Mammalian Taste Receptor Cells Research Articles

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation. Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d. We recently reported on a successful method for the extended culture of rodent taste cells. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation. Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 2011.

Mutually exclusive expression of Gαia and Gα14 reveals diversification of taste receptor cells in zebrafish

A comprehensive reevaluation of the G protein alpha subunit genes specifically expressed in taste buds in the tongue epithelium of rodents revealed that Gq and G14 of the Gq class and Gi2 and Ggust (Gt3, also known as gustducin) of the Gi class are expressed in mammalian taste buds. Meanwhile, a database search of fish genomes revealed the absence of a gene encoding an ortholog of the mammalian Ggust gene, which mediates sweet, umami, and bitter taste signals in mammalian taste receptor cells (TRCs). Histochemical screening identified two G protein alpha subunit genes, zfGia and zfG14, expressed in subsets of TRCs in zebrafish. The expression patterns of zfGia and zfG14 in taste buds were mutually exclusive, and the expression of known T1R and T2R genes in zebrafish was restricted to a subset of zfGia-expressing TRCs. These findings highlight the existence of a novel subset of TRCs in zebrafish that is absent in mammals and suggest that unidentified G protein-coupled receptors are expressed in zfG14-expressing TRCs and in zfGia-expressing TRCs where known T1R and T2R genes were not expressed in zebrafish. The existence of not only generalized but also specialized subsets of TRCs may imply a strong connection between the evolution of the peripheral gustatory system and the evolution of particular species.

Modeling and simulation of ion channels and action potentials in taste receptor cells

Based on patch clamp data on the ionic currents of rat taste receptor cells, a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components, including voltage-gated Na(+) currents and outward delayed rectifier K(+) currents. Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants. The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed. Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.

Acetic acid activates PKD1L3–PKD2L1 channel—A candidate sour taste receptor

The polycystic kidney disease (PKD) 1L3–PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca2+ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3–PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.

A Taste Receptor Required for the Caffeine Response In Vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.

Water taste: the importance of osmotic sensing in the oral cavity

The sense of taste plays critical roles in nutrient identification and toxin avoidance. The ability to respond to hypoosmotic stimuli in mammalian taste receptor cells may reflect the importance of osmotic sensing by the gustatory system. Transduction for hypoosmotic stimuli involves water influx through aquaporins followed by activation of volume-regulated anion channels. The ability of these transduction elements to be regulated by natriferic hormones at the mRNA and protein level in other transporting epithelia suggest that the gustatory system may respond to extrinsic signals related to the restoration of salt and water balance. Plasticity in the peripheral gustatory system is consistent with the activity in the taste system being reflective of underlying nutritional status. Clearly, more research is needed to determine the link between nutrition, taste and the control of food and water intake.

Regulation of ENaC by intracellular chloride in mammalian taste receptor cells

The transduction of sodium salts (e.g. “salty taste”) has been shown to involve permeation of sodium ions through epithelial sodium channels (ENaC) in the gustatory system. Though numerous studies have demonstrated complex regulation of ENaC in other sodium transporting epithelia, there have been few studies that have focused on the regulation of ENaC in the taste system. To understand the regulatory capacity of the gustatory system we have begun to investigate the ability of ENaC to be regulated by intracellular Cl ions (Schrieber et al. J. Memb. Biol. 199:85, 2004). To identify expression of various transport proteins for Cl in the gustatory system we have used RT-PCR and immunocytochemistry. Taste cells express a variety of Cl transport proteins including several members of the ClC family of Cl channels, the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium-potassium-chloride cotransporter, NKCC. Patch clamp recording on mouse taste cells revealed that both the magnitude of ENaC currents and their sensitivity to the diuretic amiloride is inversely correlated with intracellular Cl concentration. In solutions high in extracellular Cl, inhibition of Cl transport through NKCC with bumetanide leads to increases in ENaC-mediated sodium influx in taste cells. We are currently investigating the role of other Cl transport proteins in ENaC regulation in taste cells. Supported by NIH DC02507 (TAG).

Characterization and Long-Term Maintenance of Rat Taste Cells in Culture

Taste cells have a limited life span and are replaced from a basal cell population, although the specific factors involved in this process are not well known. Short- and long-term cultures of other sensory cells have facilitated efforts to understand the signals involved in proliferation, differentiation, and senescence, yet few studies have reported successful primary culture protocols for taste cells. Furthermore, no studies have demonstrated both proliferation and differentiation in vitro. In this study, we have developed an in vitro culture system to maintain and utilize rat primary taste cells for more than 2 months without losing key molecular and biochemical features. Gustducin, phospholipase C-beta2 (PLC-beta2), T1R3, and T2R5 mRNA were detected in the cultured cells by reverse transcriptase-polymerase chain reaction. Western blot analysis demonstrated gustducin and PLC-beta2 expression in the same samples, which was confirmed by immunocytochemistry. Labeling with bromo-2-deoxyuridine (BrdU) demonstrated proliferation, and a subset of BrdU-labeled cells were also immunoreactive for either gustducin or PLC-beta2, indicating differentiation of newly generated cells in vitro. Cultured cells also exhibited increases in intracellular calcium in response to several taste stimuli. These results indicate that taste cells from adult rats can be generated and maintained under the described conditions for at least 2 months. This system will enable further studies of the processes involved in proliferation, differentiation, and function of mammalian taste receptor cells in an in vitro preparation.

Potential mechanisms for functional changes in taste receptor cells following sodium deficiency in mammals

Sodium is an essential nutrient for life, and its level in the body is tightly regulated. When sodium deficient, some mammals alter their behavior towards salt by avidly consuming it, even at concentrations animals typically choose to avoid. This change in acceptance is accompanied by a reduction in the response of the gustatory chorda tympani nerve to sodium solutions. More specifically, the response rate of the sodium-specialist units to NaCl stimulation is reduced following sodium deficiency or adrenalectomy. The initial transduction of the chemical signal is mediated, in part, by Na + influx through epithelial Na + channels in the apical membrane of taste cells that synapse with the specialist neurons. Circulating hormones like angiotensin II and adrenocorticotropin hormone, which are released in response to sodium deficiency and adrenalectomy, could regulate the activity of Na + channels through G-protein linked second-messenger systems. These putative pathways are of interest because they have been described in mammalian taste receptor cells. The present review will summarize evidence linking some hormones of fluid homeostasis with the apparent attenuation of input from sodium-specialist neurons.

Differential Expression of α-Gustducin in Taste Bud Populations of the Rat and Hamster

The G-protein subunit alpha-gustducin, which is similar to rod transducin, has been implicated in the transduction of both sweet- and bitter-tasting substances. In rodents, there are differences in sensitivity to sweet and bitter stimuli in different populations of taste buds. Rat fungiform taste buds are more responsive to salts than to sweet stimuli, whereas those on the palate respond predominantly to sweet substances. In contrast, hamster fungiform taste buds are more sensitive to sweet-tasting stimuli. Taste buds in the vallate and foliate papillae of both species are sensitive to bitter compounds. These differences in sensitivity should be reflected in the numbers of gustducin-containing cells in different taste bud populations. We examined taste buds in the rat and hamster for immunoreactivity to an antibody against alpha-gustducin. Immunofluorescence of labeled taste cells was examined by confocal microscopy, and the cells were counted. Gustducin-positive cells were seen in all taste bud regions; they were spindle-shaped, with circular cross-sections and apical processes that extended to the taste pore. Cells with this characteristic shape in rat vallate taste buds are Type II (light) cells. In the rat, taste buds of the fungiform papillae had fewer gustducin-positive cells (3.1/taste bud) than those of other regions, including the posterior tongue and palate (>8.9/taste bud). Hamster fungiform taste buds contained twice as many gustducin-expressing cells (6.8/taste bud) as those of the rat. These data support the hypothesis that alpha-gustducin is involved in the transduction of both sweet- and bitter-tasting stimuli by mammalian taste receptor cells.

A cyclic-nucleotide-suppressible conductance activated by transducin in taste cells.

Taste can be divided into four primary sensations: salty, sour, sweet and bitter. Salty and sour are directly transduced by apical channels, whereas sweet and bitter utilize cyclic nucleotide second messengers. We have shown that rod transducin is present in mammalian taste receptor cells, where it is activated by a bitter receptor and in turn activates a phosphodiesterase. Here we introduce into frog taste cells peptides derived from transducin's phosphodiesterase-interaction region, which cause an inward whole-cell current in a subset of cells. We find that the peptides' effects are reversibly suppressed by IBMX and forskolin, indicative of a transducin-activated phosphodiesterase. Cyclic nucleotides suppress the whole-cell current, indicating that cyclic nucleotides may regulate taste-cell conductance. IBMX modifies taste-cell responses to two taste stimuli, implicating phosphodiesterase in taste transduction. Submicromolar cyclic nucleotides directly suppress the conductance of inside-out patches derived from the taste-cell plasma membrane, independently of protein phosphorylation. The channels are unusual in that they are suppressed, rather than activated by cyclic nucleotides. We propose that transducin, via phosphodiesterase, decreases cyclic nucleotide levels to activate the cyclic-nucleotide-suppressible conductance, leading to Ca2+ influx and taste-cell depolarization.

Voltage-dependent sodium currents recorded from dissociated rat taste cells.

Voltage-dependent sodium currents were analyzed in detail from dissociated mammalian taste receptor cells using the whole-cell patch clamp technique. Approximately 50-75% of all taste receptor cells expressed sodium currents. These currents activated close to -50 mV (holding potential = -80 mV) with maximal currents most often occurring at -10 mV. The distribution of maximal inward currents across all cells appeared to display two peaks, at -254 pA and -477 pA, possibly due to differences in sodium channel density. Inward currents were eliminated by replacing 90% of external sodium with N-methyl-D-l-glucamine. The current-voltage relationship of the activated current, as measured by a tail current analysis, was linear, suggesting an ohmic nature of the open channel conductance. The relationship between the time to the peak activated current and the step potential was well fit by a double exponential curve (tau1 = 6.18, tau2 = 37.8 msec). Development of inactivation of the sodium current was dependent upon both voltage- and temporal-parameters. The voltage dependence of the time constant (tau) obtained from removal of inactivation, development of inactivation, and decay of the sodium current displayed a bell-shaped curve with a maximum of 55 msec at -70 mV. In addition to fast inactivation (half maximal at -50 mV), these currents also displayed a slow inactivation (half maximal at -65 mV). Voltage-dependent sodium currents were reversibly inhibited by nanomolar concentrations of tetrodotoxin (Kd = 10(-8) M). There was no evidence of a TTX-insensitive sodium current. This description broadens our understanding of gustatory transduction mechanisms with a particular relevance to the physiological role of receptor cell action potentials.

A method for isolating and patch-clamping single mammalian taste receptor cells

Individual taste receptor cells were isolated from the tonque of the mouse by enzymatic treatment followed by mechanical dissociation. The cells were morphologically identical with taste cells from amphibians. Whole-cell voltage-clamp recordings indicated that the murine taste cells possess a variety of voltage-dependent inward and outward currents. Delayed rectifier currents were blocked by denatonium benzoate, one of the most bitter compounds known. This preparation should permit a detailed electrophysiological investigation of taste transduction in mammals at the level of taste receptor cells.