Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.     

Amiloride-sensitive NaCl taste responses are associated with genetic variation of ENaC alpha-subunit in mice

An epithelial Na(+) channel (ENaC) is expressed in taste cells and may be involved in the salt taste transduction. ENaC activity is blocked by amiloride, which in several mammalian species also inhibits taste responses to NaCl. In mice, lingual application of amiloride inhibits NaCl responses in the chorda tympani (CT) gustatory nerve much stronger in the C57BL/6 (B6) strain than in the 129P3/J (129) strain. We examined whether this strain difference is related to gene sequence variation or mRNA expression of three ENaC subunits (alpha, beta, gamma). Real-time RT-PCR and in situ hybridization detected no significant strain differences in expression of all three ENaC subunits in fungiform papillae. Sequences of the beta- and gammaENaC subunit genes were also similar in the B6 and 129 strains, but alphaENaC gene had three single nucleotide polymorphisms (SNPs). One of these SNPs resulted in a substitution of arginine in the B6 strain to tryptophan in the 129 strain (R616W) in the alphaENaC protein. To examine association of this SNP with amiloride sensitivity of CT responses to NaCl, we produced F(2) hybrids between B6 and 129 strains. Amiloride inhibited CT responses to NaCl in F(2) hybrids with B6/129 and B6/B6 alphaENaC R616W genotypes stronger than in F(2) hybrids with 129/129 genotype. This suggests that the R616W variation in the alphaENaC subunit affects amiloride sensitivity of the ENaC channel and provides evidence that ENaC is involved in amiloride-sensitive salt taste responses in mice.     

Effects of amiloride on gustatory neural responses to salts in the frog.

In frogs, the glossopharyngeal nerve (GL) innervates taste receptors on almost the entire tongue. The mandibular branch (MBF) and palatine branch (PN) of the facial nerve innervate taste receptors on a very small area at the base of the tongue and on the palate, respectively. In the present study, effects of amiloride, an epithelial sodium channel blocker, on the tonic responses of the GL, MBF and PN in frogs to NaCl, LiCl, KCl and CaCl(2) were investigated. In three nerves, amiloride at 0.5 mM, a relatively high concentration, did not affect the responses to 0.15 (concentration just above threshold)-0.5 M NaCl, 0.5 M LiCl and 0.3 M KCl, whereas it almost completely inhibited the response to 1.0 mM CaCl(2). Amiloride may exert an inhibitory action on the response to CaCl(2) by a competitive antagonism between Ca(2+) and a monovalent cation of amiloride, because the response to Ca(2+) is competitively inhibited by other cations such as Na(+) and Mg(2+). The lack of inhibitory effect of amiloride on the responses in the GL, MBF and PN to NaCl suggests that amiloride-sensitive sodium channels in the apical membrane of taste receptor cells are not involved in sodium taste transduction in frogs.     

Co-localization of epithelial sodium channels and glutamate receptors in single taste cells

Umami taste is elicited by monosodium glutamate (MSG), a compound consisting of two potent taste stimuli, Na(+) and glutamate. In rat fungiform taste cells, amiloride-sensitive epithelial sodium channels (ENaCs) mediate Na(+) transduction, while glutamate is transduced by a combination of ionotropic and metabotropic glutamate receptors. We used giga-seal whole-cell recording to determine if responses to glutamate and Na(+) occur in the same taste cells. Approximately 68% of the cells tested responded to amiloride, indicating that they express functional ENaCs. Responses to glutamate occurred in about 58% of the cells tested. Interestingly, responses to glutamate occurred in the subset of cells that also responded to amiloride, indicating that glutamate receptors are located preferentially in the same taste cells that also express ENaCs. Further experiments showed that amiloride did not suppress responses to glutamate under voltage-clamp conditions. Taken together, the data suggest that although ENaCs are not involved directly in glutamate transduction, their co-localization with glutamate receptors provides a substrate for the cellular integration of these independent pathways. Copyright Copyright 1999 S. Karger AG, Basel     

Amiloride-sensitive and amiloride-insensitive responses to NaCl + acid mixtures in hamster chorda tympani nerve

Component signaling in taste mixtures containing both beneficial and dangerous chemicals depends on peripheral processing. Unidirectional mixture suppression of chorda tympani (CT) nerve responses to sucrose by quinine and acid is documented for golden hamsters (Mesocricetus auratus). To investigate mixtures of NaCl and acids, we recorded multifiber responses to 50 mM NaCl, 1 and 3 mM citric acid and acetic acid, 250 μM citric acid, 20 mM acetic acid, and all binary combinations of each acid with NaCl (with and without 30 μM amiloride added). By blocking epithelial Na(+) channels, amiloride treatment separated amiloride-sensitive NaCl-specific responses from amiloride-insensitive electrolyte-generalist responses, which encompass all of the CT response to the acids as well as responses to NaCl. Like CT sucrose responses, the amiloride-sensitive NaCl responses were suppressed by as much as 50% by citric acid (P = 0.001). The amiloride-insensitive electrolyte-generalist responses to NaCl + acid mixtures approximated the sum of NaCl and acid component responses. Thus, although NaCl-specific responses to NaCl were weakened in NaCl-acid mixtures, electrolyte-generalist responses to acid and NaCl, which tastes KCl-like, were transmitted undiminished in intensity to the central nervous system. The 2 distinct CT pathways are consistent with known rodent behavioral discriminations.     

Strain differences in amiloride inhibition of NaCl responses in mice,Mus musculus

Summary The effects of lingual treatment with amiloride, an inhibitor of salt taste responses in several mammalian species, on NaCl responses of the chorda tympani nerve were compared between four inbred strains of mouse (BALB/cCrSlc, DBA/2CrSlc, C57BL/6CrSlc and C3H/HeSlc). In C57BL and C3H mice amiloride significantly suppressed responses of the chorda tympani nerve to NaCl at a concentration 0.1 M or more whereas in BALB and DBA mice the drug did not significantly affect the responses to NaCl at any concentration, suggesting a lack of the amiloride-sensitive receptor component for NaCl in the latter two strains.A two-bottle preference test demonstrated that all strains of mouse usually showed no preference for NaCl at any concentration and avoided NaCl at 0.3 M or more, although some differences were observed in that C57BL and C3H mice showed aversive responses to 0.1 and 0.15 M NaCl, whereas BALB and DBA mice were indifferent to these solutions.The results suggest that there exist prominent differences between mouse strains in the amiloride-sensitive component of their salt receptor systems. However, in mice the taste information derived from the amiloride-sensitive receptor component probably has no remarkable effect on behavioral responses to NaCl except for a possible contribution to decreasing aversion thresholds for NaCl by increasing overall taste information about NaCl.     

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.     

A psychophysical and electrophysiological analysis of salt taste in Trpv1 null mice

Current evidence suggests salt taste transduction involves at least two mechanisms, one that is amiloride sensitive and appears to use apically located epithelial sodium channels relatively selective for Na(+) and a second that is amiloride insensitive and uses a variant of the transient receptor potential vanilloid receptor 1 (TRPV1) that serves as a nonspecific cation channel. To provide a functional context for these findings, we trained Trpv1 knockout (KO) and wild-type (WT) C57BL/6J mice (n = 9 or 10/group) in a two-response operant discrimination procedure and measured detection thresholds to NaCl and KCl with and without amiloride. The KO and WT mice had similar detection thresholds for NaCl and KCl. Amiloride shifted the NaCl sensitivity curve to the same degree in both groups and had virtually no effect on KCl thresholds. In addition, a more detailed analysis of chorda tympani nerve (CT) responses to NaCl, with and without benzamil (Bz, an amiloride analog) treatment revealed that the tonic portion of the CT response of KO mice to NaCl + Bz was absent, but both KO and WT mice displayed some degree of a phasic response to NaCl with and without Bz. Because these transients constitute the entire CT response to NaCl + Bz in Trpv1 KO mice, it is possible that these signals are sufficient to maintain normal NaCl detectabilty in the behavioral task used here. Additionally, there may be other amiloride-insensitive salt transduction mechanisms in taste receptor fields other than the anterior tongue that maintain normal salt detection performance in the KO mice.     

Proton currents through amiloride-sensitive Na channels in hamster taste cells. Role in acid transduction.

The activity of taste cells maintained in the intact hamster tongue was monitored in response to acid stimulation by recording action currents from taste receptor cells with an extracellular "macro" patch pipette: a glass pipette was pressed over the taste pore of fungiform papillae and perfused with citric acid, hydrochloric acid, or NaCl. Because this technique restricted stimulus application to the small surface area of the apical membranes of the taste cells, many nonspecific, and potentially detrimental, effects of acid stimulation could be avoided. Acid stimulation reliably elicited fast transient currents (action currents of average amplitude, 9 pA) which were consistently smaller than those elicited by NaCl (29 pA). The frequency of action currents elicited by acid stimuli increased in a dose-dependent manner with decreasing pH from a threshold of about pH 5.0. Acid-elicited responses were independent of K+, Na+, Cl-, or Ca2+ at physiological (salivary) concentrations, and were unaffected by anthracene-9-carboxylic acid, tetraethylammonium bromide, diisothiocyanate-stilbene-2,2'-disulfonic acid, vanadate, or Cd2+. In contrast, amiloride (< or = 30 microM) fully and reversibly suppressed acid-evoked action currents. At submaximal amiloride concentrations, the frequency and amplitude of the action currents were reduced, indicating a reduction of the taste cell apical conductance concomitant with a decrease in cell excitation. Exposure to low pH elicited, in addition to transient currents, an amiloride-sensitive sustained d.c. current. This current is apparently carried by protons instead of Na+ through amiloride-sensitive channels. When citric acid was applied while the taste bud was stimulated by NaCl, the action currents became smaller and the response resembled that produced by acid alone. Because of the strong interdependence of the acid and salt (NaCl) responses when both stimuli are applied simultaneously, and because of the similarity in the concentration dependence of amiloride block, we conclude that amiloride-sensitive Na+ channels on hamster taste receptor cells are permeable to protons and may play a role in acid (sour) taste.     

Amiloride-sensitive signals and NaCl preference and appetite: a lick-rate analysis

Rats prefer hypotonic and isotonic NaCl solutions to water in long-access drinking paradigms. To focus on the role of taste signals in NaCl preference, licking patterns of rats with 30-s exposure to NaCl solutions (0-0.5 M) were examined when they were either water deprived, sodium depleted, or not deprived (NaCl mixed in dilute sucrose). In all three conditions, rats displayed a preference for NaCl. The addition of 100 microM amiloride, a sodium channel blocker, to NaCl did not change rats' licking when they were sodium replete but dramatically reduced licking when they were deplete. Transection of the chorda tympani (CT) nerve, an afferent pathway for amiloride-sensitive Na(+) signals, had no effect on NaCl preference in nondeprived rats and only a modest effect on those that were Na(+) deplete. Amiloride was found to exert significant suppression of NaCl intake in Na(+)-depleted rats with transection of the CT, supporting the existence of other afferent pathways for transmission of amiloride-sensitive Na(+) signalling. Together, these studies argue for the involvement of different neural signalling mechanisms in NaCl preference in the presence and absence of explicit Na(+) need.     

Taste discrimination between NaCl and KCl is disrupted by amiloride in inbred mice with amiloride-insensitive chorda tympani nerves

The amiloride-sensitive salt transduction pathway is thought to be critical for the discrimination between sodium and nonsodium salts in rodents. In rats, lingual application of amiloride appears to render NaCl qualitatively indistinguishable from KCl. In this study, we tested four strains of mice for salt discriminability. In one strain (C57BL/6J), chorda tympani nerve (CT) responses to NaCl are attenuated by amiloride, and in the other three strains (BALB/cByJ, 129P3/J, DBA/2J) they are not. Under water-restriction conditions, these mice (7 mice/strain) were trained in a gustometer to lick for water from one reinforcement spout in response to a five-lick presentation of NaCl and to lick from another in response to KCl [salt concentration was varied (0.1-1 M) to render intensity irrelevant]. Mice were then tested with the stimuli dissolved in amiloride hydrochloride, and the latter was used as the reinforcer as well. Each concentration of amiloride (0.1-100 microM) was used on 2 separate days with control sessions interposed. Mice from all four strains were able to discriminate NaCl from KCl reliably. Amiloride impaired this discrimination in a dose-dependent fashion. Moreover, performance on NaCl trials appeared to be more affected by amiloride than that on KCl trials in all four strains. Thus, in contrast to the predictions based on CT recordings, discrimination in all four strains appeared to depend on the amiloride-sensitive transduction pathway, which, in the case of BALB/cByJ, 129P3/J, and DBA/2J (and perhaps C57BL/6 as well), may exist in taste buds innervated by nerves other than the CT.     

Transecting the gustatory branches of the facial nerve impairs NH(4)Cl vs. KCl discrimination in rats

Ammonium and potassium chloride share a common taste quality and an amiloride-insensitive route of transduction. An amiloride-sensitive pathway might also be partially activated by these salts, although very few studies have reported effects of amiloride on nonsodium salt perception. This experiment was designed to determine 1) whether rats could discriminate KCl from NH(4)Cl and, if discrimination was evident, whether performance was impaired with 2) amiloride or 3) gustatory nerve transection. Rats were trained to discriminate KCl from NH(4)Cl (n = 8) and NaCl from NH(4)Cl (n = 8). Amiloride (100 microM) impaired NaCl vs. NH(4)Cl but not KCl vs. NH(4)Cl performance, whereas both groups showed significant impairments after transection of the chorda tympani (CT) and greater superficial petrosal (GSP) branches of the facial nerve. This suggests that rats can discriminate between KCl and NH(4)Cl and that this discrimination does not rely on an amiloride-sensitive mechanism but does depend on the CT and/or GSP nerves. This experiment supports the hypothesis that the facial nerve is important for salt taste recognition and discrimination.     

Detection of NaCl and KCl in TRPV1 knockout mice

Both amiloride-sensitive and -insensitive mechanisms contribute to NaCl taste transduction. The amiloride-sensitive mechanism relies on the epithelial Na(+) channel ENaC, which is widely expressed on the apical membrane of fungiform taste cells. The amiloride-insensitive mechanism, which predominates in circumvallate and foliate taste buds, was recently reported to involve a variant of the nonselective cation channel TRPV1. We performed 2-bottle preference and threshold experiments with TRPV1 knockout mice and wild-type (C57BL/6J) controls to test for NaCl preference and detection thresholds in the presence and absence of amiloride. Surprisingly, TRPV1 knockout mice not only detected NaCl in the presence of amiloride but they preferred NaCl over water at concentrations avoided by the wild-type mice. NaCl detection thresholds were between 2 and 3 mM for both genotypes. Amiloride increased the detection thresholds of wild-type mice but not knockout mice. The knockout mice also preferred 100 mM KCl compared with wild-type controls, suggesting that TRPV1 receptors may mediate a general aversive response to salts. Analyses of consumption data also revealed that TRPV1 knockout mice ingested more of the NaCl, with and without amiloride, and KCl solutions than the wild-type mice. However, comparisons of preference ratios and consumption volumes indicated that both wild-type and TRPV1 knockout mice avoided citric acid in quite a similar manner, suggesting that TRPV1 receptors do not mediate the detection of citric acid. These data, taken together, suggest that additional mechanisms must contribute to the amiloride-insensitive NaCl response.     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.     

The active ion transport properties of canine lingual epithelia in vitro. Implications for gustatory transduction

The electrophysiological properties of the dorsal and ventral canine lingual epithelium are studied in vitro. The dorsal epithelium contains a special ion transport system activated by mucosal solutions hyperosmotic in NaCl or LiCl. Hyperosmotic KCl is significantly less effective as an activator of this system. The lingual frenulum does not contain the transport system. In the dorsal surface it is characterized by a rapid increase in inward current and can be quantitated as a second component in the time course of either the open-circuit potential or short-circuit current when the mucosal solution is hyperosmotic in NaCl or LiCl. The increased inward current (hyperosmotic response) can be eliminated by amiloride (10(-4) M). The specific location of this transport system in the dorsal surface and the fact that it operates over the concentration range characteristic of mammalian salt taste suggests a possible link to gustatory transduction. This possibility is tested by recording neural responses in the rat to NaCl and KCl over a concentration range including the hyperosmotic. We demonstrate that amiloride specifically blocks the response to NaCl over the hyperosmotic range while affecting the KCl response significantly less. The results suggest that gustatory transduction for NaCl is mediated by Na entry into the taste cells via the same amiloride-sensitive pathway responsible for the hyperosmotic response in vitro. Further studies of the in vitro system give evidence for paracellular as well as transcellular current paths. The transmural current-voltage relations are linear under both symmetrical and asymmetrical conditions. After ouabain treatment under symmetrical conditions, the short-circuit current decays to zero. The increase in resistance, though significant, is small, which suggests a sizeable shunt pathway for current. Flux measurements show that sodium is absorbed under symmetrical conditions. Mucosal solutions hyperosmotic in various sugars also induce an amiloride-sensitive inward current. In summary, this work provides evidence that the sodium taste receptor is most probably a sodium transport system, specifically adapted to the dorsal surface of the tongue. The transport paradigm of gustation also suggests a simple model for electric taste and possible mechanisms for sweet taste.     

The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Amiloride is known to inhibit the taste response of vertebrates to salt by blocking the amiloride-sensitive sodium channel. In this study, we investigated electrophysiologically the effect of amiloride on the taste response of the fleshfly Boettcherisca peregrina. When 0.5 mM amiloride was included in taste solutions, the response of the salt receptor cell (salt response) to sodium chloride (NaCl) was not depressed but those of the sugar receptor cell (sugar responses) to sucrose, glucose, fructose, l-valine (l-Val) and l-phenylalanine (l-Phe) were strongly depressed. An inhibitory effect of amiloride on the concentration-response relationship for both sucrose and l-Phe was clearly revealed, but not at high concentrations of sucrose. After pretreatment of a chemosensory seta with 0.15 mM amiloride for 10 min, the salt response to NaCl was not affected. On the other hand, the sugar responses to sucrose, fructose, l-Val and l-Phe were depressed just after amiloride pretreatment. The sugar response to adenosine 5’-diphosphate (ADP) mixed with 0.5 mM amiloride was not depressed, but the response to ADP alone was depressed after amiloride pretreatment. It was therefore observed that amiloride depressed the responses to all stimulants that react with each of the receptor sites of the sugar receptor cell.     

Taste Disks are Induced in the Lingual Epithelium of Salamanders during Metamorphosis

Morphological changes of oral cavity during metamorphosis withspecial reference to the taste organ were examined in Ezo salamanders(Hynobius retardatus) and axolotls (Ambystoma mexicanum), andcompared with those in bullfrogs (Rana catesbeiana). The non-distensibletongue of salamanders changed the structure progressively duringmetamorphosis: a small area of the rostrum protruded and developedcaudally with recession of the flat area of the tongue. Theprotrusion that developed on the tongue had numerous papillae,as seen in the frog tongue. The apical region of the papillaeoccasionally had a cell mass similar to the taste disk of frogs(termed a taste disk-like cell mass). On the flat area of thetongue, the barrel-shaped taste buds of larval salamanders weretransformed into taste buds with a wider receptor area. Thebarrel-shaped taste buds decreased progressively during metamorphosis,while taste disk-like cell masses increased. Neuronal labelingwith an antibody to neuron-specific enolase and fluorescentcarbocyanine dye showed that the taste disk-like cell massesin metamorphosed salamanders were innervated by the glossopharyngealnerve (nerve IX). Nerve IX responded to taste stimulation aswell as mechanical stimulation applied to the rostral tongue.During metamorphosis the salamanders undergo transformationand rearrangement of taste organs on the tongue possibly asan adaptation to the terrestrial environment. Chem. Senses 22:535–545, 1997.     

Oral amiloride treatment decreases taste sensitivity to sodium salts in C57BL/6J and DBA/2J mice

Sodium taste transduction is thought to occur via an amiloride-sensitive, sodium-selective pathway and an amiloride-insensitive, cation nonselective, anion-dependent pathway(s). It has been shown by others that amiloride, an epithelial sodium channel (ENaC) blocker, significantly reduces the chorda tympani nerve response to lingually applied NaCl in C57BL/6 (B6) mice but not in DBA/2 (D2) mice, suggesting that the latter strain might not possess functional ENaCs in taste receptor cells. We psychophysically measured and compared taste detection thresholds of NaCl and sodium gluconate (NaGlu) prepared with and without 100 microM amiloride in these two strains (eight/strain). Mice were trained and tested in a two-response operant signal detection procedure conducted in a gustometer. Surprisingly, no strain effect was found for the detection thresholds of both salts (approximately 0.05-0.06 M). Moreover, these thresholds were increased by almost an order of magnitude by amiloride adulteration of the solutions. This marked effect of amiloride on sodium detection thresholds suggests that ENaCs are necessary for normal sensitivity to sodium salts in both strains. In addition, because NaGlu is thought to stimulate primarily the amiloride-sensitive pathway, especially at low concentrations, the similarity of NaCl and NaGlu thresholds (r > 0.81 both strains) suggests that ENaCs are also sufficient to support the detection of sodium in weak solutions by B6 and D2 mice.     

Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction

There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar-activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na-preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.