Sue C. Kinnamon

ATP Signaling Is Crucial for Communication from Taste Buds to Gustatory Nerves

Taste receptor cells detect chemicals in the oral cavity and transmit this information to taste nerves, but the neurotransmitter(s) have not been identified. We report that adenosine 5′-triphosphate (ATP) is the key neurotransmitter in this system. Genetic elimination of ionotropic purinergic receptors (P2X2 and P2X3) eliminates taste responses in the taste nerves, although the nerves remain responsive to touch, temperature, and menthol. Similarly, P2X-knockout mice show greatly reduced behavioral responses to sweeteners, glutamate, and bitter substances. Finally, stimulation of taste buds in vitro evokes release of ATP. Thus, ATP fulfils the criteria for a neurotransmitter linking taste buds to the nervous system.

Mechanisms of taste transduction.

Taste cells use a wide variety of mechanisms for transduction. Ionic stimuli, such as salts and acids, interact directly with ion channels to depolarize taste cells. More complex stimuli, such as sugars and amino acids, utilize apically located receptors for transduction. Recent molecular biological results suggest that the metabotropic glutamate receptor mGluR4 may function in glutamate taste transduction. New biochemical studies have identified a bitter-responsive receptor that activates gustducin. Unexpected results with knockout mice suggest that gustducin may be directly involved in both bitter and sweet transduction. Electrophysiological experiments indicate that both inositol trisphosphate and cyclic nucleotides function in both bitter and sweet transduction events.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction

BackgroundTaste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C β2, and the G protein subunits α-gustducin, β3, and γ13. However, the identity of the IP3 receptor subtype in this pathway is not known. In the present study we used immunocytochemistry on rodent taste tissue to identify the IP3 receptors expressed in taste cells and to examine taste bud expression patterns for IP3R3.ResultsAntibodies against Type I, II, and III IP3 receptors were tested on sections of rat and mouse circumvallate papillae. Robust cytoplasmic labeling for the Type III IP3 receptor (IP3R3) was found in a large subset of taste cells in both species. In contrast, little or no immunoreactivity was seen with antibodies against the Type I or Type II IP3 receptors. To investigate the potential role of IP3R3 in bitter taste transduction, we used double-label immunocytochemistry to determine whether IP3R3 is expressed in the same subset of cells expressing other bitter signaling components. IP3R3 immunoreactive taste cells were also immunoreactive for PLCβ2 and γ13. Alpha-gustducin immunoreactivity was present in a subset of IP3R3, PLCβ2, and γ13 positive cells.ConclusionsIP3R3 is the dominant form of the IP3 receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway

Rat taste buds contain three morphologically distinct cell types that are candidates for taste transduction. The physiologic roles of these cells are, however, not clear. Inositol 1,4,5‐triphosphate (IP3) has been implicated as an important second messenger in bitter, sweet, and umami taste transductions. Previously, we identified the type III IP3 receptor (IP3R3) as the dominant isoform in taste receptor cells. In addition, a recent study showed that phospholipase Cβ2 (PLCβ2) is essential for the transduction of bitter, sweet, and umami stimuli. IP3R3 and PLCβ2 are expressed in the same subset of cells. To identify the taste cell types that express proteins involved in PLC signal transduction, we used 3,3′diaminobenzidine tetrahydrochloride immunoelectron microscopy and fluorescence microscopy to identify cells with IP3R3. Confocal microscopy was used to compare IP3R3 or PLCβ2 immunoreactivity with that of some known cell type markers such as serotonin, protein gene‐regulated product 9.5, and neural cell adhesion molecule. Here we show that a large subset of type II cells and a small subset of type III cells display IP3R3 immunoreactivity within their cytoplasm. These data suggest that type II cells are the principal transducers of bitter, sweet, and umami taste transduction. However, we did not observe synapses between type II taste cells and nerve fibers. Interestingly, we observed subsurface cisternae of smooth endoplasmic reticulum at the close appositions between the plasma membrane of type II taste cells and nerve processes. We speculate that some type II cells may communicate to the nervous system via subsurface cisternae of smooth endoplasmic reticulum in lieu of conventional synapses. J. Comp. Neurol. 468:311–321, 2004.

Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25.

BackgroundTaste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC) signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP) from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells.ResultsDepolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells from those expressing T1R3 or TRPM5. These data indicate that G protein-coupled taste receptors and conventional synaptic signaling mechanisms are expressed in separate populations of taste cells.ConclusionThe taste receptor cells responsible for the transduction of bitter, sweet, and umami stimuli are unlikely to communicate with nerve fibers by using conventional chemical synapses.

Proton currents through amiloride-sensitive Na+ channels in isolated hamster taste cells: Enhancement by vasopressin and cAMP

Amiloride has been suggested to inhibit responses to a variety of taste stimuli, including salty, sweet, and sour (acid). To test for the involvement of amiloride-sensitive Na+ channels in the transduction of acid stimuli, fungiform taste receptor cells were examined using patch-clamp techniques. Approximately one-half of all cells had amiloride-sensitive Na+ currents (INa) with a Ki value near 0.2 microM amiloride. After blocking voltage-gated conductances, cells having amiloride sensitivity were tested for responses to acid stimuli. Over three-fourths of cells showed an inward proton current (IH+) with an extrapolated reversal potential near approximately +150 mV, which was completely blocked by amiloride (30 microM). Treatment of isolated taste cells with arginine8-vasopressin caused equivalent increases in both INa and IH+; each effect was mimicked by 8-Br-cAMP. Taken together, these results indicate that protons permeate amiloride-sensitive Na+ channels in hamster fungiform taste cells and contribute to acid transduction.

Epithelial Na+ channel subunits in rat taste cells: Localization and regulation by aldosterone

Amiloride‐sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit‐specific antibodies directed against α, β, and γ subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for β and γ subunits. Dual localization experiments showed that immunoreactivity for the taste cell‐specific G protein, gustducin, occurs in a subset of rENaC positive taste cells.

Amiloride-sensitive channels in type I fungiform taste cells in mouse

BackgroundTaste buds are the sensory organs of taste perception. Three types of taste cells have been described. Type I cells have voltage-gated outward currents, but lack voltage-gated inward currents. These cells have been presumed to play only a support role in the taste bud. Type II cells have voltage-gated Na+ and K+ current, and the receptors and transduction machinery for bitter, sweet, and umami taste stimuli. Type III cells have voltage-gated Na+, K+, and Ca2+ currents, and make prominent synapses with afferent nerve fibers. Na+ salt transduction in part involves amiloride-sensitive epithelial sodium channels (ENaCs). In rodents, these channels are located in taste cells of fungiform papillae on the anterior part of the tongue innervated by the chorda tympani nerve. However, the taste cell type that expresses ENaCs is not known. This study used whole cell recordings of single fungiform taste cells of transgenic mice expressing GFP in Type II taste cells to identify the taste cells responding to amiloride. We also used immunocytochemistry to further define and compare cell types in fungiform and circumvallate taste buds of these mice.ResultsTaste cell types were identified by their response to depolarizing voltage steps and their presence or absence of GFP fluorescence. TRPM5-GFP taste cells expressed large voltage-gated Na+ and K+ currents, but lacked voltage-gated Ca2+ currents, as expected from previous studies. Approximately half of the unlabeled cells had similar membrane properties, suggesting they comprise a separate population of Type II cells. The other half expressed voltage-gated outward currents only, typical of Type I cells. A single taste cell had voltage-gated Ca2+ current characteristic of Type III cells. Responses to amiloride occurred only in cells that lacked voltage-gated inward currents. Immunocytochemistry showed that fungiform taste buds have significantly fewer Type II cells expressing PLC signalling components, and significantly fewer Type III cells than circumvallate taste buds.ConclusionThe principal finding is that amiloride-sensitive Na+ channels appear to be expressed in cells that lack voltage-gated inward currents, likely the Type I taste cells. These cells were previously assumed to provide only a support function in the taste bud.

Taste receptor signalling – from tongues to lungs

Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G‐protein‐coupled receptors (GPCRs) and second‐messenger signalling mechanisms. This review will focus on GPCR signalling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signalling effectors that include Gβγ activation of phospholipase Cβ2, 1,4,5‐inositol trisphosphate mediated release of Ca2+ from intracellular stores and Ca2+‐dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α‐gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signalling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signalling effectors as taste cells.