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Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298

Address correspondence to Dr. Vijay Lyall, Department of Physiology, Sanger Hall 3-002, 1101 E. Marshall St., Richmond, VA 23298-0551. Fax: (804) 828-7382; E-mail: vlyall{at}hsc.vcu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 HCO₃^-/CO₂ 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 HCO₃^-/CO₂ 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; r² = 0.98). At constant pH_o, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO₃^-/CO₂ decreased resting TRC pH_i, and MK-507 or MK-417 attenuated the decrease in pH_i in TRCs perfused with HCO₃^-/CO₂ 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 NH₄Cl or sodium acetate at constant pH_o. Diethylpyrocarbonate and Zn²⁺, modification reagents for histidine residues in proteins, attenuated the CO₂-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.

Key Words: salt taste • sour taste • intracellular pH • taste mixture interaction • epithelial sodium channel

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During a meal taste receptors cells (TRCs)^* are normally stimulated by mixtures of taste stimuli. Since individual TRCs generally respond to more than one class of stimuli (Gilbertson et al., 2001), their outputs reflect the interplay of intracellular processes that link the various transduction mechanisms in complex nonlinear ways. Such interactions are the source of modifications in perceived intensity and quality arising in the peripheral taste organs. Mixture interactions may give rise to either mixture suppression or enhancement. The rat chorda tympani (CT) nerve responses to Na⁺ salts are suppressed when presented together with potassium benzoate (Sato and Beidler, 1979) or potassium gluconate (Stewart et al., 1996). In contrast, CT responses to NaCl are enhanced in mixtures of mannitol or cellobiose (Lyall et al., 1999). In foods and beverages human TRCs are exposed to pHs ranging from 2.3 to 8.6 (Feldman and Barnett, 1995). Protons are not only the primary sour stimulus source (DeSimone et al., 1995, 2001a; Lyall et al., 2001, 2002), but also may play a regulatory role in the transduction of other taste modalities. Beidler (1954) and Ogawa (1969) reported that rat CT nerve responses to NaCl are inhibited at pHs below 3–4, suggesting that taste mixture interactions occur between sour taste and salt taste modalities. However, at present the physiological mechanisms involved in mixture interactions between sour taste and salt taste modalities have not been delineated.

Amiloride inhibits a significant part of the rat CT responses to NaCl, indicating that in fungiform TRCs amiloride-sensitive epithelial sodium channels (ENaCs) contribute most to the NaCl-induced increase in CT response (Lindemann, 1996, 2001; Herness and Gilbertson, 1999; Kretz et al., 1999; Lin et al., 1999; Stewart et al., 1997). Several studies indicate that protons regulate amiloride-sensitive ENaCs in a variety of epithelial cells (Lyall and Biber, 1995; Lyall et al., 1995; Garty and Palmer, 1997; Chalfant et al., 1999; Zeiske et al., 1999; Konstas et al., 2000; Howard et al., 2001). The , ß, and subunits of cloned ENaC expressed in Xenopus laevis oocytes demonstrated decreased Na⁺ current when intracellular pH (pH_i) was decreased, but not when extracellular pH (pH_o) was decreased (Chalfant et al., 1999). A reduction in pH_i reduced the single-channel open probability of ENaC without altering single channel conductance (Chalfant et al., 1999; Zeiske et al., 1999). We have shown previously that weak organic acids as well as fully dissociated strong acids produce a sustained decrease in TRC pH_i (DeSimone et al., 2001a; Lyall et al., 2001, 2002). Given that acid stimulation decreases pH_i, it is reasonable to hypothesize that intracellular protons also modulate the activity of TRC ENaC over a wide range of pH. If TRC ENaC is also subject to regulation by H⁺ ions, changes in pH could modulate CT responses to NaCl. Intracellular second messengers, cAMP and Ca²⁺, regulate salt taste (Gilbertson et al., 1993; Lin et al., 1999; Alam et al., 2002; Russell et al., 2002) and sour taste (Gilbertson et al., 1993; Lyall et al., 2002) modalities. It is likely that changes in one or more second messengers (Liu and Simon, 2001) during acid stimulation may also participate in mixture interactions and alter salt responses in bimodal TRCs.

In this paper we investigated mixture interactions between sour taste and salt taste modalities by monitoring CT responses to NaCl under lingual voltage-clamp conditions and the temporal changes in intracellular Na⁺ ([Na⁺]_i) and pH_i in polarized fungiform TRCs over a wide range of pHs. Our results demonstrate that changes in pH_i regulate apical Na⁺ influx via amiloride-sensitive ENaCs in TRCs and hence modulate NaCl CT responses.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Vivo Studies
CT nerve recordings.
Female Sprague-Dawley rats (150–200 g) were anesthetized by intraperitoneal injection of pentobarbital (60 mg/Kg) and supplemental pentobarbital (20 mg/Kg) was administered as necessary to maintain surgical anesthesia. Body temperatures were maintained at 36–37°C with a circulating water heating pad. The left CT nerve was exposed laterally as it exited the tympanic bulla (Ye et al., 1993, 1994; DeSimone et al., 1995; Stewart et al., 1998) and placed onto a 32G platinum/iridium wire electrode. An indifferent electrode was placed in nearby tissue. Neural responses were differentially amplified with a custom built, optically coupled isolation amplifier. For display, responses were filtered using a band pass filter with cutoff frequencies of 40 Hz to 3 KHz and fed to an oscilloscope. Responses were then full-wave rectified and integrated with a time constant of 1 s. Integrated neural responses and current and voltage records were recorded on a Soltec (model 3314) chart recorder and also captured on disk using Labview software and analyzed off-line (DeSimone et al., 2001b; Lyall et al., 2001, 2002). Stimulus solutions 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 chamber was fitted with separate Ag-AgCl electrodes for measurement of current and potential. These electrodes served as inputs to a voltage-current clamp amplifier that permitted the recording of neural responses with the chemically stimulated receptive field under zero current-clamp (0CC) or voltage-clamp (Ye et al., 1993, 1994). The clamp-voltages were referenced to the mucosal side of the tongue. The voltage sensitivity index (VSI) was calculated as the difference between the CT response at -60 and 60 mV (CT_{-60 mV}-CT _{60 mV}) relative to 0CC voltages (Ye et al., 1993, 1994). The anterior lingual surface was stimulated with a rinse solution containing KCl and with a salt stimulus solution containing KCl + NaCl (Table I). Amiloride (100 µM) or benzamil (5 µM) was used to block Na⁺ ion entry via the apical Na⁺ channels (Ye et al., 1993, 1994; Stewart et al., 1998).

Data Analysis
The numerical value of an integrated CT response was obtained in the quasi-steady-state part of the response as the area under the integrated CT response curve for a time interval of 1 min measured from the end of a typical 2 min stimulation period (Lyall et al., 1999, 2001, 2002; DeSimone et al., 2001a). The changes in the area under the integrated NaCl CT response curves under different conditions were normalized to the responses observed in each animal to 300 mM NH₄Cl (CT_NaCl/CT_NH4Cl) and were expressed as the mean ± SEM of N; where N represents the number of animals in each group; M ± SEM (N). Student's t test was employed to analyze the differences between sets of data.

In Vitro Studies
pH_i measurement.
Rats were anesthetized with isoflurane and killed by cervical dislocation. The tongues were rapidly removed and stored in ice-cold Ringer's solution (R; Table II, set 1). The lingual epithelium was isolated by collagenase treatment (Lyall et al., 1997; Stewart et al., 1998). A small piece of the anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber (Chu et al., 1995) as described before (Lyall et al., 2001, 2002; DeSimone et al., 2001a). The tissue was intermittently perfused with Ringer's solution (R; Table II, set 1) containing 25 µM of the pH-sensitive fluoroprobe BCECF-AM (Molecular Probes) at 4°C for 2 h. Before the experiment was started, the tissue was perfused on both sides with control solution for 15 min. The control solution was Ringer's solution without Na-pyruvate (RC; Table II, set 1). 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 (The Lee Company). The TRCs in the taste bud were visualized from the basolateral side through a 40x objective (ZEISS; 0.9 NA) with a ZEISS Axioskop 2 plus upright fluorescence microscope and imaged with a set up consisting of: a cooled CCD camera (Imago, TILL Photonics; Applied Scientific Instrumentation) attached to an image intensifier (VS4–1845; Videoscope), an epifluorescent light source (TILL Photonics Polychrome IV), a 515 nm dichroic beam splitter (Omega Optical), and a 535 nm emission filter (20 nm band pass; Omega Optical). The cells were alternately excited at 490 and 440 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 FIR (fluorescence intensity ratio; F₄₉₀/F₄₄₀) were analyzed using TILLvisION v3.1 imaging software. Each ROI contained 2–3 receptor cells. Thus, the fluorescence intensity recorded for a ROI represents the mean value from 2–3 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 490 and 440 nm were corrected from images of a taste bud without the dye. The changes in TRC pH_i were calibrated by bilateral perfusion of high K⁺ calibrating solutions containing 10 µM nigericin (CS; Table II, set 5) adjusted to pHs between 6.5 and 8.0 (DeSimone et al., 2001b; Lyall et al., 2001, 2002).

Data analysis.
The changes in TRC pH_i were expressed as the mean ± SEM of N; where N represents the number of ROIs within the taste bud; M ± SEM (N). In TRCs loaded with sodium green the changes in [Na⁺]_i were expressed relative to the fluorescence intensity (F₄₉₀) under control conditions. The F₄₉₀ under control conditions for each ROI was taken as 100%. For TRCs loaded with SBFI, the relative changes in FIR (F₃₄₀/F₃₈₀) were compared between different ROIs under different conditions. The data were also presented as the mean ± SEM from different tissue preparations. In this case N represented the number of taste buds. Student's t test was employed to analyze the differences between sets of data.

Solutions
The composition of various Ringer's solutions used in the in vitro experiments is given in Table II. In some experiments the apical or basolateral perfusion solutions contained either MK-417 or MK-507 (Merck), membrane-permeable inhibitors of carbonic anhydrases (Fanous et al., 1999; Lyall et al., 2001). The drugs were used at lower concentrations when applied from the basolateral side.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Vivo Studies
Effect of external pH (pH_o) on NaCl CT responses
Under physiological conditions TRCs are exposed to wide variations in pH (Feldman and Barnett, 1995). We investigated if changes in pH alter neural responses to NaCl stimulation. The CT responses were monitored while the tongue was stimulated with 100 mM NaCl solutions adjusted to pH 2.0, 7.0, and 10.3. In Fig. 1 the lingual surface was perfused with a rinse solution R7 (pH 7; Table I, set 1) and the CT activity was taken as baseline. Perfusing the tongue with NaCl stimulus solution S7 (pH 7; Table I, set 1) increased CT activity (upward deflection in the CT trace; a and b). Replacing S7 with a similar NaCl stimulus solution S2, containing 10 mM HCl (pH 2.0; Table I, set 1) decreased the CT response (b and c). Upon reperfusing S7 the CT activity increased to its original steady-state level (c and d) and rinsing with R7 returned the CT activity to baseline (d and e). In the next step, replacing R7 with a similar rinse solution R2, containing 10 mM HCl (pH 2.0, Table I, set 1) reversibly increased the CT activity (e–g). This increase in CT response is due to the presence of HCl in the rinse solution, R2 (DeSimone et al., 1995; Stewart et al., 1998; Lyall et al., 2001, 2002). The NaCl CT responses were normalized to the response observed in each animal to 300 mM NH₄Cl (CT_NaCl/CT_NH4Cl). In five experiments, replacing S7 with S2 decreased the magnitude of the NaCl CT response from 0.71 ± 0.03 to 0.60 ± 0.04 ( = -0.11 ± 0.01), a decrease of 16.1 ± 1.9% (P < 0.01) with respect to its activity in S7. In the same experiments replacing R7 with R2 increased CT activity by 0.17 ± 0.01 (n = 5). If there was no interaction between HCl and NaCl at the level of TRCs, one would predict that replacing S7 with S2 would produce the same magnitude of increase in the CT response as observed by replacing R7 with R2. However, under these conditions there was an overall decrease in the magnitude of the CT response (b and c). In additional experiments, the magnitude of the CT response to S10.3 (pH 10.3; Table I, set 1) was also decreased upon switching from S10.3 to S7 or by replacing S10.3 with S2 (unpublished data). However, under these conditions, it is difficult to estimate the actual pH-induced inhibition in the CT response because at low pH there is an upward shift in the baseline.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Effect of pHo on the NaCl CT responses. The integrated CT responses were recorded under open-circuit conditions (i.e., under zero current clamp; 0CC). The tongue was initially perfused with rinse (R7) solution containing 10 mM KCl + 200 mM mannitol and then with the NaCl stimulus solution (S7) containing 10 mM KCl + 100 mM NaCl (a and b). Both R7 and S7 were buffered to pH 7.0 with HEPES. In the second step, S7 was replaced with the NaCl stimulus solution S2 (b and c). In the final step, R7 was replaced with the rinse solution R2 (e and f). Both R2 and S2 were adjusted to pH 2.0 with 10 mM HCl (without HEPES).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Effect of pHo on the voltage sensitivity of the NaCl CT responses. The lingual surface was perfused with rinse (R) solutions containing 10 mM KCl + 200 mM mannitol and stimulus (S) solutions containing 10 mM KCl + 100 mM NaCl adjusted to pH 2.0 (R2 and S2; left), 7.0 (R7 and S7; middle), and 10.3 (R10.3 and S10.3; right). Integrated CT responses were recorded under open-circuit conditions (i.e., under zero current clamp; 0CC), at -60 mV, and at 60 mV lingual voltage-clamp. The NaCl CT responses were voltage-sensitive. The CT responses were enhanced at -60 mV and suppressed at 60 mV voltage-clamp relative to 0CC. Also shown are the CT responses to 0.3 M NH4Cl test pulses before and after the experiment.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Effect of benzamil on the pHo-induced changes in NaCl CT responses. The lingual surface was perfused with rinse (R) solutions (10 mM KCl + 200 mM mannitol) and stimulus (S) solutions (10 mM KCl + 100 mM NaCl) adjusted to pH 2.0, 7.0, and 10.3 in the presence and absence of 5 µM benzamil. Integrated CT responses were recorded under 0CC (1), at -60 mV (2), and at 60 mV (3) lingual voltage clamp. The area under the CT response curve was taken as the magnitude of the CT response. In each animal the NaCl CT responses were normalized to the CT response obtained with 300 mM NH4Cl (CTNaCl/CTNH4Cl). The values are presented as M ± SEM of N (see text for details).

Also shown in Fig. 2 (right) are the CT responses when both the rinse and the stimulus solutions were applied at pH 10.3. Replacing rinse solution R7 (pH 7.0; Table I, set 1) with rinse solution R10.3 (pH 10.3; Table I, set 1) did not alter the baseline CT response. Subsequently, perfusing the tongue with the NaCl stimulus solution S10.3 (pH 10.3; Table I, set 1) increased the CT response above baseline under 0CC. However, the magnitude of the CT response (S10.3-R10.3 at 0CC) was increased by 50% relative to CT response at pH 7.0 (S7-R7 at 0CC). At pH 10.3, the NaCl CT response demonstrated greater voltage-sensitivity relative to pH 7.0 (compare middle panel). The data from several animals are also summarized in Fig. 3. At pH 10.3 the mean normalized CT response to 100 mM NaCl at 0CC was 0.82 ± 0.06 (N = 5; hatched bar 1) and was significantly greater than the corresponding value at pH 7.0 (0.68 ± 0.02; P < 0.01; unpaired). In four animals the CT responses were also recorded under lingual voltage-clamp conditions. The mean normalized CT response (0.80 ± 0.07) increased to 1.16 ± 0.08 at -60 mV (hatched bar 2) and decreased to 0.51 ± 0.04 at 60 mV (hatched bar 3). Thus increasing the pH from 7.0 to 10.3 increased mean NaCl CT response by 20.6% and increased the VSI from 0.44 ± 0.02 (N = 5) to 0.65 ± 0.05 (N = 4), an increase of 47.7%. When the NaCl CT responses were monitored over the entire range of pH between 2.0 and 10.3, the normalized NaCl CT responses demonstrated a linear relationship with pH_o (Fig. 4, filled circles; slope = 0.06; r² = 0.99).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Relationship between pHo versus NaCl CT responses and between pHo and TRC pHi. The area under the CT response curve was taken as the magnitude of the CT response. The NaCl CT responses were normalized to the response observed in each animal to 300 mM NH4Cl (CTNaCl/CTNH4Cl). The values are presented as M ± SEM of N, where N represents the number of observations at each pHo (in each case N was between 2 and 9). The NaCl CT responses were linearly related to pHo with a slope of 0.06 (filled circles; r2 = 0.99). The changes in TRC pHi were also linearly related to pHo with a slope of 0.035 (open circles; intercept = 6.92; r2 = 0.95). TRC pHi values are presented as mean ± SEM of N, where N represents the number of observations at each pHo (in each case N was between 4 and 6).

As described in detail previously (Ye et al., 1993, 1994), we tested the responses to two reference stimuli, namely, 0.3 M NaCl and NH₄Cl before and after the experiment. The data from an experiment were accepted if the CT responses to the above two reference stimuli did not differ by >10% before and after the experiment. In the data shown in Fig. 2, the responses to 0.3 M NH₄Cl before and after stimulating the tongue repeatedly with NaCl solutions buffered to pH 2, 7, and 10.3 did not differ by >3%.

Effect of Benzamil on NaCl CT Responses
Consistent with our previous studies (Stewart et al., 1998), the addition of 5 µM benzamil to R7 (pH 7.0) did not alter the baseline CT activity. Subsequent perfusion of S7 + benzamil produced an increase in NaCl CT response. However, the magnitude of the response in the presence of benzamil was significantly smaller as compared with control. The effect of benzamil on several animals is also summarized in Fig. 3. At pH 7.0, the mean normalized CT response to 100 mM NaCl at 0CC in the presence of benzamil was 0.32 ± 0.03 (N = 4; black bar 1). The CT response increased to 0.37 ± 0.06 at -60 mV (black bar 2) and decreased to 0.22 ± 0.02 at +60 mV (P > 0.05; black bar 3). The VSI was 0.15 ± 0.04. Thus at pH 7.0, benzamil decreased the mean normalized NaCl CT response by 53.4% and decreased the VSI by 67.4% relative to control. These effects of benzamil are consistent with its known role as a blocker of ENaCs (Lyall et al., 1993, 1995; Lyall and Biber, 1995; Stewart et al., 1998).

Data shown in Fig. 5 demonstrate that at pH 7.0, the benzamil-induced inhibition of NaCl CT response (left) are comparable in magnitude to the inhibition observed by decreasing the pH of the NaCl stimulus solution from 7.0 to 2.0 in the absence of benzamil in the same animal (right). The mean normalized CT responses at pH 2.0 under 0CC (0.31 ± 0.02) were not significantly different from CT responses at pH 7.0 in the presence of benzamil (0.32 ± 0.03; P > 0.05; N = 4). Similarly, the mean VSI at pH 2.0 (0.16 ± 0.03) was not significantly different from its corresponding value (0.14 ± 0.04) at pH 7.0 in the presence of benzamil. Data summarized in Fig. 3 also show that in the continuous presence of benzamil, applying the stimulus at pH 2.0 (switching from R2 to S2) did not demonstrate any further suppression of the NaCl CT response. Furthermore, in the presence of benzamil, no significant changes were observed in the VSI at pH 2.0. In additional experiments, 100 µM amiloride inhibited the NaCl CT response at pH 7.0 and in the continuous presence of amiloride, applying the stimulus at pH 2.0, did not produce further suppression in the NaCl CT response (unpublished data). These results indicate that a decrease in pH inhibits most of the amiloride- and benzamil-sensitive component of the NaCl CT response. The data further indicate that the amiloride-insensitive component of the CT response is pH-insensitive.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Comparative effects of benzamil and pHo on NaCl CT responses. The lingual surface was perfused with rinse (R) solution and the stimulus (S) solution at pH 7.0 containing 5 µM benzamil (left) or with both rinse (R2) and stimulus (S2) solutions adjusted to pH 2.0 without benzamil (right) in the same animal. Integrated CT responses were recorded under 0CC, at -60 mV, and at 60 mV lingual voltage-clamp. The area under the CT response curve was taken as the magnitude of the CT response.

Effect of Membrane Permeable Weak Acids on NaCl CT Responses
To investigate if changes in NaCl CT responses are due to the changes in TRC pH_i, further experiments were done in which both the rinse and stimulus solutions were maintained at constant pH_o by buffering the solutions with weak organic acids, acetic acid (AA; Table I, sets 2 and 3) or CO₂ (Table I, set 4) and their corresponding potassium salts, potassium acetate (KA) or KHCO₃ (Lyall et al., 2001).

Studies with Acetic Acid
Data summarized in Fig. 6 A show that switching from the rinse solution HRH6.1 (pH 6.1; Table I, set 2) to the NaCl stimulus solution HSH6.1 (pH 6.1; Table I, set 2) increased the NaCl CT response. For this experiment the area under the NaCl CT response curve for a time interval of 1 min measured from the end of the 2-min stimulation period was taken as 100%. In the second step, switching from HRH6.1 to HRAA6.1 (pH 6.1; Table I, set 2), increased the CT response. This increase in CT response is presumably due to the passive influx of membrane-permeable undissociated AA in the rinse solution across the apical membranes of TRCs (Lyall et al., 2001). Subsequently, stimulating with the NaCl stimulus solution HSAA6.1 (pH 6.1; Table I, set 2) increased the CT response further. This increase in CT response is due to the presence of NaCl in HSAA6.1. However, the magnitude of the NaCl CT response in the presence of AA (HSAA6.1-HRAA6.1) was 40% less than the corresponding NaCl CT response in the presence of HEPES (HSH6.1-HRH6.1). Some experiments were also performed at a lower AA concentration. In this case both the rinse and stimulus solutions contained 175 mM KA and the solutions were buffered to pH 6.1 with either 10 mM HEPES (LRH6.1 and LSH6.1) or 10 mM AA (LRAA6.1 and LSAA6.1) (Table I, set 3). The data from several animals are summarized in Fig. 6 B. In the presence of 175 mM KA + 10 mM AA (pH 6.1) the mean normalized NaCl CT responses decreased from 0.71 ± 0.02 (N = 9) to 0.62 ± 0.06 (N = 3), an inhibition of 12.7 ± 4.0% of control. However, in the presence of 525 mM KA + 30 mM AA (pH 6.1) the NaCl CT response decreased to 0.45 ± 0.03, an inhibition of 36.3 ± 3.8% of control (P < 0.005; N = 5; paired). These results indicate that AA induces a dose-dependent inhibition of NaCl CT responses.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Effect of intracellular acid loading with acetic acid on NaCl CT responses. (A) CT responses were recorded while the lingual surface was perfused with rinse (R) solutions containing 525 mM KA + 200 mM mannitol and with stimulus (S) solutions containing 525 mM KA + 100 mM NaCl. Both rinse (HRH) and stimulus (HSH) solutions contained, in addition, 30 mM HEPES and the pH was adjusted to 6.1 with KOH. In the second part of the experiment, both the rinse (HRAA) and the stimulus (HSAA) solutions were adjusted to pH 6.1 by adding 30 mM AA. (B) Summarizes data from several animals. The area under the CT response curve was taken as the magnitude of the CT response. In each animal the NaCl CT responses were normalized to the CT response obtained with 300 mM NH4Cl (CTNaCl/CTNH4Cl). The values are presented as M ± SEM of N. The value of N is given in parentheses. Asterisk indicates P < 0.005 (paired).

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Effect of intracellular acid loading with CO2 on NaCl CT responses. (A) CT responses were recorded while the lingual surface was perfused with rinse (R) solutions containing 72 mM KCl + 200 mM mannitol and with stimulus (S) solutions containing 72 mM KCl + 100 mM NaCl. Both rinse (RH) and stimulus (SH) solutions contained, in addition, 10 mM HEPES and the pH was adjusted to 7.4 with KOH. In the second part of the experiment, both the rinse (RCO2) and the stimulus (SCO2) solutions were buffered to pH 7.4 with 72 mM KHCO3/10% CO2. (B) The lingual surface was treated with 50 mM MK-507 for 15 min, and the CT responses were recorded using the same protocol as in A. (C) Summarizes data from several animals. The area under the CT response curve was taken as the magnitude of the CT response. In each animal the NaCl CT responses were normalized to the CT response obtained with 300 mM NH4Cl (CTNaCl/CTNH4Cl). The values are presented as M ± SEM of N. The value of N is given in parentheses. Asterisk indicates P < 0.01 (unpaired).

Studies with DEPC and Zn²⁺
Histidine residues in the cytoplasmic NH₂ terminus of the -rENaC have been suggested to be potential candidates for its pH-sensitivity (Chalfant et al., 1999). To investigate if changes in pH_i exert their effects by interacting directly with histidine residues in TRC ENaC, we tested the effect of DEPC and Zn²⁺, modification reagents for histidine residues in proteins (Mankelow and Henderson, 2001; Seebungkert and Lynch, 2001), on NaCl CT responses. Fig. 8 A shows that, similar to the data in Fig. 7 A, stimulating the tongue with 100 mM NaCl solution buffered with HCO₃^-/CO₂ (pH 7.4) produced NaCl CT response (SCO₂7.4-RCO₂7.4) that was 40% smaller than the corresponding CT response obtained with stimulating the tongue with NaCl solution buffered with HEPES (SH7.4-RH7.4). After this, we applied 20 mM DEPC (dissolved in the rinse solution RH7.4) topically to the lingual surface for 10 min. DEPC had no effect on the NaCl CT response obtained by stimulating the tongue with NaCl solution buffered with HEPES (SH7.4-RH7.4; Fig. 8 B). However, in the continuous presence of DEPC, stimulating the tongue with NaCl solutions buffered with HCO₃^-/CO₂ did not show any inhibition of NaCl CT response, i.e., the CO₂ response (RCO₂7.4-RH7.4) and the NaCl response (SCO₂7.4-RCO₂7.4) were additive (Fig. 8 B). The effects of DEPC were completely reversible. Perfusing the tongue with RH7.4 without DEPC for 10 min (Fig. 8 C) again produced NaCl CT response in HCO₃^-/CO₂ buffer (SCO₂7.4-RCO₂7.4) that was 40% smaller than the corresponding response (SH7.4- RH7.4) in HEPES buffer. Similar results were obtained with 20 mM ZnCl₂ (unpublished data). The data from several animals are also summarized in Fig. 7 C. In three animals the magnitude of the CT responses obtained by stimulating the tongue with NaCl solutions buffered to pH 7.4 with HEPES were not different (P > 0.05) from corresponding CT responses obtained by stimulating with NaCl solutions buffered with HCO₃^-/CO₂ after DEPC treatment. These results suggest that agents that modify histidine residues in TRC ENaC subunits prevent the CO₂-induced decrease in NaCl CT responses.

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Effect of DEPC on CO2-induced NaCl CT responses. (A) CT responses were recorded while the lingual surface was perfused with rinse (RH) solution containing 72 mM KCl + 200 mM mannitol and with stimulus (SH) solution containing 72 mM KCl + 100 mM NaCl. Both the rinse (RH7.4) and stimulus (SH7.4) solutions contained, in addition, 10 mM HEPES and the pH was adjusted to 7.4 with KOH. In the second part of the experiment, both the rinse (RCO27.4) and the stimulus (SCO27.4) solutions were buffered to pH 7.4 with 72 mM KHCO3/10% CO2. (B) The lingual surface was treated with 20 mM DEPC for 10 min and the CT responses were recorded using the same protocol as in A. (C) The lingual surface was perfused with rinse solution without DEPC (RH7.4) for 10 min and the CT responses were recorded using the same protocol as in A.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Effect of CO2 on the NaCl CT responses without preadaptation. The integrated CT responses were recorded under open-circuit conditions (i.e., under zero current-clamp; 0CC). The tongue was initially perfused with the rinse solution RH7.4 and then with the NaCl stimulus solution SH7.4 (a-b). Both RH7.4 and SH7.4 were buffered to pH 7.4 with HEPES. In the second step, SH7.4 was replaced with the NaCl stimulus solution SCO27.4 (b-c). In the final step, RH7.4 was replaced with RCO27.4 (e and f). Both RCO27.4 and SCO27.4 were buffered to pH 7.4 with HCO3-/CO2.

Effect of Acid Stimulation on TRC pH_i
Studies with HCl
Fig. 10 A shows that in polarized TRCs perfused with control Ringer's solution (RC; pH 7.4; Table II, set 1), decreasing the apical solution pH to 2.0 (RHCl; Table II, set 1) reversibly decreased resting pH_i from 7.46 ± 0.01 to 7.10 ± 0.02 (pH_i = -0.35 ± 0.03; N = 5; P < 0.001, paired) and an increase in the apical solution pH from 7.4 to 10.3 (RTris; Table II, set 1) reversibly increased pH_i (Fig. 10 B) from 7.34 ± 0.01 to 7.48 ± 0.01 (pH_i = 0.14 ± 0.02; N = 6; P < 0.001). Varying the pH_o between 10.3 and 2.0 decreased TRC pH_i from 7.35 ± 0.01 to 7.05 ± 0.01 (N = 6), indicating that during acid taste transduction, changes in TRC pH_i remain within the physiological range. The mean changes in TRC pH_i in several tissues induced by variations in pH_o are also plotted in Fig. 4 (slope = 0.035; r² = 0.95). The data show that the NaCl CT responses are linearly related to changes in TRC pH_i.

View larger version (22K): [in this window] [in a new window]   FIGURE 10. Effect of pHo on TRC pHi. The pHi was monitored while the polarized TRCs were initially perfused on both sides with control Ringer's solution (pH 7.4) and when the apical membrane was perfused with a similar solution at pH 2.0 (A) or with a similar solution at pH 10.3 (B). The pHi values are presented as mean ± SEM of six ROIs within the taste bud.

Studies with CO₂
Fig. 11 shows that in polarized TRCs initially perfused with Ringer's solution buffered to pH 7.4 with HEPES, switching to a similar solution buffered with NaHCO₃/CO₂ (RCO₂; pH 7.4; Table II, set 2) in the apical compartment induced a rapid and reversible decrease in TRC pH_i (a and b) from 7.16 ± 0.01 to 6.85 ± 0.01 (pH_i = 0.31 ± 0.01; N = 6; P < 0.001; paired). In three polarized TRC preparations CO₂ decreased TRC pH_i by 0.26 ± 0.03 pH unit (P < 0.025; paired). In the experiment shown in Fig. 11, perfusing the basolateral side with 10 mM MK-417, a membrane permeable blocker of carbonic anhydrases, increased TRC pH_i (c and d) from 7.11 ± 0.01 to 7.24 ± 0.01 (pH_i = 0.13 ± 0.03; P < 0.025). However, in the continuous presence of MK-417, switching from HEPES-buffered solution (RC) to the HCO₃/CO₂-buffered solution (RCO₂) decreased pH_i (d and e) from 7.24 ± 0.01 to 7.11 ± 0.01 (pH_i = 0.13 ± 0.01; N = 6; P < 0.001). Thus MK-417 inhibited the dissolved CO₂-induced decrease in TRC pH_i by 61.6%. Since in our in vivo studies (compare Fig. 7 B) the drug was applied topically to the lingual surface, we also tested if the apical application of MK-417 also inhibits CO₂-induced decreases in TRC pH_i. In another tissue, switching from HEPES-buffered solution to the HCO₃/CO₂-buffered solution in the apical compartment decreased pH_i from 7.17 ± 0.002 to 6.94 ± 0.002 (pH_i = 0.23 ± 0.002; N = 6; P < 0.001). After this, the apical membrane was perfused with HEPES-buffered solution (pH 7.4) containing 40 mM MK-417 for 15 min. Upon switching to a similar solution buffered with HCO₃/CO₂ (pH 7.4; also containing MK-417) in the apical compartment decreased TRC pH_i from 7.07 ± 0.003 to 6.98 ± 0.001 (pH_i = 0.09 ± 0.003; N = 6; P < 0.001). Thus, in the presence of apical 40 mM MK-417, the dissolved CO₂-induced decrease in TRC pH_i was inhibited by 60%. Similar results were obtained with MK-507 (unpublished data).

View larger version (20K): [in this window] [in a new window]   FIGURE 11. Effect of CO2 on TRC pHi. The pHi was monitored while the polarized TRCs were initially perfused on both sides with control Ringer's solution (pH 7.4) and when the apical membrane was perfused with a similar solution buffered with 72 mM NaHCO3/10% CO2 at pH 7.4 (a and b). In the second part of the figure, the basolateral cell membrane was perfused with control Ringer's solution containing 10 mM MK-417 (c and d). In the continuous presence of basolateral MK-417 the apical solution was once again switched from a HEPES-buffered solution to a similar solution buffered with HCO3-/CO2 at pH 7.4 (d and e). The pHi values are presented as mean ± SEM of six ROIs within the taste bud.

View larger version (32K): [in this window] [in a new window]   FIGURE 12. Effect of acetic acid (AA) on TRC pHi. The pHi was monitored while the polarized TRCs were initially perfused on the basolateral side with control Ringer's solution (pH 7.4) and the apical side was perfused with a solution containing 150 mM NaAc (150 NaAc replacing 150 mM NaCl) buffered to pH 6.3 with MOPS. In the first step, the apical solution was switched to a similar solution buffered to pH 6.3 with AA (a–c). In the second step, the apical solution containing 150 mM NaAc + 600 mM mannitol (Man) was replaced with an isosmotic solution containing 450 mM NaAc (both solutions were buffered to pH 6.3 with AA; d–f). The pHi values are presented as mean ± SEM of five ROIs within the taste bud.

SBFI Loading
Fig. 13 A shows the transmitted image of a fungiform papilla containing a single taste bud mounted in the microscopy chamber. The taste bud was viewed from the basolateral side with a 40x water immersion objective. Fig. 13, B and C, show the image of the same taste bud excited at 340 and 380 nm, respectively. The resulting ratio image (F₃₄₀/F₃₈₀) is shown in Fig. 13 D. Fig. 13 shows that SBFI is specifically taken up by the TRCs within the papilla and is excluded from the surrounding epithelial cells. However, squamous epithelial cells on the papillary periphery also absorb the dye. Measurements of F₃₄₀/F₃₈₀ changes were made exclusively from the dye-loaded TRCs and reflect relative changes in TRC [Na⁺]_i. Similar to SBFI, the single wavelength dye, sodium-green, was also specifically taken up by the TRCs within the papilla (unpublished data). We have demonstrated previously that the pH-sensitive fluoroprobe, BCECF, is also taken up specifically by the TRCs within the fungiform papilla (Lyall et al., 2001, 2002). Therefore, both changes in [Na⁺]_i and pH_i reflect changes within TRCs.

View larger version (76K): [in this window] [in a new window]   FIGURE 13. SBFI loading of TRCs. An isolated piece of rat anterior lingual epithelium containing a single fungiform papilla was mounted in a special microscopy chamber and was perfused with control solution containing SBFI-AM. The taste bud was viewed from the basolateral side with a 40x magnification. Bar, 10 µm. The figure shows the transmitted image of the taste bud (A), and the fluorescence image of the same taste bud excited at 340 nm (B) and excited at 380 nm (C). The fluorescence intensity ratio image, F340/F380, is shown in D.

View larger version (29K): [in this window] [in a new window]   FIGURE 14. Effect of apical Na+ and amiloride on TRC [Na+]i. (A) The relative changes in [Na+]i were monitored in polarized TRCs loaded with SBFI while they were initially perfused on both sides with control Ringer's solution (pH 7.4) and when the apical membrane was perfused with a Na+-free solution (150 mM NaCl was replaced with an equivalent amount of Tris-HCl). The relative changes in [Na+]i are presented as mean ± SEM changes in FIR (F340/F380) of three ROIs within the taste bud. (B) The relative changes in [Na+]i were monitored in polarized TRCs loaded with sodium green while they were initially perfused on both sides with control Ringer's solution (pH 7.4) and when the apical membrane was perfused with (i) control Ringer's solution containing 100 µM amiloride (a and b) and (ii) a Na+-free solution (150 mM NaCl was replaced with an equivalent amount of NMDG-HCl) also containing 100 µM amiloride (b and c). The relative changes in [Na+]i are presented as mean ± SEM of the percentage change in fluorescence intensity (F490) of four ROIs within the taste bud.

View larger version (23K): [in this window] [in a new window]   FIGURE 20. Effect of DEPC and pH on Na+ influx across the apical membrane of TRCs. In polarized TRCs loaded with sodium green the Na+ influx across the apical cell membranes was measured as relative changes in [Na+]i during a unilateral step change in apical Na+ concentration from 150 mM to zero. (A) The Na+ influx was measured using 0 and 150 mM Na+-solutions adjusted to pH 7.4 (a–c) and to pH 4.4 (d–f). In the second part of the experiment (B) the basolateral membrane was perfused with Ringer's solution containing 5 mM DEPC (pH 7.4) for 10 min and apical Na+ influx was estimated again using 0 and 150 mM Na+-solutions adjusted to pH 4.4 (g–i). The relative changes in [Na+]i are presented as percentage mean ± SEM changes in fluorescence at 490 nm (F490) of six ROIs within the taste bud.

View larger version (16K): [in this window] [in a new window]   FIGURE 15. Effect of pHo on TRC [Na+]i. The relative changes in [Na+]i were monitored in polarized TRCs loaded with sodium green while they were initially perfused on both sides with control Ringer's solution (pH 7.4) and when the apical membrane was perfused with a similar solution at pH 3.0 (1 mM HCl). The relative changes in [Na+]i are presented as mean ± SEM of the percentage change in fluorescence intensity (F490) of four ROIs within the taste bud.

Effect of Intracellular Acid Loading on TRC [Na⁺]_i
Studies with basolateral NH₄Cl pulses
Fig. 16 shows