Regulation of the Benzamil-Insensitive Salt Taste Receptor by Intracellular Ca²⁺, Protein Kinase C, and Calcineurin

Abstract

The regulation of the benzamil (Bz)-insensitive salt taste receptor was investigated by intracellular Ca²⁺ ([Ca²⁺]_i), protein kinase C (PKC), and the Ca²⁺-dependent serine-threonine phosphatase, calcineurin (PP2B), by monitoring chorda tympani taste nerve responses to 0.1 M NaCl solutions containing Bz (5 × 10⁻⁶ M) and resiniferatoxin (RTX; 0–10 × 10⁻⁶ M) in Sprague–Dawley rats and in wild-type (WT) and transient receptor potential vanilloid-1 knockout (TRPV1 KO) mice. In rats and WT mice, RTX increased the NaCl + Bz chorda tympani responses between 0.25 × 10⁻⁶ and 1 × 10⁻⁶ M and inhibited the responses above 1 × 10⁻⁶ M. Decreasing taste receptor cell (TRC) [Ca²⁺]_i with BAPTA loading, activation of PKC with 4α-phorbol-12,13-didecanoate (PMA), or inhibition of PP2B by cyclosporin A or FK-506, enhanced the magnitude of the Bz-insensitive NaCl chorda tympani responses in the presence of RTX and either minimized or completely eliminated the decrease in the chorda tympani response >1 × 10⁻⁶ M RTX. In contrast, increasing TRC [Ca²⁺]_i with ionomycin inhibited Bz-insensitive NaCl chorda tympani responses in the presence of RTX. No effect of the cited modulators was observed on the chorda tympani responses in WT mice and rats in the presence of TRPV1 blocker SB-366791 (1 × 10⁻⁶ M) or in TRPV1 KO mice. ³²P-labeling demonstrated direct phosphorylation of TRPV1 or TRPV1t in anterior lingual epithelium by PMA, cyclosporin A, or FK-506. PMA also enhanced the RTX-sensitive unilateral apical Na⁺ flux in polarized fungiform TRC in vitro. We conclude that TRPV1 or its variant TRPV1t is phosphorylated and dephosphorylated by PKC and PP2B, respectively, and either sensitizes or desensitizes the Bz-insensitive NaCl chorda tympani responses to RTX stimulation.

INTRODUCTION

In rodents the chorda tympani taste nerve responses to lingual stimulation with NaCl are composed of two components. The amiloride- and benzamil (Bz)-sensitive component constitutes 60–70% of the total NaCl chorda tympani response and is derived from Na⁺ entry into a subset of TRCs that express the epithelial Na⁺ channel (ENaC) in their apical cell membrane. The amiloride- and Bz-insensitive component constitutes about 30–40% of the NaCl chorda tympani response. It is derived from Na⁺ entry into a subset of TRCs via a nonspecific cation channel that is a variant of the pain receptor, transient receptor potential vanilloid-1 (TRPV1), designated as TRPV1t (DeSimone and Lyall 2006, 2008; Lyall et al. 2004). Na⁺ entry depolarizes a subset of TRCs within the taste buds and decreases receptor potential, resulting in the release of the taste-specific neurotransmitter adenosine 5′-triphosphate (ATP). ATP binds to the P₂X₂/P₂X₃ receptors on the taste nerve fibers that synapse with TRCs within the taste buds (Finger et al. 2005) and increase taste nerve activity.

In rodents, the magnitude of the Bz-insensitive NaCl chorda tympani response is modulated by vanilloids (resiniferatoxin [RTX] and capsaicin), ethanol, nicotine, temperature, external pH, cetylpyridinium chloride (Lyall et al. 2004, 2005a,b, 2007) and by naturally occurring Maillard peptides and Maillard reacted peptides conjugated with different sugar moieties (Katsumata et al. 2008). The Bz-insensitive NaCl chorda tympani response is inhibited by capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamide (SB-366791) (Katsumata et al. 2008; Lyall et al. 2004). Unlike rats and wild-type (WT) mice, in TRPV1 knockout (KO) mice no spontaneous Bz-insensitive NaCl chorda tympani responses are observed above the rinse baseline level for the entire NaCl concentration range (0.1–1.0 M) (Treesukosol et al. 2007). In addition, the above-cited agonists do not induce an increase in the Bz-insensitive NaCl chorda tympani response above the rinse baseline level in TRPV1 KO mice (Katsumata et al. 2008; Lyall et al. 2004, 2005a,b, 2007). Agonists that modulate the Bz-insensitive NaCl chorda tympani responses in rats and WT mice also modulate human salt taste (Katsumata et al. 2008), suggesting that TRPV1t contributes significantly to human salt taste perception. All of the TRPV1t modulators tested to date produce enhancement of the Bz-insensitive NaCl chorda tympani responses at low concentrations but inhibit the responses at higher concentrations. The biphasic chorda tympani response profiles for the cited modulators are observed over a wide range of agonist concentrations. At present the intracellular effectors that regulate the Bz-insensitive NaCl chorda tympani responses, and thus TRPV1t, have not been identified. We hypothesize that TRPV1t agonists mediate the sensitization and desensitization of the Bz-insensitive NaCl chorda tympani responses by altering one or more intracellular effectors in TRCs that alter the phosphorylation state of TRPV1t. Since TRPV1t shares many similarities with TRPV1, we hypothesize that common intracellular effectors regulate the activity of both ion channels.

Herein we investigated the regulation of native TRPV1t in TRCs by changes in intracellular Ca²⁺ ([Ca²⁺]_i) and by Ca²⁺-triggered intracellular signaling cascades involving phosphorylation/dephosphorylation of TRPV1t by Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) or calcineurin, a Ca²⁺-dependent serine-threonine phosphatase (PP2B). In addition, we tested the effect of TRPV1t phosphorylation mediated by protein kinase C (PKC) or cyclic 3′,5′ adenosine monophosphate (cAMP)–activated protein kinase (PKA) on the Bz-insensitive NaCl chorda tympani response. The Bz-insensitive NaCl chorda tympani responses were monitored in anesthetized Spraque–Dawley rats and in WT and TRPV1 KO mice in vivo in the absence and presence of RTX, a potent TRPV1t modulator. The chorda tympani recordings were made under the conditions in which the activity of one or more of the above-cited intracellular effectors was either enhanced or diminished in TRCs in vivo by the topical lingual application of agonists or antagonists of these effectors. The results summarized herein demonstrate that both the constitutive activity and the RTX-induced sensitization and desensitization of native TRPV1t in TRCs are modulated by [Ca²⁺]_i, PKC, and PP2B.

METHODS

Chorda tympani nerve recordings

The activity of native TRPV1t in TRCs was evaluated by generating dose–response relationships between varying RTX concentrations and the magnitude of the Bz-insensitive NaCl chorda tympani responses under various experimental conditions. Animals were housed in the Virginia Commonwealth University animal facility in accordance with institutional guidelines. All animal protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Female Sprague–Dawley rats (150–200 g) were anesthetized by intraperitoneal (ip) injection of pentobarbital (60 mg/kg) and supplemental pentobarbital (20 mg/kg) was administered as necessary to maintain surgical anesthesia. The animal's corneal reflex and toe-pinch reflex were used to monitor the depth of surgical anesthesia. Body temperatures were maintained at 37°C with a Deltaphase Isothermal PAD (Model 39 DP: Braintree Scientific, Braintree, MA). The left chorda tympani nerve was exposed laterally as it exited the tympanic bulla and placed onto a 32G platinum/iridium wire electrode. Stimulus solutions maintained at room temperature were injected into a Lucite chamber (3 ml; 1 ml/s) affixed by vacuum to a 28-mm² patch of anterior dorsal lingual surface. The chorda tympani responses were recorded under zero lingual current-clamp and analyzed as described previously (Katsumata et al. 2008).

Chorda tympani responses were also monitored in wild-type (C57BL/6J) and homozygous TRPV1 KO mice (B6.129S4-Trpv1^tmijul; The Jackson Laboratory, Bar Harbor, ME). Mice (30–40 g) were anesthetized by ip injection of pentobarbital (30 mg/kg) and supplemental pentobarbital (10 mg/kg) was administered as necessary to maintain surgical anesthesia. The rest of the procedure was the same as that used in rats. At the end of each experiment animals were humanely killed by an ip overdose of pentobarbital (∼195 mg/kg body weight for rats and 150 mg/kg weight for mice).

Compositions of the various stimulating solutions used in the chorda tympani experiments are given in Table 1. The various drugs and their targets used in this study are summarized in Table 2. The anterior lingual surface was stimulated with a rinse solution (R; pH 6) and with a salt solution (N; pH 6) containing 0 to 10 × 10⁻⁶ M RTX. RTX specifically modulates the Bz-insensitive NaCl chorda tympani response. In previous studies, the RTX-induced increase in the Bz-insensitive NaCl chorda tympani response varied with pH. The relationship between pH and the magnitude of the chorda tympani response was bell shaped. The maximum increase in the chorda tympani response was observed around pH 6 (Lyall et al. 2004). Benzamil (Bz) was used to block Na⁺ entry through apical epithelial Na⁺ channel (ENaC). Mannitol was added to maintain both R and N solutions at the same osmotic pressure. Mannitol by itself does not elicit a chorda tympani response (Lyall et al. 2006). RTX versus Bz-insensitive NaCl chorda tympani dose–response curves were obtained before and after treating the anterior tongue with various drugs listed in Table 2.

TABLE 1.

Composition of stimulating solutions for chorda tympani experiments

Solution	Composition	pH
Rinse (R)	0.01 M KCl + 0.2 M mannitol + 0.01 M HEPES	6
Salt stimuli (N)	0.01 M KCl + 0.1 M NaCl + 0.01 M HEPES	6
R + RTX	R + 0.1 × 10−6 to 10 × 10−6 M RTX	6
N + RTX	N + 0.1 × 10−6 to 10 × 10−6 M RTX	6
R + CaCl2	R + 0 to 0.3 M CaCl2
N + RTX + CaCl2	N + 0.1 × 10−6 M RTX + 0 to 0.3 M CaCl2
Control-1	0.3 M NH4Cl
Control-2	0.3 M NaCl
Ringer's solution	0.14 M NaCl + 0.005 M KCl + 0.001 M CaCl2 + 0.001 M MgCl2 + 0.01 Na-pyruvate + 0.01 glucose + 0.01 HEPES	7.4
0 Na+ Ringer's	0.15 M NMDG-Cl + 0.005 M KCl + 0.001 M CaCl2 + 0.001 M MgCl2 + 0.01 pyruvic acid + 0.01 glucose + 0.01 HEPES	7.4

NMDG, N-methyl-d-glucamine.

TABLE 2.

Summary of the effects of various drugs on intracellular effectors that modulate the benzamil (Bz)-insensitive NaCl chorda tympani response

Drug	(M)	Intracellular Target	RTX–Chorda Tympani Response, Vmax	n
BAPTA-AM	5 × 10−3 to 30 × 10−3	↓[Ca2+]i ↓calcineurin	↑	3
Cyclosporin A	250 × 10−6	↓calcineurin	↑	3
FK-506	250 × 10−6	↓calcineurin	↑	2
Ionomycin + Ca2+	150 × 10−6 + 0.01 × 10−3	↑ [Ca2+]i ↑ calcineurin	↓	3
PMA	50 × 10−6 to 250 × 10−6	↑ PKC	↑	4
R031-8220	50 × 10−6	↓PKC	↓PMA	3
4α-Phorbol-12,13-didecanoate	250 × 10−6	↔ PKC	↔	3
PKCɛ pseudosubstrate inhibitor peptide	25 × 10−6	↓PKCɛ	↓PMA	3
8-CPT-cAMP	250 × 10−6	↑ PKA	↔	3
KN-93	250 × 10−6	↓CaMK II	↔	3
KN-92	250 × 10−6	↔ CaMK II	↔	3
SB-366791	1 × 10−6	↓TRPV1t	↓	12
Bz	5 × 10−6	↓ENaC	↔	36

(↑) increase; (↓) decrease; (↔) no change. V_max, maximum value of the chorda tympani response at 1 × 10⁻⁶ M RTX; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); 8-CPT-cAMP, 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate; ENaC, amiloride- and Bz-sensitive Na⁺ channel; KN-93, N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]-phenyl]-N-(2-hydroxy-ethyl)-4-methoxy-benzenesulfonamide phosphate; KN-92, [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methyl-benzylamine]; PMA, phorbol 12-myristate 13-acetate; R031-822, 2-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(methylindol-3-yl)maleimide methane-sulfonate; SB-366791, N-(3-methoxyphenyl)-4-chlorocinnamid. All drugs were obtained from Sigma, except PKCɛ pseudo substrate inhibitor peptide was obtained from Calbiochem. n = number of animals used for each treatment. Bz and SB were added to the salt stimuli and produced their effects on the Bz-insensitive NaCl chorda tympani responses immediately. However, all other drugs were dissolved directly in 3 ml of dimethylsulfoxide (DMSO) and were topically applied to the tongue for ≥30 min. DMSO by itself has no effect of the chorda tympani responses to salt stimuli (Lyall et al. 1999).

To decrease TRC [Ca²⁺]_i in situ, the anterior tongue was treated with a membrane-permeable Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakisacetoxy-methyl ester (BAPTA-AM) (Lyall et al. 2006). Following its entry into cells, nonspecific esterases hydrolyze BAPTA-AM to BAPTA-acid. Intracellular BAPTA-acid chelates free Ca²⁺, decreases resting TRC [Ca²⁺]_i, and prevents any subsequent increase in its concentration during taste transduction. Alternately, TRCs were loaded in vivo with Ca²⁺ using the Ca²⁺ ionophore, ionomycin. In addition, all rinse and stimulating solutions were supplemented with 0.01 M CaCl₂ (Table 2) (Lyall et al. 2006). RTX–chorda tympani dose–response profiles were obtained before and after topical lingual application of KN-93, a specific blocker of CaMK II (Liu and Simon 2003) or specific inhibitors of PP2B (cyclosporin A or FK-506) (Klee et al. 1998; Pearce et al. 2008). As a control for KN-93, we used the kinase-inactive analog KN-92 (Table 2). RTX–chorda tympani response profiles were also obtained before and after topical lingual application of PMA, a specific activator of PKC or an inactive phorbol ester, 4α-phorbol-12,13-didecanoate. In some experiments, the tongue was pretreated with the specific PKC inhibitor, R031-8220 or pseudosubstrate inhibitor peptide, a specific blocker of the Ca²⁺-independent isoenzyme of PKC, PKCɛ (Walsh et al. 1994) before applying PMA to the tongue. In some experiments the tongue was treated with 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP), a membrane-permeable cAMP analog (Table 2).

Typically, stimulus solutions remained on the tongue for 1–2 min. Control stimuli consisting of 0.3 M NH₄Cl and 0.3 M NaCl (Table 2) applied at the beginning and at the end of experiment were used to assess preparation stability (see Fig. 1, A and B). The preparation was considered stable only if the difference between the magnitude of the control stimuli at the beginning and at the end of the experiment was <10%. The following stimulus series were used in the chorda tympani experiments

FIG. 1.

Effect of resiniferatoxin (RTX), benzamil (Bz), and SB-366791 (SB) on the NaCl chorda tympani responses in wild-type (WT) mice and transient receptor potential vanilloid-1 (TRPV1) knockout (KO) mice and rats. Chorda tympani responses were monitored while the mouse tongue was first rinsed with a rinse solution (R; Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0–10 × 10⁻⁶ M RTX in WT (A) and TRPV1 KO (B) mice. The chorda tympani responses to control 0.3 M NH₄Cl lingual stimulations are also shown at the beginning and end of the experiment for both WT and KO mice. In each case the NH₄Cl chorda tympani responses were nearly identical before and after the experiment. The effects of RTX on the tonic Bz-insensitive NaCl chorda tympani responses from 3 WT and 3 KO mice are summarized in C. Chorda tympani responses were also monitored in rats while their tongues were first rinsed with a rinse solution (R; Table 1) and then with the stimulating salt solutions, N, N + Bz, and N + Bz + RTX (1 × 10⁻⁶ M) (Table 1). The effects of RTX on the tonic Bz-insensitive NaCl chorda tympani responses from 3 rats are shown in D. Tonic responses to RTX were compared in 3 WT mice and 3 rats. Significant differences were found for RTX concentration (P < 0.0005 for both, 2-way ANOVA; Bonferroni corrected). However, no significant difference was observed in the RTX response in WT mice and rats (P > 0.05). The effects of Bz, RTX, and SB on the tonic Bz-insensitive NaCl chorda tympani responses from 3 rats are summarized in E. In each animal the Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point in C and D or each bar in E presents the means ± SE values of the normalized chorda tympani response from 3 animals. In E, in the presence of N + Bz the mean normalized tonic NaCl chorda tympani response was significantly decreased from N (P = 0.0001) and in the presence of N + Bz + SB-366791, it further decreased to baseline (P > 0.05 with respect to zero; paired 2-sample t-test). RTX elicited no tonic chorda tympani response above baseline whether Bz + SB were added in the salt stimulus alone (open bar) or when Bz + SB-366791 were present in both rinse (R + Bz + SB) and the salt stimulus (hatched bar).

The R → (N + Bz + RTX) → R step was repeated for each concentration of RTX between 0.1 × 10⁻⁶ and 10 × 10⁻⁶ M. At the end of the RTX concentration series, the control stimuli were again applied (R → 0.3 M NH₄Cl → R → 0.3 M NaCl → R).

The data were digitized and analyzed off-line. In chorda tympani experiments the tonic (steady-state) part of the NaCl neural responses was quantified. In some experiments we also quantified the transient (phasic) part of the neural response. We also quantified the transient (phasic) response to the application of rinse (R) to a tongue already superfused with R. To quantify the phasic part of the chorda tympani response, the height of the stimulus-induced maximum chorda tympani response relative to baseline response was divided by the mean steady-state (tonic) response to 0.3 M NH₄Cl. To quantify the tonic part of a response the area under the response versus time curve was taken over the final 30 s of the response. To normalize this result, this area was divided by the area under the 0.3 M NH₄Cl response curve over the final 30 s of the tonic response period. The normalized data were reported as means ± SE of the number of animals. Since we are comparing the normalized chorda tympani responses before and after TRPV1t modulators in the same chorda tympani preparation, a paired t-test was used to evaluate statistical significance. In experiments in which repeated measurements to sequential drug applications were obtained, the data were analyzed using two-way ANOVA and all statistical significant values were Bonferroni-adjusted.

For clarity the points on the graphs of the mean normalized phasic and tonic responses versus the logarithm of the RTX concentration were connected respectively by smooth curves. The curves were generated using a fitting function that models the characteristic biphasic property of the RTX concentration versus the magnitude of the chorda tympani response. The biphasic property has been observed with every agonist of amiloride- and Bz-insensitive NaCl chorda tympani response thus far examined (Katsumata et al. 2008; Lyall et al. 2004, 2005b, 2007). The fitting function used was

(1)

where

(2)

and

(3)

Here R is the response; x is the logarithm of the RTX concentration expressed in moles/liter; and a, b, d, m, n, and r are parameters chosen by least-squares criteria (Katsumata et al. 2008).

In vitro phosphorylation of TRPV1/TRPV1t by PMA, cyclosporin A and FK-506

To test the hypothesis that PMA, cyclosporin A, and FK-506 alter TRPV1t activity by changing the phosphorylation state of the channel, direct evidence for TRPV1t/TRPV1 phosphorylation was obtained using an in vitro method (Lee et al. 2005). Rat lingual epithelium containing fungiform papillae was isolated as described before (Lyall et al. 2004). Lingual epithelium was labeled for 4 h with ³²P. After chemical treatment with PMA (10 × 10⁻⁶ M), cyclosporin A, or FK-506 (50 × 10⁻⁶ M) for 10 min, the lingual epithelium was washed with ice-cold PBS, and then lysed with cold immuno-precipitation buffer containing 0.2 × 10⁻³ M sodium orthovanadate, 0.2 × 10⁻³ M phenylmethylsulfonylfluoride (PMSF), and 0.5% NP-40, with a protease inhibitor cocktail (Boehringer Mannheim). After removal of the large aggregates, the soluble cell lysates were immunoprecipitated with polyclonal anti-VR1 antibody [VR1(C-15):sc-12503; Santa Cruz Biotechnology] and protein A-agarose. The precipitates were washed three times with ice-cold immuno-precipitation buffer for 10 min at 55°C. The samples were centrifuged at 4°C for 10 min, separated by 8% SDS-PAGE, and transferred to polyvinylidene fluoride membranes for immunoblotting and autoradiography.

Detection of PKCɛ using PCR

To confirm the presence of PKCɛ in TRCs and in the anterior lingual epithelium containing fungiform taste papillae we used both polymerase chain reaction (PCR) and Western blotting techniques. Taste buds were harvested from rat fungiform papillae, aspirated with a micropipette, and individually transferred onto coverslips, avoiding contaminating cells and debris as described before (Lyall et al. 2004). RNA was prepared using the RNeasy Protect Kit (Qiagen, Valencia, CA) and the cDNA was generated using the M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA) according to the manufacturers' protocols. PCR screening of the fungiform cDNA for the presence of rat PKCɛ (NM_017171) was performed with Taq DNA Polymerase (Roche, Indianapolis, IN) using primer pair

and

with an annealing temperature of 62.5°C. This primer pair should result in a PCR product of 543 bp. As a negative control the reaction was run without the cDNA template. The PCR products were analyzed by agarose gel electrophoresis and bands of the predicted size were excised and then purified using the MinElute Gel Extraction kit (Qiagen). The isolated band was cloned into PCRII TOPO using the TOPO-TA Cloning Kit (Invitrogen) and sequenced from both the T7 and SP6 promoters at the VCU Nucleic Acids Research Facilities. The generated sequences were compared with that of NM_017171 using Vector NTI Advanced 9.0 (Invitrogen) and found to be identical to the published sequence.

Detection of PKCɛ using Western blotting

Rat anterior lingual epithelium was isolated as described before (Lyall et al. 2004). For Western blotting three rat tongues were used for the experiment. The lingual epithelium was dissected into small fragments and solubilized on ice for 1 h in medium containing 0.02 M Tri-HCl (pH 8.0), 1 × 10⁻³ M DTT, 0.1 M NaCl, 0.5% sodium dodecyl sulfate, 0.75% deoxycholate, 1 × 10⁻³ M PMSF, 10 × 10⁻⁶ g/ml of leupeptin, and 100 × 10⁻⁶ g/ml of aprotinin. The proteins were resolved by SDS-PAGE and electrophoretically transferred onto nitrocullose membranes. The membranes were incubated for 12 h with anti PKCɛ antibody (1:1,000) and then for 1 h with horseradish peroxidase–conjugated secondary antibody (1:2,000). The protein bands were identified with enhanced chemiluminescence reagent.

Intracellular Na⁺ ([Na⁺]_i) measurement in polarized fungiform taste receptor cells

We used Na⁺ imaging to directly demonstrate the effect of RTX and PMA on the unilateral Na⁺ flux in polarized fungiform TRCs. Rats were anesthetized with isoflurane and killed by cervical dislocation. The tongues were rapidly removed and stored in ice-cold Ringer solution (Table 1). The lingual epithelium was isolated by collagenase treatment. A small piece of the anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber as described before (Lyall et al. 2007). The tissue was intermittently perfused with Ringer solution containing 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclo-pentadecane-7,13-diylbis(5-methoxy-6,12-benzo-furandiyl)]-bistetrakis-(acetyloxy) (SBFI-AM; 10 × 10⁻⁶ M) in the presence of 0.15% pluronic (both from Molecular Probes) at room temperature for 4 h. Before the experiment was started, the tissue was perfused on both sides with control solution for 15 min. The tissue was continuously perfused at the rate of 1 ml/min and the solution changes in the apical or basolateral compartment were made using three-way miniature LFAA solenoid valves (Lee, Westbrook, CT). The TRCs in the taste bud were visualized from the basolateral side through a ×40 objective (Zeiss; 0.9 NA) with a Zeiss Axioskop 2 plus upright fluorescence microscope and imaged with a setup consisting of: a cooled charge-coupled device camera (Imago, TILL Photonics; Applied Scientific Instrumentation) attached to an image intensifier (VS4-1845; Videoscope), an epifluorescent light source (TILL Photonics Polychrome IV), a 439-nm dichroic beam splitter (Omega Optical), and a 510-nm emission filter (20-nm band-pass; Omega Optical). The cells were alternately excited at 340 and 380 nm and imaged at 10-s intervals. Small regions of interest (ROIs) in the taste bud (diameter, 2–3 μm) were chosen in which the changes in fluorescence intensity ratio (FIR: F₃₄₀/F₃₈₀) were analyzed using TILLvisION v3.1 imaging software. Each ROI contained two to three receptor cells. Thus the fluorescence intensity recorded for a ROI represents the mean value from two to three receptor cells within the ROI. In a typical experiment the FIR measurements were made in an optical plane in the taste bud containing at least six ROIs (∼18 cells). The background and autofluorescence at 340 and 380 nm were corrected from images of a taste bud without the dye. All experiments were done at room temperature (∼22°C). The relative changes in FIR were compared between different ROIs under different conditions. The data were also presented as the means ± SE from different tissue preparations. In this case n represented the number of taste buds. Student's t-test was used to analyze the differences between sets of data.

RESULTS

Effect of RTX on the Bz-insensitive NaCl chorda tympani responses in WT and TRPV1 KO mice

Consistent with previous studies (Lyall et al. 2004), in a WT mouse, stimulating the tongue with N + Bz solutions containing increasing concentrations of RTX (Table 1) initially produced an increase in tonic NaCl chorda tympani response between 0.25 × 10⁻⁶ and 1 × 10⁻⁶ M (Fig. 1A). Above 1 × 10⁻⁶ M RTX, the tonic response was less than its maximum value. In addition to the tonic component, RTX also clearly modulates the phasic component of the salt response. In three WT mice, RTX produced a bell-shaped dose–response relationship for both tonic (Fig. 1C; ○) and phasic (see Supplemental Fig. S1A; ○) components of the salt response.1 The maximum increase in the mean normalized phasic and tonic chorda tympani responses occurred at approximately the same RTX concentration (1 × 10⁻⁶ M RTX) as estimated from the fitted curves. These results demonstrate that both phasic and tonic components of the Bz-insensitive NaCl chorda tympani response produce similar RTX concentration–response relations.

In TRPV1 KO mice (Fig. 1B), which lack the Bz-insensitive component of the NaCl chorda tympani response, RTX concentrations between 0.1 × 10⁻⁶ and 10 × 10⁻⁶ M elicited only transient responses that were concentration independent and were indistinguishable from the mechanical rinse artifact (see Supplemental Fig. S1A; •). In three KO mice, no increase in tonic (Fig. 1C; •) chorda tympani response to N + Bz was observed above baseline. In WT mice, RTX dissolved in the rinse solution (R; Table 1) also elicited only transient responses that were concentration independent and were indistinguishable from the mechanical rinse artifact (data not shown). These results suggest that within the range of concentrations (0.1 × 10⁻⁶ to 10 × 10⁻⁶ M) used in this study, RTX by itself does not elicit a chorda tympani response.

Similar to WT mice, in Sprague–Dawley rats, RTX also elicited a biphasic response in both tonic (Fig. 1D) and phasic (see Supplemental Fig. S1B) Bz-insensitive NaCl chorda tympani responses. The maximum increase in the two components of the NaCl chorda tympani response was again observed at 1 × 10⁻⁶ M RTX.

Effect of SB-366791 on the Bz-insensitive NaCl chorda tympani responses in the absence and presence of RTX

Rat NaCl chorda tympani responses were monitored under control condition and in the presence of Bz alone, SB-366791 alone, or Bz + SB-366791 relative to the rinse (R; Table 1). Consistent with previous studies (Katsumata et al. 2008), in three animals, Bz inhibited 68.6% of the tonic Bz-insensitive NaCl chorda tympani response and in the presence of Bz + SB-366791 the response was inhibited to rinse baseline. Although not reported previously, Bz also inhibited the phasic chorda tympani NaCl response and Bz + SB-366791 reduced it further to the level of the rinse artifact (see Supplemental Fig. S1C). These results show that in the presence of Bz, both phasic and tonic NaCl chorda tympani responses are blocked by SB-366791.

In the presence of SB-366791 (1 × 10⁻⁶ M), RTX (1 × 10⁻⁶ M) produced no significant increase in the normalized mean tonic chorda tympani response (N + Bz + SB + RTX) above baseline (Fig. 1E). RTX elicited no tonic chorda tympani response above baseline, irrespective of whether Bz + SB were added in the salt stimulus alone (Fig. 1E; open bar) or when Bz + SB-366791 were present in both rinse (R + Bz + SB) and the salt stimulus (Fig. 1E; hatched bar). Similarly, RTX (1 × 10⁻⁶ M) failed to enhance phasic NaCl response in the presence of N + Bz + SB-366791 (1 × 10⁻⁶ M) (see Supplemental Fig. S1C). These results show that SB-366791 inhibits the effect of RTX on both phasic and tonic Bz-insensitive NaCl chorda tympani responses. Thus, unlike the phasic and tonic chorda tympani responses to acidic stimuli (DeSimone and Lyall 2006), both components of the neural response to NaCl have the same physiological mechanism.

RTX failed to elicit a chorda tympani response in rats and in WT mice when the Bz-insensitive NaCl chorda tympani response is blocked by SB-366791 or in TRPV1 KO mice that lack the Bz-insensitive NaCl chorda tympani response. Taken together, the above-cited results suggest that RTX specifically modulates the Bz-insensitive NaCl response by interacting with the TRPV1 channel or its variant TRPV1t. Thus RTX and SB-366791 can be used as specific modulators of the Bz-insensitive NaCl chorda tympani response to investigate its regulation in situ.

Chorda tympani responses to CaCl₂ are modulated by RTX and SB-366791

TRPV1 is a nonselective channel that shows preference for Ca²⁺ over Na⁺ (P_Ca/P_Na ∼10) (Novakova-Tousova et al. 2007). Therefore we first investigated whether chorda tympani responses to CaCl₂ are also elicited by Ca²⁺ influx through TRPV1t. During the steady-state tonic chorda tympani response to 0.1 M CaCl₂, stimulating the tongue with 0.1 M CaCl₂ + RTX (1 × 10⁻⁶ M) (at the second arrow) reversibly enhanced the tonic response relative to CaCl₂ alone (Fig. 2A). Similarly, stimulating the tongue with 0.3 M CaCl₂ + RTX (1 × 10⁻⁶ M) enhanced both phasic and tonic chorda tympani responses relative to 0.3 M CaCl₂ alone (Fig. 2B). The data shown in Fig. 2B further demonstrate that stimulating the tongue with 0.3 M CaCl₂ + SB-366791 (1 × 10⁻⁶ M) inhibited both the phasic and tonic components of the chorda tympani response relative to 0.3 M CaCl₂ alone (see also Fig. 2C). In four rats, SB-366791 blocked the tonic chorda tympani response to 0.3 M CaCl₂ by 94% (Fig. 2E). These results support the conclusion that TRPV1t is a nonselective cation channel that is not only permeable to Na⁺, K⁺, and NH₄⁺ (Lyall et al. 2004), but is also permeable to Ca²⁺. The data further suggest that Ca²⁺ influx through TRPV1t contributes to both phasic and tonic CaCl₂ chorda tympani responses.

FIG. 2.

Effect of CaCl₂ on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani responses in rats. Chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with 0.1 M CaCl₂ (A) or 0.3 M CaCl₂ (B) in the absence and presence of 1 × 10⁻⁶ M RTX. The arrows represent the time interval when the rat tongues were superfused with different solutions. In B the chorda tympani response to 0.3 M CaCl₂ was also monitored in the absence and presence of 1 × 10⁻⁶ M SB-366791 (SB). C: chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with R + CaCl₂ (0.05, 0.1, 0.2, and 0.3 M) or N + Bz + RTX (1 × 10⁻⁶ M) + CaCl₂ (0.05, 0.1, 0.2, and 0.3 M). The effects of CaCl₂ on the tonic Bz-insensitive NaCl chorda tympani responses are summarized in D. The effects of SB on the tonic CaCl₂ chorda tympani responses are summarized in E. In each animal the chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point in D and each bar in E presents the means ± SE values of the normalized chorda tympani response from 3 and 4 animals, respectively. In D the tonic chorda tympani responses in the presence of N + Bz + RTX were significantly decreased in the presence of 0.2 and 0.3 M CaCl₂ relative to 0 CaCl₂ (P = 0.0074 and 0.0392, respectively; paired 2-sample t-test). In E chorda tympani responses in the presence of SB + 0.3 M CaCl₂ were significantly decreased relative to 0.3 M CaCl₂ (P = 0.0001; paired 2-sample t-test).

Extracellular Ca²⁺ ([Ca²⁺]_o) modulates Bz-insensitive NaCl chorda tympani responses

Next we tested whether changes in [Ca²⁺]_o modulate the Bz-insensitive salt responses. Chorda tympani responses were monitored in R and N + Bz + RTX (1 × 10⁻⁶ M) solutions containing CaCl₂ (0–0.3 M) (Table 1). Addition of CaCl₂ to the rinse solution elicited a dose-dependent increase in the chorda tympani response (Fig. 2C). Importantly, CaCl₂ inhibited both tonic (Fig. 2D) and phasic (see Supplemental Fig. S1D) chorda tympani responses to N + Bz + RTX in a dose-dependent manner. These data demonstrate that the presence of Ca²⁺ in the salt solution desensitizes TRPV1t, resulting in a smaller increase in the Bz-insensitive NaCl chorda tympani responses in the presence of RTX.

Ionomycin modulates chorda tympani responses to CaCl₂

To confirm that chorda tympani responses to CaCl₂ are dependent on apical Ca²⁺ influx, we increased the permeability of the apical membrane of TRCs to Ca²⁺ in situ by topical lingual application of ionomycin (150 × 10⁻⁶ M) for 30 min. In the presence of ionomycin, adding CaCl₂ to the rinse solution (R) produced a dose-dependent increase in chorda tympani response at 0.01, 0.02, and 0.05 M CaCl₂ (Fig. 3 A). Thus in the presence of ionomycin the chorda tympani response to CaCl₂ is observed at significantly lower concentrations relative to control (Fig. 2C).

FIG. 3.

Effect of Ca²⁺ or BAPTA loading on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani responses in rats. A: a rat tongue was pretreated with 150 × 10⁻⁶ M ionomycin for 30 min. Chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with R + CaCl₂ (0.1, 0.2, and 0.3 M) or N + Bz + RTX (1 × 10⁻⁶ M) + CaCl₂ (0.1, 0.2, and 0.3 M). B: chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0–10 × 10⁻⁶ M RTX before (; Control) and after topical lingual application of 150 × 10⁻⁶ M ionomycin for 30 min (○; postionomycin + Ca²⁺). The effects of ionomycin + Ca²⁺ on the Bz-insensitive NaCl chorda tympani responses from 3 rats are summarized. In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point presents the means ± SE values of the normalized chorda tympani response from 3 animals. Tonic responses to RTX were compared in 3 rats before and after ionomycin treatment. Significant differences were found for RTX concentration (P < 0.005 for both, 2-way ANOVA; Bonferroni corrected) and their interactions (P = 0.025). C: chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution (R; Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0–10 × 10⁻⁶ M RTX before (; Control) and after topical lingual application of 30 × 10⁻³ M BAPTA-AM for 30 min (○; post-BAPTA). The effects of BAPTA on the Bz-insenstive NaCl chorda tympani responses are summarized. In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point presents the means ± SE values of the normalized chorda tympani response from 3 animals. Tonic responses to RTX were compared in 3 rats before and after BAPTA treatment. Significant differences were found for RTX concentration (P = 0.028 for both, 2-way ANOVA) and their interactions (P = 0.0018).

Modulation of the Bz-insensitive NaCl chorda tympani response by [Ca²⁺]_i

To investigate whether changes in [Ca²⁺]_i modulate Bz-insensitive NaCl chorda tympani responses, TRC resting [Ca²⁺]_i was increased in vivo above control level by topical lingual application of ionomycin + Ca²⁺ or was decreased below the control level by loading the cells with BAPTA.

Effect of increasing TRC [Ca²⁺]_i in vivo on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response.

The effect of RTX on Bz-insensitive NaCl chorda tympani responses was monitored after the topical lingual application of ionomycin. Following ionomycin treatment, adding 0.01, 0.02, and 0.05 M CaCl₂ to N + Bz + RTX inhibited the Bz-insensitive NaCl chorda tympani response in a dose-dependent manner. Thus in the presence of ionomycin, CaCl₂ inhibited the Bz-insensitive NaCl chorda tympani response at significantly lower concentrations relative to control (Fig. 2C).

Additional experiments were performed keeping CaCl₂ at a fixed concentration of 0.01 M (raw data are shown in Supplemental Fig. S2A). The results show that ionomycin-induced increase in TRC [Ca²⁺]_i does not affect the Bz-insensitive NaCl chorda tympani response in the absence of RTX. However, it attenuated the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response between 0.25 × 10⁻⁶ and 3 × 10⁻⁶ M RTX (Fig. 3B).

Effect of chelating TRC [Ca²⁺]_i in vivo on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response.

The effect of RTX (0 to 10 × 10⁻⁶ M) on the Bz-insensitive NaCl chorda tympani responses was monitored before and after topical lingual application of BAPTA-AM. Under control conditions (see Supplemental Fig. S2B; Control) RTX produced a biphasic effect on the Bz-insensitive NaCl chorda tympani response as before. Following BAPTA-AM treatment the Bz-insensitive NaCl chorda tympani response was increased in the absence of RTX. In addition, RTX produced a greater increase in the Bz-insensitive chorda tympani response at all RTX concentrations relative to control (see Supplemental Fig. S2B; post-BAPTA). The paired normalized chorda tympani response data from three animals are shown in Fig. 3C. The results show that a decrease in BAPTA-induced resting TRC [Ca²⁺]_i enhances the magnitude of the Bz-insensitive tonic chorda tympani response in the absence and presence of RTX. Importantly, the inhibition of the Bz-insensitive NaCl tonic chorda tympani response at RTX concentrations >1 × 10⁻⁶ M was itself largely inhibited. The results shown in Fig. 3, B and C demonstrate that TRPV1t activity is enhanced in the presence of RTX by a decrease and inhibited by an increase in TRC [Ca²⁺]_i.

Effect of KN-93, cyclosporin A, and FK-506 on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response.

To test whether changes in TRC [Ca²⁺]_i modulate TRPV1t activity via CaMK II, the effect of RTX on the Bz-insensitive NaCl chorda tympani responses was monitored before and after the topical lingual application of KN-93, a specific blocker of CaMK II. In three rats, KN-93 and KN-92 (each at 250 × 10⁻⁶ M) produced no effect on the Bz-insensitive NaCl chorda tympani response in the absence and presence of RTX (data not shown). These results suggest that in TRCs, native TRPV1t activity is not sensitive to modulation by CaMK II induced phosphorylation of the channel protein.

Next, we monitored the effect of RTX on the Bz-insensitive NaCl chorda tympani responses before and after the topical lingual application of cyclosporin A or FK-506, specific inhibitors of calcineurin (PP2B). At 250 × 10⁻⁶ M, both cyclosporin A or FK-506 produced qualitatively similar effects on the Bz-insensitive NaCl chorda tympani response in the absence and presence of RTX. Both drugs increased the Bz-insensitive NaCl chorda tympani response in the absence and presence of RTX. Therefore the data from these two drugs were pooled (Fig. 4 A). In tongues pretreated with ionomycin, the inhibition of the Bz-insensitive NaCl chorda tympani response in the presence of 0.02 M [Ca²⁺]_o was not observed after the topical lingual application of 250 × 10⁻⁶ M FK-506 (Fig. 4, C and D). These results suggest that inhibiting the Ca²⁺-dependent increase in PP2B activity enhances the chorda tympani response to N + Bz + RTX + CaCl₂.

FIG. 4.

Effect of cyclosporin A and FK-506 on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani responses. A: chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0–10 × 10⁻⁶ M RTX before (; Control) and after topical lingual application of 250 × 10⁻⁶ M cyclosporin A or FK-506 for 30 min (○; cyclosporin A or FK-506). In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point presents the means ± SE values of the normalized chorda tympani response from 5 animals. Tonic responses to RTX were compared in 3 rats before and after cyclosporin A/FK-506 treatment. Significant differences were found for RTX concentration (P < 0.006 for both, 2-way ANOVA; Bonferroni corrected) and their interactions (P = 0.0015). B: in ³²P-labeled isolated anterior lingual epithelium cyclosporin A or FK-506 (each at 50 × 10⁻⁶ M) phosphorylate TRPV1 or TRPV1t relative to untreated control lingual epithelium. C: a rat tongue was pretreated with 150 × 10⁻⁶ M ionomycin for 30 min. Chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R + 0.02 M CaCl₂ (Table 1) and then with the stimulating salt solution, N + Bz + RTX (1 × 10⁻⁶ M) + 0.02 M CaCl₂ (Table 1) before (Control) and after 50 × 10⁻⁶ M FK-506 (post-FK-506). D: summary of the effects of FK-506 on the Bz-insenstive NaCl chorda tympani responses. In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each bar presents the means ± SE values of the normalized chorda tympani response from 3 animals. Following ionomycin treatment, 0.02 M CaCl₂ significantly inhibited the tonic chorda tympani response to N + Bz + RTX (P = 0.0026; paired 2-sample t-test). Post-FK-506 tonic chorda tympani responses to N + Bz + RTX + 0.02 M CaCl₂ were not significantly different from N + Bz + RTX (P > 0.5).

Effect of PMA on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response.

To test whether TRPV1t activity is modulated by PKC, chorda tympani responses were monitored before and after topical lingual application of PMA, a specific activator of PKC. Under control conditions (see Supplemental Fig. S3A; Control) the effect of increasing RTX concentration on the Bz-insensitive NaCl chorda tympani response was the same as shown before. PMA was applied topically to the tongue for 45 min at concentrations of 50 × 10⁻⁶, 100 × 10⁻⁶, and 250 × 10⁻⁶ M. No effect of 50 × 10⁻⁶ or 100 × 10⁻⁶ M PMA was observed on the Bz-insensitive NaCl chorda tympani response in the absence and presence of RTX (data not shown). Following 250 × 10⁻⁶ M PMA treatment, RTX produced a greater increase in the Bz-insensitive NaCl chorda tympani response (see Supplemental Fig. S3B; post-PMA) relative to control. The paired normalized chorda tympani response data from four animals are shown in Fig. 5 A. The results show that PMA had no significant effect on the magnitude of the Bz-insensitive NaCl chorda tympani response in the absence of RTX. However, it enhanced the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response between 0.5 × 10⁻⁶ and 10 × 10⁻⁶ M RTX.

FIG. 5.

Effect of 4α-phorbol-12,13-didecanoate (PMA), R031-8220, and PKCɛ pseudosubstrate inhibitor peptide on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani responses. A: chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0–10 × 10⁻⁶ M RTX before (; Control) and after topical lingual application of 250 × 10⁻⁶ M PMA for 30 min (○; Post-PMA). The effects of PMA on the Bz-insensitive NaCl chorda tympani responses are summarized. In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each point presents the means ± SE values of the normalized chorda tympani response from 4 animals. Tonic responses to RTX were compared in 4 rats before and after PMA treatment. Significant differences were found for RTX concentration (P < 0.005 for both, 2-way ANOVA; Bonferroni corrected) and their interactions (P = 0.006). B: in ³²P-labeled isolated anterior lingual epithelium PMA phosphorylated TRPV1 or TRPV1t relative to untreated control lingual epithelium. Rat tongues were pretreated with either 50 × 10⁻⁶ M R031-8220 (C) or 25 × 10⁻⁶ M PKCɛ pseudosubstrate inhibitor peptide for 30 min (D) for 30 min. Chorda tympani responses were monitored while the rat tongue was first rinsed with a rinse solution, R (Table 1) and then with the stimulating salt solution, N + Bz (Table 1) containing 0.5 × 10⁻⁶ or 1 × 10⁻⁶ M RTX. Following this rat tongues were treated with 250 × 10⁻⁶ M PMA for an additional 30 min. The chorda tympani responses were again recorded by stimulating the tongue with N + Bz (Table 1) containing 0.5 × 10⁻⁶ or 1 × 10⁻⁶ M RTX. In each animal the tonic Bz-insensitive NaCl chorda tympani response was normalized to the tonic chorda tympani response to 0.3 M NH₄Cl. Each bar presents the means ± SE values of the normalized chorda tympani response from 3 animals. In our studies the effects of PMA on the Bz-insensitive NaCl chorda tympani responses were not reversible. Therefore PMA effects on the neural responses before the application of the drugs in C or D could not be included. Accordingly, the comparison between post-PMA effects in C and D was made with post-PMA effects observed with 0.5 × 10⁻⁶ and 1 × 10⁻⁶ M RTX in A. In C RTX at 0.5 × 10⁻⁶ or 1 × 10⁻⁶ M RTX increased the chorda tympani response to N + Bz in the presence of R031-8220 before (P = 0.0123 and 0.0029, respectively) and after PMA treatment (P = 0.0001 and 0.0001, respectively; paired 2-sample t-test) in a dose-dependent manner. However, no change in the magnitude of the N + Bz + RTX tonic chorda tympani responses was observed before and after PMA treatment (P > 0.5; paired 2-sample t-test) in C or D. In contrast, N + Bz responses in the presence of 0.5 × 10⁻⁶ and 1 × 10⁻⁶ M RTX were significantly enhanced after PMA treatment (post-PMA) relative to controls (Control) in A. E: rat fungiform taste bud cells contain PKCɛ. A cDNA library from rat fungiform (FF; lane 2) taste bud cells was screened for PKCɛ and yielded a single band of expected size (534 bp). Lane 1 = DNA ladder and lane 3 = negative (−) control. F: in anterior lingual epithelium containing fungiform taste buds PKCɛ antibody demonstrated a single band of about 85 KD on Western blots. In lane 1 and lane 2 the sample was loaded at 20 × 10⁻⁶ and 30 × 10⁻⁶ g, respectively.

The inactive phorbol ester 4α-phorbol-12,13-didecanoate, when applied topically to the tongue at 250 × 10⁻⁶ M for 45 min, did not have any effect on the Bz-insensitive NaCl chorda tympani response in the absence or presence of RTX (data not shown). In addition, pretreating the tongue with 50 × 10⁻⁶ M R031-8220, a specific PKC blocker, inhibited the effect of PMA on the Bz-insensitive NaCl chorda tympani response in the absence or presence of 0.5 × 10⁻⁶ and 1 × 10⁻⁶ M RTX (Fig. 5C).

In our studies an increase in TRC [Ca²⁺]_i inhibited the Bz-insensitive NaCl chorda tympani responses in the presence of RTX (Fig. 3B). This effect most likely involves the activation of PP2B (Fig. 4), suggesting that the effects of PMA on the chorda tympani response are most likely related to the activation of a Ca²⁺-independent isoenzyme of PKC, PKCɛ. Accordingly, pretreating the tongue with a specific PKCɛ blocker, pseudosubstrate inhibitor peptide (25 × 10⁻⁶ M) completely abolished the PMA-induced enhancement of the N + Bz + RTX chorda tympani response (Fig. 5D). In cDNA derived from a pure preparation of rat fungiform taste bud cells, using PKCɛ-specific primer pairs a single band of 534 bp was obtained in fungiform TRCs by PCR (Fig. 5E). The PCR product yielded 100% homology with rat PKCɛ (NM_017171). As a control both PLCβ₂ and α-gustducin (specific markers of a subset of TRCs involved in sweet, umami, and bitter taste transduction) were detected in fungiform TRCs by PCR using specific sense and antisense primer pairs (data not shown). We also confirmed the presence of PKCɛ in the rat anterior lingual epithelium containing fungiform taste papillae with Western blotting using a specific antibody to PKCɛ (Fig. 5F). PKCɛ antibody demonstrated a single band of about 85 kD on Western blots.

Direct phosphorylation of TRPV1/TRPV1t in anterior lingual epithelium by PMA, cyclosporin A, and FK-506

We used an in vitro phosphorylation method (Lee et al. 2005) to directly demonstrate that following treatment with cyclosporin A or FK-506 (50 × 10⁻⁶ M each; Fig. 4B) or PMA (25 × 10⁻⁶ M; Fig. 5B), a TRPV1 or TRPV1t channel is phosphorylated in isolated anterior lingual epithelium containing fungiform taste buds. TRPV1 and/or TRPV1t were identified using a commercial polyclonal antibody to the C-terminal region of TRPV1 and TRPV1t [VR1 (C15):sc-12503; Santa Cruz Biotechnology]. These results suggest that activating PKCɛ by PMA or inhibiting calcineurin by cyclosporin A or FK-506 increases TRPV1 and/or TRPV1t phosphorylation in fungiform TRCs.

Effect of cAMP on the RTX-induced changes in the Bz-insensitive NaCl chorda tympani response.

Chorda tympani responses were recorded before and after the topical lingual application of 8-CPT-cAMP (250 × 10⁻⁶ M for 30 min), a specific activator of PKA. Cyclic AMP treatment had no effect on the Bz-insensitive NaCl chorda tympani response in the absence of RTX and also it did not alter the RTX-dose–response relationship relative to control (data not shown). However, in the same animal 8-CPT-cAMP treatment increased the chorda tympani response to 0.3 M NaCl (Supplemental Fig. S3C). This is consistent with our previous findings that cAMP enhances the Bz-sensitive NaCl chorda tympani responses (DeSimone and Lyall 2008).

Effect of PMA and RTX on the unilateral apical Na⁺ influx in polarized fungiform TRCs.

A small piece of the lingual epithelium containing a single fungiform papilla with a single taste bud was loaded with SBFI (see Supplemental Fig. S4, A–C). The epithelium was initially perfused on the apical side with 0 Na⁺-Ringer solution (Table 1; pH 7.4) and on the basolateral side with Ringer solution (Table 1; pH 7.4). The unilateral apical Na⁺ flux was measured as the change in FIR (F₃₄₀/F₃₈₀) induced by the perfusion of apical Ringer solution containing 0.15 M NaCl in the absence and presence of RTX (1 × 10⁻⁶ M) (see Supplemental Fig. S4D). The epithelium was exposed to 25 × 10⁻⁶ M PMA for 15 min. Following PMA treatment, the RTX-induced increase in FIR was significantly greater than that under control conditions (Fig. 6 B). These data demonstrate that the RTX-sensitive unilateral apical Na⁺ influx in fungiform TRCs is enhanced by PMA treatment.

FIG. 6.

A: effect of PMA and RTX on unilateral Na⁺ influx in polarized fingiform TRCs. Polarized fungiform taste bud preparations were loaded with SBFI and relative changes in [Na⁺]_i were monitored in taste bud cells as changes in fluorescence intensity ratio (FIR: F₃₄₀/F₃₈₀). The mean temporal changes in FIR are shown when apical solution was changed from 0 Na⁺ Ringer solution to either Ringer solution containing 0.15 M NaCl or 0.15 M NaCl + 1 × 10⁻⁶ M RTX before and after treating the basolateral membrane with 25 × 10⁻⁶ M PMA for 15 min. Values were expressed as mean ± SE values from 9 regions of interest (ROIs) within the taste bud. RTX elicited a significantly greater change in FIR before (P = 0.01) and after PMA treatment (P = 0.0001; paired 2-sample t-test). B: salt taste transduction. Salt taste transduction involves both cellular and transcellular pathways. In a subset of taste receptor cells (TRCs), Na⁺ enters through amiloride- and Bz-sensitive epithelial Na⁺ channel (ENaC) and/or via TRPV1t and depolarizes the receptor potential that results in the release of taste specific neurotransmitter, adenosine 5′-triphosphate (ATP). The taste nerve fibers making synapses with TRCs contain P2X2/P2X3 receptors to which ATP binds and excites a subset of nerve fibers to elicit a neural response. Na⁺ entry via ENaC and TRPV1t gives rise to Bz-sensitive and Bz-insensitive NaCl chorda tympani responses, respectively. Both candidate salt taste receptor/ion channels are modulated by a variety of intracellular and extracellular effectors, resulting in alterations in the neural responses to NaCl. ENaC activity and thus Bz-sensitive NaCl chorda tympani responses are decreased by Bz, a decrease in extracellular (pH_o), and intracellular pH (pH_i), an increase in [Ca²⁺]_i, and increases in temperature and taste cell depolarization, whereas an increase in pH_o and pH_i, a decrease in [Ca²⁺]_i, hyperpolarization and increase in cAMP/PKA increase ENaC activity and the magnitude of the neural response to NaCl. TRPV1t is specifically modulated by vanilloids (RTX and capsaicin [CAP]) and is blocked by SB-366791 (SB), casazepine (CZP), and ruthenium red (RR). Its activity is enhanced by elevation in temperature and by altering its phosphorylation state by PKC or calcineurin. Since some of the TRPV1t agonists also alter human salt taste perception, this suggests a role for TRPV1t in human salt taste transduction. Although ENaC and TRPV1t are shown in the same TRC type, the cellular localization of TRPV1t has not been determined as yet. Transcellular pathway via the paracellular shunt is anion dependent. Substituting larger anions for chloride hyperpolarizes the receptor potential and decreases the magnitude of the chorda tympani responses to sodium salts relative to NaCl. Na⁺ exit occurs via basolateral Na⁺-K⁺-ATPase. In addition, Na⁺-H⁺ exchanger-1 (NHE-1) is localized in the basolateral membrane of taste cells. These mechanisms may be involved in the adaptation of the neural response and in the cell volume recovery during stimulation of the tongue with hypertonic NaCl solutions (DeSimone and Lyall 2006, 2008).

DISCUSSION

In rodents the Bz-insensitive NaCl chorda tympani response contributes 30–40% of the total NaCl response (Fig. 1A). Several TRPV1t agonists (Katsumata et al. 2008; Lyall et al. 2004, 2005a,b, 2007), including RTX, increase its activity by >100% (Fig. 1, C and D). In this study, we demonstrate that experimental manipulations that increase the phosphorylation state of TRPV1t sensitize the channel to produce a further increase in the RTX-induced enhancement of the NaCl neural response. These observations are not only important in understanding the regulation of TRPV1t but may also have a significant relevance to human salt taste. Unlike the case with rodents, the Bz-sensitive salt taste receptor (presumably ENaC) plays a minor role in human salt taste perception. The predominant component of human salt taste is amiloride- and Bz-insensitive (Feldman et al. 2003; Ossebaard and Smith 1995). Thus the identification of the amiloride- and Bz-insensitive salt taste receptor(s), their natural and synthetic agonists, and the physiological mechanisms that regulate salt taste transduction are necessary steps in the discovery of safe and effective salt taste enhancers. The salt taste enhancers can increase the salt taste sensitivity at low Na⁺ concentrations and may be helpful clinically to lower salt intake and in decreasing the incidence of salt-sensitive hypertension. Therefore discussion of the results obtained in this study will be focused on the importance of different TRPV1t agonists and antagonists in modulating the Bz-insensitive NaCl chorda tympani responses.

Desensitization of the Bz-insensitive NaCl chorda tympani response is a function of RTX concentration

Consistent with previous studies with TRPV1t modulators (Katsumata et al. 2008; Lyall et al. 2004, 2005a,b, 2007), in the nominal absence of external Ca²⁺, the tonic Bz-insensitive NaCl chorda tympani responses demonstrated no spontaneous decrease in their magnitude over time in the absence and presence of RTX (Figs. 1A, 2, A–C, 3A, and Supplemental Figs. S2, A and B, and S3, A and B). Also, no diminution in Bz-insensitive NaCl chorda tympani responses was observed during repeated applications of the same RTX concentration (Figs. 2C, 3A, and Supplemental Fig. S3C). This indicates that TRPV1t does not demonstrate tachyphylaxis when stimulated with RTX, suggesting that in the absence of external Ca²⁺ both TRPV1t and TRPV1 behave in a similar manner (Koplas et al. 1997; Novakova-Tousova et al. 2007; Plant et al. 2007). The absence of time-dependent desensitization in the neural responses and tachyphylaxis with repeated agonist stimulations are essential properties of TRPV1t, consistent with its role as a candidate mammalian salt taste receptor.

The magnitude of the Bz-insensitive NaCl chorda tympani response increased in a dose-dependent manner when the tongues were stimulated with N + Bz solutions containing RTX between 0.25 × 10⁻⁶ and 1 × 10⁻⁶ M and was decreased above this concentration (Figs. 1A, 3, B and C, 4A, and 5A). Similarly, other modulators of TRPV1t—capsaicin, cetylpyridnium chloride, elevated temperature, ethanol, nicotine, and Maillard peptides—reacted with different sugar moieties (Katsumata et al. 2008; Lyall et al. 2004, 2005a,b, 2007) produced enhancement of the Bz-insensitive NaCl chorda tympani responses within a narrow concentration range. This indicates that TRPV1t agonists can only be used to increase salt taste sensitivity within a narrow concentration range. At high concentrations TRPV1t agonists decrease salt taste sensitivity.

In our studies the desensitization of the Bz-insensitive NaCl chorda tympani responses at RTX concentrations >1 × 10⁻⁶ M was attenuated by chelating TRC [Ca²⁺]_i (Fig. 3C), inhibiting calcineurin (Fig. 4A), or by activating PKCɛ (Fig. 5A). This suggests that at high RTX concentrations, desensitization of the Bz-insensitive NaCl chorda tympani responses is related to a decrease in the phosphorylation state of TRPV1t. We hypothesize that a decrease in the phosphorylation state of TRPV1t is also related to the desensitization of the Bz-insensitive NaCl chorda tympani responses at high concentrations of capsaicin, cetylpyridnium chloride, elevated temperature, ethanol, nicotine, and Maillard peptides reacted with different sugar moieties (Katsumata et al. 2008; Lyall et al. 2004, 2005a,b, 2007). Thus increasing the phosphorylation state of TRPV1t can serve as a possible means to extend the range over which TRPV1t modulators increase salt taste sensitivity.

Modulation of the Bz-insensitive NaCl chorda tympani responses by [Ca²⁺]_i

TRPV1t is a nonselective cation channel that is permeable not only to Na⁺, K⁺, NH₄⁺ but also to Ca²⁺. In our studies, the same concentrations of RTX and SB-366791 that enhance and inhibit the Bz-insensitive NaCl chorda tympani responses, respectively, also modulate chorda tympani responses to CaCl₂ (Fig. 2, A–C and E). TRPV1t in TRCs is constitutively active. When Ca²⁺ is present in the salt stimulus, entry of Ca²⁺ into TRCs via TRPV1t not only elicits a chorda tympani response to CaCl₂ (Fig. 2, B, C, and E) but it also desensitizes Bz-insensitive NaCl chorda tympani responses in the presence of RTX (Figs. 2, C and D and 3, A and B). Consistent with this, a recent study suggested that a TRP channel, likely TRPV1 or TRPV1t, contributes to the constitutive Ca²⁺ influx in isolated mouse TRCs (Hacker and Medler 2008).

In our studies, an increase in resting TRC [Ca²⁺]_i suppressed and a decrease in [Ca²⁺]_i enhanced the sensitivity of TRPV1t to RTX stimulation (Fig. 3, B and C). Importantly, inhibition of the chorda tympani response at RTX concentrations >1 × 10⁻⁶ M was itself largely suppressed when [Ca²⁺]_i was decreased. The above-cited results are consistent with the observations that changes in [Ca²⁺]_i modulate capsaicin- and heat-induced currents via TRPV1 (Vellani et al. 2001). These results support the conclusion that both TRPV1 and TRPV1t are sensitized by lowering [Ca²⁺]_i and are desensitized by increasing [Ca²⁺]_i to capsaicin or RTX stimulation.

However, in our experiments in which the tongue was stimulated with NaCl solutions without added CaCl₂, RTX >1 × 10⁻⁶ M still desensitized the Bz-insensitive NaCl chorda tympani responses. Under these conditions, an increase in TRC [Ca²⁺]_i may occur via voltage-gated Ca²⁺ channels or store-operated Ca²⁺ channels located in the basolateral membrane of TRCs (Pérez et al. 2003). Alternately, the increase in TRC [Ca²⁺]_i could occur via its release from intracellular stores.

Modulation of the Bz-insensitive NaCl chorda tympani responses by phosporylation/dephosphorylation

It is suggested that an increase in [Ca²⁺]_i phosphorylates TRPV1 by CAMK II and its dephosphorylation by calcineurin leads to desensitization of the channel (Jung et al. 2004; Yao et al. 2005). A decrease in [Ca²⁺]_i inhibits calcineurin activity, decreasing the dephosphorylation of the channel. Thus an increase in the channel phosphorylation is the cause of the enhanced membrane currents gated by TRPV1t agonists (Vellani et al. 2001). In support of this hypothesis, cyclosporin A and FK-506 (Fig. 4), specific blockers of calcineurin, produced effects that were similar, but not identical, to those observed with loading TRCs in vivo with BAPTA (Fig. 3C). It is likely that under our experimental conditions cyclosporin A or FK-506 does not inhibit calcineurin activity by 100%, as may be expected after loading the cells with BAPTA. An increase in [Ca²⁺]_i enhances CaMK II activity, increasing the phosphorylation of the TRPV1. However, in our studies blocking CaMK II by KN-93, even at high concentrations, produced no significant effects on the Bz-insensitive NaCl chorda tympani responses in the absence and presence of RTX. These data suggest that in TRCs, TRPV1t activity is most likely not regulated by phosphorylation of the channel protein by CaMK II. The phosphorylation studies were done in lingual epithelium containing fungiform taste buds. This raises the possibility that the phosphorylation of TRPV1/TRPV1t could be occurring in cells other than the taste cells. In our studies, drugs that modulate the activity of PKC and calcineurin alter chorda tympani responses to N + Bz + RTX and directly alter unilateral apical Na⁺ fluxes in taste cell (Fig. 6A). These results suggests that phosphorylation of TRPV1/TRPV1t also occurs in taste cells.

Modulation of TRPV1t activity by PKCɛ

In our studies activation of PKC by PMA did not affect the constitutive activity of the Bz-insensitive NaCl chorda tympani response but enhanced the responses in the presence of RTX (Fig. 5A). The observations that the inactive phorbol ester, 4α-phorbol-12,13-didecanoate did not have any effect on the Bz-insensitive NaCl chorda tympani response in the absence or presence of RTX (data not shown), and that R031-8220, a specific PKC blocker, inhibited the effects of PMA (Fig. 5C), indicate that PMA effects are due to PKC activation. These results suggest that either a direct phosphorylation of the TRPV1t or of a key regulator of the TRPV1t by PKC modulates the activation threshold of the channel by RTX. It is further suggested that PMA potentiates currents by directly phosphorylating TRPV1 by activating PKC. The phosphorylation of C-terminal S800 of TRPV1 by PKC seems to be a necessary step for the modulation of TRPV1 function. However, phosphorylation of S502 or T704 is additionally required for the maximum activation of TRPV1 by PMA (Bhave et al. 2003; Mandadi et al. 2006; Numazaki et al. 2002). In our studies, PMA enhanced Bz-insensitive NaCl chorda tympani responses in the presence of RTX without a shift in the RTX dose–response curve (Fig. 5A). In contrast, in mammalian cells expressing rat TRPV1, PMA treatment led to a leftward shift in the capsaicin dose–response curve (Bhave et al. 2003). It is likely that these differences between the two receptors may be explained by the fact that, unlike TRPV1, TRPV1t is constitutively active at resting TRC membrane potential and normal temperature in the oral cavity. In one study (Lee et al. 2005) PMA-treated HEK293 cells, transfected with TRPV1, were found to be permeable to Ca²⁺ in the absence of TRPV1 agonists. It is likely that PKC-dependent phosphorylation of the channel might decrease the temperature threshold of TRPV1. Under these conditions, it is possible that the normal body temperature could act as a primary stimulus of TRPV1 or TRPV1t.

In TRCs the role of PKCɛ is suggested by the observations that pseudosubstrate inhibitor peptide, a specific PKCɛ blocker, inhibited the Bz-insensitive responses in the presence of RTX (Fig. 5D). In addition PKCɛ was demonstrated to be present in the anterior lingual epithelium containing fungiform papillae by Western blotting (Fig. 5F) and in cDNA made from pure fungiform taste buds (Fig. 5E). A decrease in [Ca²⁺]_i inhibits the Ca²⁺-sensitive PKC isoforms (Tanaka and Nishizuka 1994). In addition, following BAPTA loading, inhibition of calcineurin and the subsequent decrease in dephosphorylation of the channel protein is responsible for maintaining its high activity. PKCɛ, which mediates the sensitization of the heat-sensitive currents in dorsal root ganglion neurons, is not sensitive to Ca²⁺ at normal intracellular levels of Ca²⁺ (Amadesi et al. 2006; Liu and Simon 1996). This may be the reason that ionomycin-induced increase in TRC [Ca²⁺]_i did not alter the constitutive activity of TRPV1t (Fig. 3A).

In our studies, 8-CPT-cAMP had no effect on the Bz-insensitive NaCl chorda tympani response in the absence or presence of RTX (data not shown). However, in the same experiments, cAMP treatment increased the chorda tympani response to control 0.3 M NaCl stimulations (see Supplemental Fig. S3C), which is consistent with the observations that cAMP enhances NaCl responses by activating ENaC (DeSimone and Lyall 2008). Although in some studies PKA simulation has been shown to sensitize TRPV1 (Bhave et al. 2002; Distler et al. 2003; Mohapatra and Nau 2003; Rathee et al. 2002), in other studies PKA activation did not result in an increase in TRPV1 currents (Lee et al. 2000; Plant et al. 2007). Our studies suggest that in TRCs TRPV1t is constitutively active and phosphorylation of the channel by PKA does not further modulate the channel activity.

The effects of various drugs on the intracellular effectors and intracellular targets that modulate TRPV1t activity in TRCs are summarized in Table 2. In summary, our results suggest that TRPV1t activity in TRCs is regulated by phosphorylation–dephosphorylation of the channel protein. An increase in TRC [Ca²⁺]_i activates calcineurin and induces dephosphorylation of TRPV1t and desensitization of the Bz-insensitive NaCl chorda tympani responses to RTX stimulation. On the other hand, a decrease in TRC [Ca²⁺]_i inhibits calcineurin activity, decreasing the dephosphorylation of the channel, and sensitizes the Bz-insensitive NaCl chorda tympani responses to RTX stimulation. Our studies with KN-93 and KN-92 suggest that, unlike TRPV1, in TRCs the TRPV1t activity does not depend on phosphorylation of the channel protein by CaMK II. Also, our studies with 8-CPT-cAMP suggest that in TRCs the TRPV1t activity does not depend on phosphorylation of the channel protein by PKA. In contrast, phosphorylation of TRPV1t via PKC increases the sensitivity of the channel to RTX. PKC effects are most likely not related to changes in TRC [Ca²⁺]_i since PKCɛ the PKC isoform is insensitive to changes in resting Ca²⁺ levels.

A model shown in Fig. 6B summarizes the role of receptor/ion channels and the downstream intracellular effectors involved in salt taste transduction. Salt taste transduction is initiated by sodium entry into a subset of TRCs through ENaC and TRPV1t. Sodium entry depolarizes the receptor cell potential, which results in the release of ATP that, in turn, binds to P2X2/P2X3 receptors on the chorda tympani nerve and excites a subset of nerve fibers to elicit a neural response that is either Bz-sensitive or Bz-insensitive. TRPV1t activity is modulated by RTX, capsaicin, cetylpyridinium chloride, ethanol, nicotine, Maillard reacted peptides, and elevated temperature. The spontaneous activity of TRPV1t and its sensitivity to further stimulation by agonists is regulated by the phosphorylation state of TRPV1t by PKC and calcineurin.

GRANTS

This work was supported by National Institute on Deafness and Other Communication Disorders Grants DC-000122 to J. A. DeSimone and DC-005981 to V. Lyall and a Campbell Soup Company grant to J. A. DeSimone.