immunohistochemical analysis with a specific antiserum it was possible to visualize the receptor protein in distinct sensilla types surrounding the ovipositor tip. The expression of HR13 implies a chemosensory responsiveness of these sensilla types to pheromones possibly provided by PBP2. Due to the close vicinity of sensillar HR13 cells and pheromone producing cells in the ovipositor we propose that the HR13 cells might mediate abdominal responses to the emitted pheromones.

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 PLCβ2 promoter-GFP mice and specific antibodies, we have demonstrated that most of these cells express gustducin, PLCβ2, 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.

Identification of a nonmammalian Golf subtype: functional role in olfactory signaling of airborne odorants in Xenopus laevis.

Attempts to identify the Gα subtypes in the two compartments of the olfactory system from Xenopus, which are supposed to be specialized for detecting aquatic and volatile odorous compounds, revealed that a Gαo1 subtype is characteristic for the “water nose,” the lateral diverticulum, whereas a novel Gαs subtype predominates in the “air nose,” the medial diverticulum. The newly identified Gαs‐type is more closely related to Gαolf of rat and human than to the known Gαs‐isoform of Xenopus; it is therefore considered the first identified nonmammalian Gαolf subtype. Sequence comparison of Gαolf from amphibia and mammals revealed a particular conservation within the α‐helical domains, which are supposed to control the GDP/GTP‐exchange rate. The selective expression of different Gα subtypes in the two anatomically separated and functionally specialized nasal compartments parallels the expression of distinct classes of olfactory receptors. Moreover, biochemical analysis revealed that stimulation with appropriate odorous compounds elicits the formation of inositol trisphosphate in the lateral diverticulum. In contrast, cyclic adenosine monophosphate signals were induced in the medial diverticulum, and this response appears to be mediated by the novel Gαolf subtype. The data indicate that olfactory sensory neurons in each of the nasal cavities are equipped not only with defined sets of receptor types but also with a distinct molecular machinery for the chemo‐electrical transduction process. J. Comp. Neurol. 439:400–410, 2001.

Candidate chemosensory cells in the porcine stomach

A continuous chemosensory monitoring of the ingested food is of vital importance for adjusting digestive processes according to diet composition. Although any dysfunction of this surveillance system may be the cause of severe gastrointestinal disorders, information about the cellular and molecular basis of chemosensation in the gastrointestinal tract is limited. The porcine alimentary canal is considered as an appropriate model for the human gastrointestinal tract. Therefore, in this study we have investigated the gastric mucosa of swine for cells which express gustatory transduction elements such as TRPM5 or PLCβ2, and thus may represent candidate “chemosensors”. It was found that the porcine stomach indeed contains cells expressing gustatory marker molecules; however, the morphology and topographic distribution of putative chemosensory cells varied significantly from that in mice. Whereas in the murine stomach these cells were clustered at a distinct region near the gastric entrance, no such compact cell cluster was found in the pig stomach. These results indicate substantial differences regarding the phenotype of candidate chemosensory cells of mice and swine and underline the importance of choosing the most suitable model organisms.