Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular Na(+) activity ([Na(+)](i)) in polarized rat fungiform taste receptor cells (TRCs) using fluorescence imaging and by chorda tympani (CT) taste nerve recordings. CT responses were monitored during lingual stimulation with ethanol solutions containing NaCl or KCl. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial Na(+) channel [ENaC]) or the vanilloid receptor-1 (VR-1) antagonists capsazepine or SB-366791, which also block the Bz-insensitive salt taste receptor, a VR-1 variant. CT responses were recorded at 23 degrees C or 42 degrees C (a temperature at which the VR-1 variant salt taste receptor activity is maximally enhanced). In the absence of permeable cations, ethanol induced a transient decrease in TRC volume, and stimulating the tongue with ethanol solutions without added salt elicited only transient phasic CT responses that were insensitive to elevated temperature or SB-366791. Preshrinking TRCs in vivo with hypertonic mannitol (0.5 M) attenuated the magnitude of the phasic CT response, indicating that in the absence of mineral salts, transient phasic CT responses are related to the ethanol-induced osmotic shrinkage of TRCs. In the presence of mineral salts, ethanol increased the Bz-insensitive apical cation flux in TRCs without a change in cell volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a transient phasic component and a sustained tonic component. Ethanol increased the Bz-insensitive NaCl CT response. This effect was further enhanced by elevating the temperature from 23 degrees C to 42 degrees C, and was blocked by SB-366791. We conclude that in the presence of mineral salts, ethanol modulates the Bz-insensitive VR-1 variant salt taste receptor.     

Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t)

Maillard reacted peptides (MRPs) were synthesized by conjugating a peptide fraction (1000-5000 Da) purified from soy protein hydrolyzate with galacturonic acid, glucosamine, xylose, fructose, or glucose. The effect of MRPs was investigated on human salt taste and on the chorda tympani (CT) taste nerve responses to NaCl in Sprague-Dawley rats, wild-type, and transient receptor potential vanilloid 1 (TRPV1) knockout mice. MRPs produced a biphasic effect on human salt taste perception and on the CT responses in rats and wild-type mice in the presence of NaCl + benzamil (Bz, a blocker of epithelial Na+ channels), enhancing the NaCl response at low concentrations and suppressing it at high concentrations. The effectiveness of MRPs as salt taste enhancers varied with the conjugated sugar moiety: galacturonic acid = glucosamine > xylose > fructose > glucose. The concentrations at which MRPs enhanced human salt taste were significantly lower than the concentrations of MRPs that produced increase in the NaCl CT response. Elevated temperature, resiniferatoxin, capsaicin, and ethanol produced additive effects on the NaCl CT responses in the presence of MRPs. Elevated temperature and ethanol also enhanced human salt taste perception. N-(3-methoxyphenyl)-4-chlorocinnamid (a blocker of TRPV1t) inhibited the Bz-insensitive NaCl CT responses in the absence and presence of MRPs. TRPV1 knockout mice demonstrated no Bz-insensitive NaCl CT response in the absence or presence of MRPs. The results suggest that MRPs modulate human salt taste and the NaCl + Bz CT responses by interacting with TRPV1t.     

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.     

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.     

Amiloride blocks salt taste transduction of the glossopharyngeal nerve in metamorphosed salamanders

Studies in the last two decades have shown that amiloride-sensitive Na(+) channels play a role in NaCl transduction in rat taste receptors. However, this role is not readily generalized for salt taste transduction in vertebrates, because functional expression of these channels varies across species and also in development in a species. Glossopharyngeal nerve responses to sodium and potassium salts were recorded in larval and metamorphosed salamanders and compared before and after the oral floor was exposed to amiloride, a blocker of Na(+) channels known to be responsible for epithelial ion transport. Pre-exposure to amiloride (100 microM) did not affect salt taste responses in both axolotls (Ambystoma mexicanum) and larval Ezo salamanders (Hynobius retardatus). In contrast, in metamorphosed Ezo salamanders the nerve responses to NaCl were significantly reduced by amiloride. In amphibians amiloride-sensitive components in salt taste transduction seem to develop during metamorphosis.     

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.     

Intracellular pH modulates taste receptor cell volume and the phasic part of the chorda tympani response to acids

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 microM cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid-sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid-sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.     

Effects of osmolarity on taste receptor cell size and function

Osmotic effects onsalt taste were studied by recording from the rat chorda tympani (CT)nerve and by measuring changes in cell volume of isolated rat fungiformtaste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO didnot induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions alsocontained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO.The enhanced CT responses to NaCl were reversed when the saccharideswere removed and were completely blocked by addition of 100 µMamiloride to the stimulus solution. Exposure of TRCs to hyperosmoticsolutions of mannitol or cellobiose induced a rapid and sustaineddecrease in cell volume that was completely reversible, whereasexposure to hypertonic urea or DMSO did not induce sustained reductionsin cell volume. These data suggest that the osmolyte-induced increasein the CT response to NaCl involves a sustained decrease in TRC volumeand the activation of amiloride-sensitive apicalNa⁺ channels.

     

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.     

Effect of chorda tympani nerve transection on salt taste perception in mice

Effects of gustatory nerve transection on salt taste have been studied extensively in rats and hamsters but have not been well explored in the mouse. We examined the effects of chorda tympani (CT) nerve transection on NaCl taste preferences and thresholds in outbred CD-1 mice using a high-throughput phenotyping method developed in our laboratory. To measure taste thresholds, mice were conditioned by oral self-administration of LiCl or NaCl and then presented with NaCl concentration series in 2-bottle preference tests. LiCl-conditioned and control NaCl-exposed mice were given bilateral transections of the CT nerve (LiCl-CTX, NaCl-CTX) or were left intact as controls (LiCl-CNT, NaCl-CNT). After recovery from surgery, mice received a concentration series of NaCl (0-300 mM) in 48-h 2-bottle tests. CT transection increased NaCl taste thresholds in LiCl-conditioned mice and eliminated avoidance of concentrated NaCl in control NaCl-exposed mice. This demonstrates that in mice, the CT nerve is important for detection and recognition of NaCl taste and is necessary for the normal avoidance of high concentrations of NaCl. The results of this experiment also show that the method of high-throughput phenotyping of salt taste thresholds is suitable for detecting changes in the taste periphery in mouse genetic studies.     

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.     

Non-Competitive but pH-Dependent Action of SB-366791 on Proton-Induced Activation of TRPV1 Receptors

The transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel gated by numerous chemical and physical stimuli (protons, capsaicin, heat, etc). TRPV1 receptors are important integrators of multiple noxious and inflammatory signals in vertebrates. Modulation of TRPV1 receptors activity is considered to be a promising strategy for pain treatment. SB-366791 is a TRPV1 antagonist that demonstrates good analgesic effects in various models of pain. Molecular mechanisms of the SB-366791 action on TRPV1 are not clear. It antagonizes capsaicin activation in a competitive manner, but the data on its action in the case of activation by protons are controversial. Here we studied effects of SB-366791 when TRPV1 receptors are activated by acidification. We carried out patch-clamp experiments (voltage-clamp mode) on cultured CHO cells stably expressing rat TRPV1 receptors. The whole-cell proton-evoked currents were reduced in the presence of SB-366791. Concentration dependencies of the inhibitory effect of SB-366791 were studied at different pH values. Stronger acidification reduced the maximum effect of SB-366791, while the IC₅₀ values were virtually unaffected. Thus, SB-366791 acts in a non-competitive but pH-dependent way. Probably, there is an allosteric interplay between proton- and capsaicin-binding sites.     

Effects of amiloride on salt taste in humans

Amiloride reduces several responses of the taste system to NaCl.These effects are crucial support for the ion transport theoryof salt taste. Here, three experiments tested for the inhibitoryeffect of amiloride on salt taste in normal, whole-mouth tastingin humans. There was no evidence that amiloride reduces thesaltiness of NaCl when used as a pretreatment, mixed into solutionwith NaCl, or both. In a fourth experiment, pretreating thetongue with amiloride-soaked filter paper and presenting NaClon filter paper within the treated area also had no effect onsaltiness. In addition, amiloride, a bitter compound, did notsuppress the taste of sucrose, and caffeine did not enhancethe taste of NaCl. These results suggest the ion transport theoryof NaCl taste needs to be modified or extended to account fornormal whole-mouth tasting of NaCl in humans. ¹ Present address: 1605 Harbor Court Tower, 10 East Lee St.,Baltimore, MD 21202, USA     

Behavioral and electrophysiological taste responses change after brief or prolonged dietary sodium deprivation

Dietary Na+ deprivation elicits a hormonal response to promote sodium conservation and a behavioral response to increase sodium ingestion. It has generally been accepted that the former occurs within 24 h after sodium deprivation, while the latter is delayed and may not appear until as much as 10 days later. Na+ deprivation of similar duration also decreases the sensitivity of the chorda tympani nerve (CT) to NaCl, suggesting that changes in CT responses are necessary for increased NaCl intake. However, previous work from our laboratory showed that licking responses to NaCl solutions increase after only 2 days of Na+ deprivation, suggesting rapidly occurring changes in response to NaCl taste. The present experiments examined the effects of 2 days of dietary Na+ deprivation on CT responses to NaCl and patterns of NaCl consumption and found that Na+-deficient rats licked significantly more during the first NaCl intake bout compared with control rats. CT responses to NaCl were reduced at all concentrations after brief Na+ deprivation compared with Na+-replete control rats and did not decrease further with prolonged (10 days) dietary Na+ deficiency. Moreover, amiloride, which suppressed CT responses to NaCl by approximately 30% in control rats, had virtually no effect on CT responses in Na+-deprived rats. Thus, 2 days of Na+ deprivation is sufficient to alter patterns of ingestion of concentrated NaCl and to reduce gustatory responses to NaCl. Furthermore, changes in gustatory responses to NaCl during dietary Na+ deprivation may involve the amiloride-sensitive component of the CT.     

Salt taste and salt sensitivity in black adolescents

Limited support for the postulated association between tasteand blood pressure responses to NaCl has prompted investigationsof selected subgroups of subjects more likely to display a relationship.The present study included 20 black adolescents who were diagnosedas salt sensitive (n = 10) or salt insensitive (n = 10) basedupon blood pressure responses to 14 days of dietary salt loading(10 g NaCl/day). Each was administered a battery of taste tests,including measures of detection and recognition threshold, suprathresholdintensity responses and preference tests. No significant groupdifference was observed on any taste measure. While explanationsfor the failure to note differences between these individualswith marked variabilty in systemic reactivity to NaCl are offered,the present findings question the continued study of this issue.     

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine,Ethanol and Acetylcholine

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca²⁺]_i by topical lingual application of ionomycin + CaCl₂. In contrast, CT responses to nicotine were enhanced when TRC [Ca²⁺]_i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.     

Contribution of Drosophila DEG/ENaC genes to salt taste

The ability to detect salt is critical for the survival of terrestrial animals. Based on amiloride-dependent inhibition, the receptors that detect salt have been postulated to be DEG/ENaC channels. We found the Drosophila DEG/ENaC genes Pickpocket11 (ppk11) and Pickpocket19 (ppk19) expressed in the larval taste-sensing terminal organ and in adults on the taste bristles of the labelum, the legs, and the wing margins. When we disrupted PPK11 or PPK19 function, larvae lost their ability to discriminate low concentrations of Na(+) or K(+) from water, and the electrophysiologic responses to low salt concentrations were attenuated. In both larvae and adults, disrupting PPK11 or PPK19 affected the behavioral response to high salt concentrations. In contrast, the response of larvae to sucrose, pH 3, and several odors remained intact. These results indicate that the DEG/ENaC channels PPK11 and PPK19 play a key role in detecting Na(+) and K(+) salts.