Coding of sweet,bitter, and umami tastes: different receptor cells sharing similar signaling pathways

Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.     

Inhibition of signal termination-related kinases by membrane-permeant bitter and sweet tastants: potential role in taste signal termination

Sweet and bitter taste sensations are believed to be initiated by the tastant-stimulated T1R and T2R G protein-coupled receptor (GPCR) subfamilies, respectively, which occur in taste cells. Although such tastants, with their significantly diverse chemical structures (e.g., sugar and nonsugar sweeteners), may share the same or similar T1Rs, some nonsugar sweeteners and many bitter tastants are amphipathic and produce a significant delay in taste termination (lingering aftertaste). We report that such tastants may permeate rat taste bud cells rapidly in vivo and inhibit known signal termination-related kinases in vitro, such as GPCR kinase (GRK)2, GRK5, and PKA. GRK5 and perhaps GRK2 and GRK6 are present in taste cells. A new hypothesis is proposed in which membrane-permeant tastants not only interact with taste GPCRs but also interact intracellularly with the receptors' downstream shutoff components to inhibit signal termination. amphipathic tastants; tastant permeation; desensitization; lingering aftertaste     

Stimulation of the extracellular Ca²⁺-sensing receptor by denatonium

The extracellular Ca(2+)-sensing receptor (CASR) is a promiscuous G-protein-coupled receptor closely related to the taste receptors T1R1-T1R3. Here we analyzed the possibility that apart from being stimulated by external Ca(2+) and amino acids, the substances effective as tastants, CASR might serve as a receptor for other sapid compounds. CASR was heterologously expressed in HEK-293 cells, and their responsivity to a variety of bitter and sweet substances was examined. Among them, solely denatonium was found to stimulate Ca(2+) signaling in CASR-positive HEK-293 cells. Apparently, these Ca(2+) responses were specific, as those were inhibited by the CASR antagonist NSP-4123. Altogether, our findings indicate that denatonium stimulates CASR by shifting a dose-response curve for the principal CASR agonist Ca(2+) to lower concentrations.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

The influence of taste on swallowing apnea, oral preparation time, and duration and amplitude of submental muscle contraction

Prior research has documented a modulating effect of taste on swallowing. We hypothesized that presentation of tastant stimuli would be a significant variable in swallowing-respiratory coordination, duration of oral bolus preparation, and submental muscle contraction. Twenty-three healthy females were presented with 1-cm(3) gelatin samples flavored with 4 tastants of increasing intensities. Visual analogue scale ratings of perceived intensity of each were used to identify relative equivalent concentrations across the 4 tastants. Data were then collected during ingestion of 5 trials of the 4 equivalent tastants using measurements of nasal airflow and submental surface electromyography (sEMG) to record biomechanical measures. Chi-square analysis failed to identify a statistically significant influence of taste on the phase location of swallowing apnea. Repeated measures analysis of variance demonstrated significant taste effects for oral preparation time, submental sEMG amplitude, and duration (P < 0.02). Sweet tastants were prepared for a shorter time when compared with bitter tastants. Swallow duration for sour, salty, and bitter tastants were longer than sweet and neutral tastants. Sour tastants resulted in the greatest amplitude of submental muscle contraction during swallowing. This study supports existing research that found that sour substances were swallowed with more effort when compared with other tastes.     

The steroid glycoside H.g.-12 from Hoodia gordonii activates the human bitter receptor TAS2R14 and induces CCK release from HuTu-80 cells

Steroid glycosides extracted from the succulent plant Hoodia gordonii are suggested to have appetite-suppressant effects both in animals and humans. Yet, the mechanisms underlying the putative satiety action of Hoodia steroid glycosides are not fully understood. We found that H.g.-12, a steroid glycoside purified from H. gordonii extract, initiated cholecystokinin (CCK) secretion both ex vivo in rat intestine and in vitro in the human enteroendocrine (EC) cell line HuTu-80. CCK is known to exert central effects on appetite suppression via the vagus nerve which afferents terminate in the gut wall. Recent data show that G protein-coupled receptors signaling bitter taste (T2Rs) are expressed in both rodent and human gastrointestinal tract. It was further demonstrated that bitter sensing is functional in mouse STC-1 EC cells and leads to CCK secretion via increased intracellular Ca2(+) concentrations. Based on the bitter taste of H. gordonii purified extracts, we assessed whether H.g.-12 could activate human bitter receptors. The steroid glycoside activated selectively TAS2R7 and TAS2R14, both heterologously expressed in HEK 293 cells. Removing an essential structural feature from the steroid glycoside inhibited H.g.-12-induced Ca2(+) increase in TAS2R14-expressing HEK cells and abolished H.g.-12-induced CCK secretion from human EC cells. Similarly, a nonspecific bitter receptor antagonist abolished H.g.-12-induced CCK secretion in HuTu-80 cells. These results point to a potential route of action by which components of Hoodia might influence appetite control. Our data also provide additional evidence that bitter taste-sensing mechanisms are coupled to hormone release from EC cells in the intestine. Moreover, we identified a natural agonist of TAS2R7 and TAS2R14 for further studies on the role of bitter receptors in satiety control and food intake.     

The receptors for mammalian sweet and umami taste

Sweet and umami (the taste of monosodium glutamate) are the main attractive taste modalities in humans. T1Rs are candidate mammalian taste receptors that combine to assemble two heteromeric G-protein-coupled receptor complexes: T1R1+3, an umami sensor, and T1R2+3, a sweet receptor. We now report the behavioral and physiological characterization of T1R1, T1R2, and T1R3 knockout mice. We demonstrate that sweet and umami taste are strictly dependent on T1R-receptors, and show that selective elimination of T1R-subunits differentially abolishes detection and perception of these two taste modalities. To examine the basis of sweet tastant recognition and coding, we engineered animals expressing either the human T1R2-receptor (hT1R2), or a modified opioid-receptor (RASSL) in sweet cells. Expression of hT1R2 in mice generates animals with humanized sweet taste preferences, while expression of RASSL drives strong attraction to a synthetic opiate, demonstrating that sweet cells trigger dedicated behavioral outputs, but their tastant selectivity is determined by the nature of the receptors.     

Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli

Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. Here, we report that each of the two subunits in the heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.     

Sweet successes.

Mapping of the chromosomal location of genes essential for sweet and bitter taste and identification of the relevant G protein-coupled receptors reveals unanticipated complexity in taste signaling pathways. The distribution of sweet and bitter receptors suggests complete cellular segregation of these taste modalities. Sweet compounds may be distinguished through differential expression of sweet receptors. Novel heterologous expression systems to test bitter and sweet modalities now provide the tools necessary for understanding taste coding.     

Taste receptor cell-based biosensor for taste specific recognition based on temporal firing

Taste receptor cells are the taste sensation elements expressing sour, salty, sweet, bitter and umami receptors, respectively. There are cell-to-cell communications between different types of cells. Nevertheless, the mechanism of taste sensation and taste information coded by taste receptor cell is not well understood at present and it is a long-standing issue. In order to explore taste sensation and analyze taste-firing responses from another point of view, we present a promising biomimetic taste receptor cell-based biosensor. The temporal firing responses to different tastants are recorded. Meanwhile, we investigate the firing rate and temporal firing of taste receptor cells. The experimental results are consistent with that from patch clamp and molecular biology experiment. Firing rate is dependent on the concentration of stimulus. PCA analysis (principal component analysis) of the temporal firing responses shows that the responses from different types of taste receptor cells can be distinguished. Furthermore, exogenous ATP is applied to mimic the effects of transmitter ATP (adenosine triphosphate) released from type II cells onto type III cells. Both enhanced and inhibitory effects on spontaneous firing are observed. This novel biomimetic hybrid biosensor provides a potential solution to investigate the taste sensation and coding mechanisms in a non-invasive way.     

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

A serotonin-sensitive sensor for investigation of taste cell-to-cell communication

Taste receptor cells are the taste sensation elements for sour, salty, sweet, bitter and umami sensations. It was demonstrated that there are cell-to-cell communications between type II (sour) and type III (sweet, bitter and umami) taste cells. Serotonin (5-HT) is released from type III cells, which is the only type of taste cells that has synaptic process with sensory afferent fibers. Then, taste information is transmitted via fibers to the brain. During this process, 5-HT plays important roles in taste information transmission. In order to explore a sensor to detect 5-HT released from taste cell or taste cell networks, we develop a 5-HT sensitive sensor based on LAPS chip. This sensor performs with a detection limit of 3.3 × 10(-13)M and a sensitivity of 19.1 mV per concentration decade. Upon the stimuli of sour and mix (bitter, sweet and umami) tastants, 5-HT released from taste cells could be detected flexibly, benefit from the addressability of LAPS chip. The experimental results show that the local concentration of 5-HT is around several nM, which is consistent with those from other methods. In addition, immunofluorescent imaging technique is utilized to confirm the functional existence of both type II and III cells in a cluster of isolated taste cells. Different types of taste cells are labeled with corresponding specific antibody. This 5-HT sensitive LAPS chip provides a potential and promising way to detect 5-HT and to investigate the taste coding and information communication mechanisms.     

A novel family of mammalian taste receptors

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.     

The human taste receptor hTAS2R14 responds to a variety of different bitter compounds

The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, (-)-alpha-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of (-)-alpha-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances.     

Monitoring ATP release from individual cells with a biosensor

Adenosine triphosphate (ATP) is a neurotransmitter/neuromodulator in both central and peripheral nervous systems. Particularly in the taste bud, a peripheral taste organ, ATP serves as an afferent neurotransmitter. To examine the mechanism that mediates ATP secretion in taste cells, we elaborated an approach for monitoring ATP in an extracellular medium by employing a biosensor, that is, cells responsive to ATP. Two lines of ATP-sensitive cells, HEK-293 and COS-1, which endogenously express P2Y receptors, were employed. In addition, HEK-293 cells transfected with P2X₃ receptors were also used. By most relevant parameters (threshold response, inactivation kinetics of ATP responses, and refractory period), COS-1 cells were more suitable as an ATP sensor than HEK-293 cells, both native and transfected. For the HEK-293 cell-based biosensor, one of pitfalls was that they were highly responsive to mechanical disturbances, e.g., solution flux elicited by application of a chemical stimulus, owing to the expression of mechanosensitive Ca²⁺-permeable cation channels. In COS-1 cells, ATP-dependent Ca²⁺ transients were generated mostly due to Ca²⁺ release, the feature allowing one to control the activity of ATP-releasing cells electrophysiologically and to monitor the ATP secretion by Ca²⁺ responses of the ATP-biosensor. By using this technique, it was demonstrated that individual taste cells of a mouse released ATP in response to membrane depolarization.     

Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes

BACKGROUND: Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium. METHODS: We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies. RESULTS: In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond. CONCLUSIONS: This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.     

Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the -subunits of the G protein gustducin (G_gust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca²⁺ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca²⁺] ([Ca²⁺]_i) in a dose- and time-dependent manner. Chelating extracellular Ca²⁺ with EGTA blocked the increase in [Ca²⁺]_i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca²⁺]_i induced by bombesin, but did not attenuate the [Ca²⁺]_i increase elicited by DB or PTC. These results indicate that Ca²⁺ influx mediates the increase in [Ca²⁺]_i induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca²⁺ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca²⁺]_i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca²⁺]_i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca²⁺]_i and cholecystokinin release through Ca²⁺ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells. type 2 family taste receptors; gastrointestinal peptides; phospholipase C ₂; Ca²⁺ fluxes; enteroendocrine cells; cholecystokinin secretion