INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Entry of acids into taste receptor cells (TRCs) as neutral molecules (acetic acid, citric acid, and dissolved CO₂) or as H⁺ ions (HCl) produces a sustained decrease in intracellular pH (pH_i) (DeSimone et al. 2001; Lyall et al. 2001). Relative decreases in TRC pH_i evoked by all acid types were correlated with their relative chorda tympani (CT) responses. Moreover, a pharmacological blocker of the CO₂-evoked decrease in TRC pH_i also attenuated the CT responses to CO₂. Overall, the data indicate that a decrease in TRC pH_i serves as the proximate stimulus for sour taste (Lyall et al. 2001). While it is clear that undissociated weak acids and dissolved CO₂ can cross the apical membrane of TRCs by passive diffusion as neutral molecules, the entry pathway for H⁺ ions, the form of the stimulus presented by mineral acids, remains obscure. Given that mineral acids are stimuli only at pH values below 4 (Frank et al. 1983), we hypothesize that, in TRCs involved in sour taste, intracellular second messengers either modulate the apical H⁺ entry pathway or activate pH compensatory mechanisms, or both. A second-messenger-mediated increase in apical H⁺ entry due to a strong acid, such as HCl, should therefore produce a greater decrease in TRC pH_i and ultimately a larger CT response to HCl. In contrast, activation of pH compensatory mechanisms in TRC membranes should produce a transient decrease in pH_i followed by spontaneous recovery. Activation of pH recovery mechanisms should therefore produce a rapid adaptation in the CT response to HCl. By direct measurement of pH_i in polarized TRCs and CT recordings, we demonstrate that the apical H⁺ entry pathway is modulated by cAMP and that both cAMP and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) regulate pH compensatory mechanisms in TRC membranes. Thus for strong acids both the proximate event in sour taste transduction, a decrease in TRC pH_i, and subsequent events leading to sensory adaptation, are modulated by intracellular second messengers.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

pH_i measurement

Rats were anesthetized with isoflurane and killed by cervical dislocation. The tongues were rapidly removed and stored in ice-cold Ringer solution, containing (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 Na-pyruvate, 10 glucose, and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) pH 7.4. 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 as described before (Lyall et al. 2001). The tissue was intermittently perfused with Ringer solution containing 25 µM of the pH-sensitive fluoroprobe BCECF-AM (Molecular Probes, Eugene, OR) 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 solution without Na-pyruvate. The tissue was perfused at the rate of 1 ml/min. The TRCs in the taste bud were visualized from the basolateral side through a ×40 objective (Zeiss; 0.9 NA) with a Zeiss Axioskop microscope and imaged with a setup consisting of a cooled charge-coupled device (CCD) camera (Imago, TILL Photonics, Applied Scientific Instrumentation, Eugene, OR) attached to an image intensifier (VS4-1845 Videoscope, Washington, DC), 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 (2-3 µm² diam) were chosen in which the changes in fluorescence intensity ratio (FIR; F₄₉₀/F₄₄₀) were analyzed using TILLvisION v3.1 imaging software. 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 between pH 6.5 and 8.0 containing 10 µM nigericin as described before (Lyall et al. 2001). All experiments were done at room temperature (22 ± 1°C).

The apical membrane of polarized TRCs was stimulated with a control solution containing 1 mM (pH_o 3), 10 mM (pH_o 2), or 30 mM (pH_o 1.5) HCl without HEPES. The rate of spontaneous pH_i recovery was monitored using a variant of the standard NH₄Cl prepulse technique (Roos and Boron 1981) in which the TRC basolateral membranes were exposed to a 15-mM NH₄Cl pulse (Lyall et al. 1997; Stewart et al. 1998). In some experiments the basolateral membrane was exposed to a control solution containing, in addition, 10 mM CaCl₂, 50 µM 5-(N-methyl-N-isobutyl)-amiloride (MIA), 250 µM 8-cholorophenylthio (CPT)-cAMP, or 20 µM ionomycin (Sigma, St. Louis, MO).

CT nerve recordings

Female Sprague-Dawley rats (150-200 g) were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg), and supplemental pentobarbital (60 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 (DeSimone et al. 1995; Stewart et al. 1998; Ye et al. 1993) 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 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 chart recorder and also captured on disk using Labview software and analyzed off-line (Lyall et al. 2001). 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 current (0 CC) or voltage clamp (Ye et al. 1993, 1994). The clamp voltages were referenced to the mucosal side of the tongue. The anterior lingual surface was stimulated with a rinse solution (10 mM KCl) and with 1-, 10-, 20-, or 30-mM HCl solutions. Amiloride (100 µM) was used to block H⁺ ion entry via the apical Na⁺ channels (Gilbertson et al. 1993). In some experiments 20 mM 8-CPT-cAMP or 150 µM ionomycin dissolved in dimethyl sulfoxide (DMSO) were applied topically to the lingual surface for 1 h. In experiments using ionomycin the rinse solution and HCl solution contained 10 mM CaCl₂. DMSO alone had no effect on CT responses as previously shown (Lyall et al. 1999). The numerical value of an integrated CT response was obtained as the area under the integrated CT response curve (Lyall et al. 1999).

In isolated lingual preparations, 8-CPT-cAMP and ionomycin were applied at micromolar concentrations on the basolateral side in vitro and induced their effects within minutes. However, in the in vivo experiments these drugs were necessarily applied topically to the lingual surface at millimolar concentrations. The fact that higher concentrations of the drugs and longer exposure times were required to observe significant effects on CT responses is consistent with previous results that indicate the presence of a significant diffusion barrier in the taste pore region (Kloub et al. 1998; Lyall et al. 2001).

Data analysis

The changes in TRC pH_i were expressed as means ± SE of N, where N represents the number of ROIs in the taste bud. Student's t-test was employed to analyze the differences between sets of data.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

BCECF loading

Figure 1A 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 ×40 water immersion objective. Figure 1B shows the image of the taste bud excited at 490 nm. The figure shows that BCECF is specifically taken up by the TRCs within the papilla and is excluded from surrounding epithelial cells. However, squamous epithelial cells on the papillary periphery also absorb dye. Measurements of fluorescence changes were made exclusively from the dye-loaded TRCs (Lyall et al. 2001).

View larger version (76K): [in this window] [in a new window]   Fig. 1. BCECF loading of taste receptor cells (TRCs). An isolated piece of rat lingual epithelium containing a single fungiform papilla was mounted in the special microscopy chamber and was perfused with control solution containing BCECF-AM. The taste bud was viewed from the basolateral side (bar = 10 µm) with a ×40 magnification. The figure shows the transmitted image of the taste bud and the fluorescent image of the same taste bud excited at 490 nm.

Effect of HCl on TRC pH_i and CT responses

In polarized TRCs the acidic stimuli were applied to the apical side while monitoring changes in pH_i in situ from the basolateral side. Consistent with previous studies (DeSimone et al. 2001; Lyall et al. 1997, 2001; Stewart et al. 1998), stimulating the lingual surface with HCl decreased TRC pH_i (Fig. 2). Increasing the concentration of HCl in the apical solution to 1 mM (pH_o 3.0), 10 mM (pH_o 2.0), and 30 mM (pH_o 1.5) decreased TRC resting pH_i in six ROIs within the taste bud by 0.27 ± 0.03, 0.36 ± 0.03, and 0.59 ± 0.02 (SE) pH unit, respectively. The changes in pH_i were sustained and were completely reversible. The changes in TRC pH_i (pH_i) for a given change in pH_o (pH_o) were small and remained within the physiological range. In Fig. 2, the mean pH_i/pH_o for 1-, 10-, and 30-mM HCl concentration was 0.061, 0.067, and 0.10, respectively. The decrease in TRC pH_i after stimulation with 1 mM HCl (pH_o 3.0) was normalized to 100%. The increase in the magnitude of TRC pH_i change after stimulation with 10 and 30 mM HCl was expressed relative to 1 mM HCl. Figure 3 shows the plot of pH_o versus normalized TRC pH_i responses observed in Fig. 2 (). Stimulation of the lingual surface with HCl produced a similar dose-dependent increase in the magnitude of TRC pH_i decrease in two other tissues.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effect of HCl stimulation on TRC pHi. Initially the tissue was perfused on both sides with control solution (pH 7.4). Small regions of interest (ROIs; 2-3 µm2) within the taste bud were monitored for changes in pHi. The lingual surface was stimulated with control solution (without HEPES) containing 1 mM (pHo 3.0), 10 mM (pHo 2.0), and 30 mM (pHo 1.5) HCl. The pHi values are presented as means ± SE of 5 ROIs within the taste bud.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Relation between pHo, TRC pHi, and chorda tympani (CT) responses. The changes in TRC pHi and the area under the integrated CT response curve following HCl stimulation normalized, respectively, to the pHi or CT response observed with 1 mM HCl (pHo 3.0). The decrease in TRC pHi and increase in the area under the integrated CT response curves after stimulation with 10 and 30 mM HCl were then each expressed relative to 1 mM HCl. Note the scaled responses are described by the same function of extracellular pH (pHo), indicating that the decrease in pHi and the CT response change in proportion to one another.

In anesthetized rats, stimulating the tongue with HCl (Fig. 4) increased CT responses relative to rinse (10 mM KCl). The CT responses were dose dependent; the magnitude of CT responses increased with increasing concentration of HCl. In two additional animals, similar increases in CT responses were observed when the lingual surface was stimulated with increasing concentrations of HCl. The numerical value of the integrated CT response curve was obtained as the area under the integrated CT response curve for a time interval of 1 min from the onset of the chemically evoked neural activity. The area under the integrated CT response curve after stimulation with 1 mM HCl (pH_o 3.0) was normalized to 100%. The increase in the area of the integrated CT responses after stimulation with 10 and 30 mM HCl were expressed relative to 1 mM HCl. Figure 3 also shows the plot of pH_o versus normalized CT response (). The data show that pH_o versus normalized CT responses and the normalized changes in TRC pH_i in vitro have similar profiles.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effect of HCl stimulation on CT nerve activity. Integrated CT responses to 1, 10, and 30 mM HCl. Rinse solution (R) was 10 mM KCl.

Effect of cAMP on TRC pH_i and CT responses

At a fixed HCl concentration, the pH_i decrease was greater after treatment of the basolateral membrane with 250 µM 8-CPT-cAMP for 15 min. In a representative experiment shown in Fig. 5, decreasing the apical pH from 7.4 to 3.0 with 1 mM HCl decreased TRC pH_i from 7.51 ± 0.03 to 7.40 ± 0.02 (n = 4). After cAMP treatment the same HCl stimulus decreased TRC pH_i from 7.52 ± 0.03 to 7.31 ± 0.04. Thus in the presence of cAMP, stimulation of the apical membrane with HCl produced a twofold greater decrease in TRC pH_i. In two other tissues, stimulating the lingual surface with 1 mM HCl in the absence of cAMP induced an average decrease in TRC pH_i by 0.06 ± 0.002 and 0.13 ± 0.03 pH unit. Following cAMP treatment, 1 mM HCl induced an average decrease in TRC pH_i of 0.12 ± 0.01 and 0.25 ± 0.01 pH unit, respectively (n = 6). The data suggest that cAMP increases H⁺ entry across the apical membranes of TRCs.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of cAMP on HCl-induced decrease in TRC pHi. The lingual surface pH was decreased from 7.4 to 3.0 with 1 mM HCl under control conditions and after treatment of the basolateral membrane with 250 µM 8-CPT-cAMP for 15 min. The pHi values are presented as means ± SE of 4 ROIs within the taste bud.

To determine whether pH recovery mechanisms are also modulated by cAMP, we monitored the rate of spontaneous TRC pH_i recovery following intracellular acid loading at constant external pH. Figure 6 shows the effect of short basolateral side pulses of NH₄Cl on TRC pH_i. Following the application of NH₄Cl TRC pH_i rapidly alkalinized (a-b) due to the entry of NH₃ and conversion of free intracellular H⁺ to NH (Roos and Boron 1981). On NH₄Cl washout, TRC pH_i acidified and became lower than its resting value (b-c). This is due to the rapid exit of NH₃ from the cells and the conversion of NH to NH₃ plus free H⁺. This decrease in pH_i was transient and recovered spontaneously toward its resting value (c-d). A spontaneous recovery from transient hyperacidity demonstrates in general the presence of pH recovery mechanisms in a given cell type (Roos and Boron 1981). The data therefore show that TRCs are among the cell types that can contain pH-recovery mechanisms (DeSimone et al. 2001; Lyall et al. 1997; Stewart et al. 1998). In Fig. 6, under control conditions the rate of pH_i recovery was 0.085 ± 0.003 pH unit/min (n = 6). Following exposure of the basolateral membrane to 8-CPT-cAMP for 15 min, there was a small decrease in resting TRC pH_i from 7.37 ± 0.02 to 7.31 ± 0.02 (e-f). In the presence of cAMP the rate of spontaneous pH_i recovery from NH₄Cl prepulse (h-i) decreased to 0.046 ± 0.005 pH unit/min, a 45.8 ± 11.4% decrease in the rate of pH_i recovery (P < 0.01; paired). Exposing the basolateral membrane to 8-CPT-cAMP for 30 min further decreased the pH_i recovery rate from the NH₄Cl pulse to 0.022 ± 0.003 pH unit/min, a 74.0 ± 4.7% decrease in the rate of pH_i recovery (P < 0.001; paired; n = 6; data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effect of cAMP on TRC pHi recovery from NH4Cl prepulse. A lingual epithelium was initially perfused on both sides with control solution containing 150 NaCl (pH 7.4). Temporal changes in TRC pHi were monitored following a short basolateral NH4Cl pulse (15 mM NH4Cl replaced 15 mM NaCl in the basolateral solutions) under control conditions and after treatment of the basolateral membrane with 250 µM 8-chlorophenylthio (CPT)-cAMP for 15 min. Exposing the basolateral membrane of TRCs to NH4Cl pulse decreased TRC pHi (a-c) that recovered spontaneously (c-d). 8-CPT-cAMP inhibited the spontaneous rate of pHi recovery (h-i vs. c-d). The pHi values are presented as means ± SE of 6 ROIs within the taste bud.

In our in vitro studies, cAMP increased apical H⁺ entry and inhibited pH recovery from intracellular acid loading. In keeping with our previous results showing that a decrease in pH_i is the proximate sour taste stimulus (Lyall et al. 2001), we predicted that an increase in TRC cAMP in vivo should increase taste nerve responses to HCl. In a representative experiment shown in Fig. 7A, in anesthetized rats, stimulating the tongue with 20 mM HCl increased CT responses (0 cc) relative to rinse (10 mM KCl). As shown previously (DeSimone et al. 1995; Stewart et al. 1998), the HCl-induced CT response was amiloride insensitive and was not altered when the tissue was voltage clamped at ±60 mV (data not shown). Topical lingual application of 20 mM 8-CPT-cAMP for 1 h increased the CT response (0 cc) to HCl by about twofold. In contrast to control conditions, the post-cAMP CT response was enhanced at 60 mV and suppressed at +60 mV voltage clamp. However, as in the control condition (DeSimone et al. 1995; Stewart et al. 1998), the response remained amiloride insensitive. Figure 7B summarizes data from several animals. The numerical value of the integrated CT response was obtained as the area under the integrated CT response curve for a time interval of 2 min from the onset of the chemically evoked neural activity. Under control conditions, CT responses to 20 mM HCl were not significantly affected (P > 0.05; n = 3) by voltage clamping the tissue at ±60 mV. In four animals the lingual application of 8-CPT-cAMP significantly increased HCl-induced CT responses under 0 cc (*P < 0.0001; paired) as compared with CT responses before cAMP treatment. Following cAMP treatment, voltage clamping the tissue to 60 mV increased CT responses (**P < 0.048; n = 3) and clamping the potential to +60 mV decreased CT response (**P < 0.016; n = 3) as compared with CT responses at 0 cc. However, in the presence of cAMP, amiloride had no effect on CT responses under 0 cc (P > 0.05; n = 3).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of cAMP on HCl-induced changes in CT nerve activity. A: the rat CT responses to 20 mM HCl were monitored under open circuit conditions (0 current clamp; 0 cc) and under lingual voltage clamp, before and after the topical application of 20 mM 8-CPT-cAMP for 1 h. The rinse solution was 10 mM KCl (R). B: mean ± SE CT responses in several animals. In each animal the CT responses under different conditions were normalized to the CT response to 20 mM HCl under 0 cc in the absence of cAMP [CT/CT(control; 0 cc)]. The lingual application of 8-CPT-cAMP significantly increased the HCl-induced CT response under 0 cc (*P < 0.0001; paired; n = 4) as compared with CT responses before cAMP treatment. In 3 tissues in the presence of cAMP, voltage clamping the tissue to 60 mV increased CT responses (**P < 0.048), and clamping the potential to +60 mV decreased CT response (**P < 0.016) as compared with CT responses at 0 cc.

Effect of ionomycin on TRC pH_i and CT responses

Exposing the basolateral membrane to 20 µM ionomycin alkalinized the resting TRC pH_i. In a representative experiment shown in Fig. 8, under control conditions the resting TRC pH_i was 7.14 ± 0.02 and increased to 7.51 ± 0.03 (pH_i = 0.37 ± 0.01; P < 0.001; paired; n = 7) in the presence of ionomycin (e-f). Although not shown in the figure, ionomycin-induced alkalinization was completely reversible, perfusing the basolateral membrane with control solution decreased TRC pH_i to 7.18 ± 0.04. In three tissues, ionomycin treatment reversibly increased TRC pH_i by 0.47 ± 0.12. In the absence of ionomycin, increasing the calcium concentration in the basolateral solution from 1 to 10 mM also induced intracellular alkalinization. In the example shown in Fig. 9, increasing basolateral calcium concentration increased resting TRC pH_i from 7.28 ± 0.05 to 7.43 ± 0.04 (n = 6). In another tissue, a similar increase in basolateral calcium concentration increased TRC pH_i from 7.23 ± 0.02 to 7.40 ± 0.04 (n = 6).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Effect of ionomycin on TRC pHi. A lingual epithelium was initially perfused on both sides with control solution (pH 7.4). Exposing the basolateral membrane of TRCs to NH4Cl pulse decreased TRC pHi (a-c) that recovered spontaneously (c-d). In the 2nd part of the experiment, exposure of basolateral membrane to 20 µM ionomycin alkalinized TRC pHi (e-f) and increased the spontaneous rate of pHi recovery from the NH4Cl pulse (h-i). The pHi values are presented as means ± SE of 7 ROIs within the taste bud.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Effect of external Ca2+ on TRC pHi. A lingual epithelium was initially perfused on both sides with control solution containing 1 mM CaCl2 (pH 7.4). TRC pHi was monitored under control conditions and after increasing the CaCl2 concentration of the basolateral solution to 10 mM. The pHi values are presented as means ± SE of 6 ROIs within the taste bud.

The data summarized in Fig. 8 further show that the ionomycin-induced increase in pH_i is due to the activation of pH recovery mechanisms in TRCs. In Fig. 8, under control conditions the rate of pH_i recovery from NH₄Cl pulse (c-d) was 0.027 ± 0.002 pH unit/min. In the presence of ionomycin, the pH_i recovery rate (h-i) increased to 0.104 ± 0.009 pH unit/min, a 388 ± 27% increase in pH_i recovery rate (P < 0.001; paired; n = 7; h-i vs. c-d). Although not shown in the figure, the ionomycin-induced increase in pH_i recovery rate was completely reversible, perfusing the basolateral membrane with control solution decreased pH_i recovery rate to 0.044 ± 0.01 pH unit/min. In all tissues examined, ionomycin-induced alkalinization was accompanied by a reversible increase in spontaneous pH_i recovery rate from acid loading.

Treatment of the basolateral membrane with 50 µM MIA, a blocker of Na⁺-H⁺ exchange (NHE) activity, inhibited ionomycin-induced alkalinization. In a representative experiment shown in Fig. 10A, in the absence of MIA, ionomycin increased TRC pH_i from 7.26 ± 0.04 to 7.58 ± 0.06 (pH_i = 0.32 ± 0.03). However, in the presence of basolateral MIA, ionomycin increased TRC pH_i from 7.21 ± 0.01 to 7.31 ± 0.02 (pH_i = 0.100 ± 0.01), an inhibition of 70 ± 9% (P < 0.001; paired; n = 6). As shown in Fig. 10B, MIA also inhibited the spontaneous pH_i recovery from NH₄Cl pulse (a-b vs. c-d). In additional experiments, the spontaneous pH_i recovery rate from NH₄Cl pulse was also blocked by the addition of amiloride or 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) to the basolateral perfusate (data not shown). Both amiloride and EIPA are also blockers of the NHE activity (Kleyman and Cragoe 1988). These data indicate that pH recovery involves the activation of a basolateral membrane NHE.

View larger version (18K): [in this window] [in a new window]   Fig. 10. Effect of 5-(N-methyl-N-isobutyl)-amiloride (MIA) on TRC pHi. A: a lingual epithelium was initially perfused on both sides with control solution (pH 7.4). The basolateral membrane of TRCs was exposed to 20 µM ionomycin in the absence and presence of 50 µM basolateral MIA. The pHi values are presented as means ± SE of 5 ROIs within the taste bud. B: the spontaneous rate of pHi recovery from the NH4Cl prepulse was monitored in the absence and presence of 50 µM basolateral MIA. The pHi values are presented as means ± SE of 6 ROIs within the taste bud.

Consistent with the data shown in Figs. 2 and 5, a decrease in lingual surface pH from 7.4 to 3.0 with HCl induced a sustained decrease in TRC pH_i in nine ROIs in the taste bud under control conditions (Fig. 11). Following ionomycin treatment, TRC pH_i alkalinized. However, in the continuous presence of ionomycin, a decrease in lingual surface pH from 7.4 to 3.0 with HCl induced a transient decrease in TRC pH_i that recovered spontaneously toward baseline. It is important to note that the magnitude of the initial HCl-induced decrease in pH_i was not different in the presence and absence of ionomycin. The data suggest that apical H⁺ entry is not modulated by an increase in TRC [Ca²⁺]_i.

View larger version (10K): [in this window] [in a new window]   Fig. 11. Effect of ionomycin on HCl-induced changes in TRC pHi. TRC pHi was monitored during a decrease in the lingual surface pH from 7.4 to 3.0 with 1 mM HCl under control conditions and after treatment of the basolateral membrane with 20 µM ionomycin for 15 min. The pHi values are presented as means of 9 ROIs within the taste bud.

In anesthetized rats, stimulation with 20 mM HCl + 10 mM CaCl₂ (Fig. 12; left trace) increased CT responses relative to rinse (R; 10 mM KCl + 10 mM CaCl₂). The topical application of 150 µM ionomycin in DMSO for 1 h did not affect the initial CT response to HCl, but it declined to 50% of its initial level within 2-3 min (middle trace). The ionomycin effects were completely reversible. Following ionomycin treatment, the tongue was suffused with the rinse solution without ionomycin for 5 min. Subsequently stimulating the tongue with 20 mM HCl induced CT responses that were similar to pre-ionomycin responses; that is, the neural responses demonstrated little or no adaptation (right trace). In three animals the ionomycin treatment decreased the CT response (expressed as the area under the integrated CT response curve for a time interval of 5 min from the onset of the chemically evoked neural activity) by 39.8 ± 4.2% (P < 0.0007; paired).

View larger version (23K): [in this window] [in a new window]   Fig. 12. Effect of ionomycin on HCl-induced changes in CT nerve activity. The CT responses were recorded under open circuit conditions (0 current clamp; 0 cc) under control conditions (left trace), after the lingual application of 150 µM ionomycin in DMSO (middle trace), and following the application of rinse solution without ionomycin for 5 min (right trace). The rinse solution contained 10 mM KCl + 10 mM CaCl2 (R) and the stimulus solution was 20 mM HCl + 10 mM CaCl2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Relationship between TRC pH_i and CT responses

It is now well-established that the proximate stimulus to which mammalian acid-sensing cells react is a decrease in pH_i. This is true for carotid body type I cells (Buckler et al. 1991), chemoreceptors of the ventrolateral medulla (Ritucci et al. 1998; Wiemann et al. 1998), the locus coeruleus (Pineda and Aghajanian 1997), and the lingual epithelium (Lyall et al. 2001). In each case a reduction in pH_i by <0.1 pH unit is sufficient to fully activate the chemoreceptors. We have previously shown that weak organic acids and CO₂ lower the intracellular pH of TRCs and stimulate CT responses to these acids in a manner that is independent of pH_o (Lyall et al. 2001). The reason is that in each case the actual acid stimulus (H⁺ ions) is formed intracellularly as demonstrated by the fact that blocking the decrease in pH_i pharmacologically inhibits the CT response (Lyall et al. 2001). The acid precursors that diffuse across the apical membranes of the TRCs are in these cases electroneutral molecules, i.e., undissociated acids or dissolved CO₂. However, transport pathways for H⁺ ions must also be present in TRCs because strong acids stimulate the taste nerves at pH_o values where these acids are fully dissociated. As shown in Figs. 2, 3, 5, and 11, H⁺ ions enter rat TRCs from the apical side as monitored by the decrease in TRC pH_i. Consistent with earlier results (Lyall et al. 2001), HCl induced relatively small changes in TRC pH_i, but as in other mammalian acid-sensing cells, this is adequate to cause a neural response (Figs. 3, 4, and 7). At the highest concentration of HCl used in these experiments (30 mM; pH_o 1.5) the maximum decrease in TRC pH_i was 0.59 ± 0.02 pH unit (n = 6). These data confirm and extend our earlier observations (Lyall et al. 2001) that, during the rigors of acid stimulation, the variations in TRC pH_i are attenuated so that they remain within the physiological range while serving as the proximate sour taste stimulus. Second, the HCl-induced changes in TRC pH_i were sustained. A sustained decrease in TRC pH_i is a common feature of mammalian acid-sensing cells (DeSimone et al. 2001; Lyall et al. 2001). The sustained changes in TRC pH_i most likely are due to the inhibition of pH recovery mechanisms (Chamber-Kersh et al. 2000; Lyall et al. 2001; Ritucci et al. 1998). Our data suggest that inhibition of pH recovery mechanisms in TRCs may be due to an intracellular increase in cAMP (Fig. 6) or a decrease in [Ca²⁺]_i (Fig. 8). This hypothesis is consistent with observations that in a variety of cells the activity of pH regulatory mechanisms such as the Na⁺-H⁺ exchangers and Cl-HCO exchangers is regulated by cAMP and [Ca²⁺]_i (Noel and Pouysségur 1995; Reuss and Stoddard 1987). This hypothesis is further supported by recent studies of Liu and Simon (2001) and our unpublished observations, in which stimulating rat TRCs with acid induced a decrease in [Ca²⁺]_i in a subset of cells. However, other factors such as changes in cell volume and the cytoskeleton may also be involved in modulating pH recovery mechanisms in TRCs during acid transduction (Ritter et al. 2001).

Our data also demonstrate that stimulating the lingual surface with increasing concentrations of HCl produced a dose-dependent increase in CT response (Fig. 4). It is important to note that CT nerve activity in anesthetized rats was monitored at normal physiological temperatures, and the HCl solutions were applied to the tongue at the rate of 1 ml/s. In contrast, our pH_i measurements were made in a small microscopy chamber in which the maximum flow rate was limited to 1 ml/min, and the measurements were made at room temperature (see METHODS). However, in our previous studies (Lyall et al. 2001), we made some CT recordings with stimulus and rinse applied at 1 ml/min. Our data demonstrated that the phasic part of the CT response was strongly influenced by the flow rate; however, the magnitude of the maximum CT response to HCl was not affected by the flow rate. At the flow rate of 1 ml/min the HCl-induced CT response profiles were similar to the TRC pH_i changes observed in vitro (Lyall et al. 2001). These data suggest that changes in TRC pH_i under our experimental conditions can be correlated with in vivo maximum CT responses when stimuli are applied at low flow rate or with the time-average CT response when the stimuli are applied at higher flow rate. A correlation between normalized time-average HCl-induced CT responses and normalized changes in TRC pH_i at three values of pH_o is shown in Fig. 3. The normalized CT responses and the normalized changes in TRC pH_i in vitro are described by the same function of pH_o. This can only be the case if the magnitude of the CT response is proportional to the decrease in pH_i. This result is therefore consistent with our earlier observations (Lyall et al. 2001) that a decrease in TRC pH_i serves as the proximate stimulus in sour taste transduction. This conclusion is further supported by the observations that acetic acid and dissolved CO₂-induced CT responses demonstrated a good temporal relationship to changes in TRC pH_i and were independent of pH_o. Second, a membrane-permeable carbonic anhydrase blocker attenuated CT responses and inhibited TRC pH_i changes induced by dissolved CO₂.

Role of cAMP and [Ca²+]_i in sour taste responses

In studies on taste transduction, cAMP appears to be one of the second messengers involved in the generation of the receptor cell response (Avenet et al. 1988). Rat TRCs sensitive to sucrose respond with an increase in cAMP (Varkevisser and Kinnamon 2000) or cGMP (Krizhanovsky et al. 2000). In contrast, the bitter stimuli, denatonium and strychinine, induce rapid and transient reductions in cAMP and cGMP in murine taste tissue (Yan et al. 2001). In patch-clamp experiments, in posterior rat TRCs cytosolic cAMP inhibited outward K⁺ currents (Herness et al. 1997) and depolarized TRCs. Similarly there is ample evidence to suggest that changes in TRC [Ca²⁺]_i play an important role in taste transduction. Synthetic sweeteners stimulate production of inositol 1,4,5-tris-phosphate (IP₃) and diacylglycerol (DAG) and increase [Ca²⁺]_i (Bernhardt et al. 1996; Ogura and Kinnamon 1999; Varkevisser and Kinnamon 2000). Similarly a phospholipase C (PLC)-₂-dependent rise in IP₃ has been observed in TRCs with the bitter stimuli, denatonium and strychnine (Yan et al. 2001). There is evidence that both cAMP and [Ca²⁺]_i also modulate sour taste. In isolated hamster TRCs, patch-clamp studies indicated that H⁺ ions pass through amiloride-sensitive Na⁺ channels and the H⁺ current is enhanced by vasopressin and cAMP (Gilbertson et al. 1993). In a recent study by Liu and Simon (2001), stimulation of TRCs with acid revealed two distinct responses. Type 1 TRCs responded by an increase in [Ca²⁺]_i, and type II TRCs responded with a decrease in [Ca²⁺]_i. More recently, hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN 1 and 4) have been identified in rat circumvallate TRCs and have been suggested to play a role in acid transduction (Bufe et al. 2000; Stevens and Lindemann 1999, 2000).

Our data demonstrate that cAMP enhances the apical entry of protons into TRCs (Fig. 5). Increasing TRC cAMP levels increased the HCl-evoked decrease in pH_i twofold, a change expected to produce, in turn, a twofold increase in the HCl-evoked CT response if a decrease in pH_i is the proximate sensory stimulus. As expected, increasing TRC cAMP levels in vivo through topical application of 8-CPT-cAMP caused a twofold increase in the HCl-evoked CT response (Fig. 7). Moreover, the cAMP-modulated apical H⁺ entry pathway is also a voltage-modulated pathway (Fig. 7). Making the submucosal side of the stimulated lingual epithelium more electronegative (hyperpolarizing the TRC apical membranes) increased the CT response to HCl following cAMP treatment while making it more electropositive (depolarizing the TRC apical membranes) decreased the CT response. This is similar to the modulation of the NaCl CT response under voltage-clamp conditions (Ye et al. 1994). However, in the present case, the cAMP-enhanced HCl response remained amiloride insensitive, indicating that the voltage sensitivity arises in a separate conductance different from the Na⁺-conducting epithelial sodium channel. This also indicates that the cAMP-enhanced conductance is not BNC1, a member of the ENaC-ASIC family, found in rat vallate taste buds (Kinnamon et al. 2000; Ugawa et al. 1998). This is unlike isolated hamster TRCs where cAMP was shown to increase H⁺ currents through amiloride-sensitive Na⁺ channels (Gilbertson et al. 1993).

Although our results show that cAMP activates an apical H⁺ conductance, it should not be assumed that as a consequence all affected cells will become depolarized. The entry of H⁺ ions definitely decreases pH_i (cf. Fig. 2) and could possibly depolarize some cells if the H⁺ conductance were the only conductance in the apical membrane. However, in the presence of other cell membrane conductances, such as ENaC, depolarization due to a decrease in apical pH_o is not an assured consequence. For example in various Na⁺-conducting epithelia when pH_o is reduced, typically the Na⁺ channels are blocked and the potential across the apical membrane becomes hyperpolarized. The net effect is to reduce the short-circuit current and therefore Na⁺ ion transport (Lyall et al. 1995). Consistent with these observations, in mouse taste cells 10 mM HCl caused some TRCs to depolarize and others to hyperpolarize (Ohtubo et al. 2001). So it is unlikely that H⁺ stimulation will in and of itself produce a sustained depolarizing response especially in polymodal cells. Once changes in pH_i occur, further modulation of basolateral conductances (e.g., K⁺ ion conductances) can lead to cell depolarization. Modulation of K⁺ ion conductances by pH_i has been shown in locus coeruleus chemosensitive neurons (Pineda and Aghajanian 1997).

At present it is unclear whether the cAMP-activated, voltage-sensitive apical proton pathway reported here can be attributed to HCN channels (HCN 1 and 4), recently shown to be present in rat circumvallate TRCs (Bufe et al. 2000; Stevens and Lindemann 1999, 2000). In addition to its effect on an apical ion conductance, cAMP has a second modulatory effect on the TRCs, i.e., cAMP inhibits TRC pH_i recovery (Fig. 6). Both cAMP actions alone would produce an enhanced decrease in pH_i on acid stimulation. Since they will inevitably occur in concert, the net effect of cAMP is to amplify the intracellular stimulus intensity and hence the CT response due to acid stimulation. Our data further indicate that the TRCs contain an MIA-sensitive NHE activity (Fig. 10). The fact that pH recovery is attenuated by cAMP treatment (Fig. 6) suggests that the type 3 isoform of NHE (NHE3) is present in TRC membranes and is most likely responsible for the observed pH_i recovery (Noel and Pouysségur 1995; Ritter et al. 2001; also our unpublished observations on TRC NHE3).

Ionomycin, a Ca²⁺ ionophore, presumably increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) and alkalinizes TRC pH_i (Figs. 8 and 9). Both ionomycin-induced alkalinization and pH_i recovery from NH₄Cl pulse were blocked by MIA (Fig. 10), suggesting that these effects are due to the activation of the type 1 isoform of NHE (NHE1) in the basolateral membrane by [Ca²⁺]_i (Noel and Pouysségur 1995; Ritter et al. 2001; also our unpublished observations on TRC NHE1). The increase in NHE1 activity is also responsible for TRC pH_i recovery following apical stimulation with HCl (Fig. 8).

The initial magnitude of the HCl-induced decrease in pH_i was not different in the presence and absence of ionomycin (Fig. 11), suggesting that apical H⁺ entry is not modulated by an increase in TRC [Ca²⁺]_i. In anesthetized animals, topical application of ionomycin also did not increase the initial CT response to HCl (Fig. 12), which is consistent with the lack of an effect of increased [Ca²⁺]_i on the HCl-induced decrease in pH_i and supports the conclusion that Ca²⁺ does not activate an apical H⁺ entry pathway. However, the CT response decreased to 50% of its initial value in 2 min. The comparable time courses of both pH_i recovery and the decay in CT response following ionomycin suggest that sensory adaptation is modulated by the rapid recovery of TRC pH_i induced by an increase in TRC [Ca²⁺]_i and activation of the basolateral NHE1 activity.

Our results suggest that stimulation of TRCs by acids involves the entry of acid equivalents (acid precursors and/or protons) across the apical membrane of TRCs and the activity of pH regulatory mechanisms in the TRC membranes. Acid-induced changes in TRC pH_i are small and remain within the physiological range, indicating that the apical membranes of TRCs have a lower permeability to organic acids and are significantly less conductive to H⁺ ions than the basolateral membranes of TRCs (Lyall et al. 2001). However, our present results show that the H⁺ conductance across the apical membrane can be regulated by intracellular cAMP. The fact that cAMP increased apical H⁺ entry in polarized TRCs and enhanced CT responses in anesthetized rats is itself further evidence supporting intracellular pH decrease as the proximate stimulus for sour taste. Although cAMP effects have been investigated on TRCs in vitro, to our knowledge, this is the first report in which topical lingual application of cAMP has been shown to directly modulate taste nerve responses in rat. Also this is the first time ionomycin has been used to successfully Ca²⁺ load TRCs in vivo. In gerbil, topical lingual application of 8-bromo-cAMP is reported to decrease the CT response to 10 mM HCl by 17% (Schiffman et al. 1994), an effect both smaller in magnitude and opposite in direction from our results in rat. Second, little is known at the level of the taste receptor cell regarding the physiological mechanisms involved in taste sensory adaptation for any of the taste submodalities. On the basis of the data presented here, at least in the case of sour taste, a Ca²⁺-activated NHE1 appears to be a functional TRC sensory adaptation mechanism. Third, the evidence that sour taste is modulated by cAMP and [Ca²⁺]_i may have implications for taste mixture interactions. For a binary mixture of bitter and sour stimuli, the bitter stimulus may cause both a fall in TRC cAMP and a rise in intracellular Ca²⁺. Assuming the TRC to be bimodal, both effects would lead to a suppression of the sour response by both decreasing the rate of entry of H⁺ ions into the TRC across the cell apical membranes (reduced cAMP) and by activating TRC pH recovery mechanisms (reduced cAMP and increased Ca²⁺) that also have the effect of reducing the acid-induced drop in pH_i. On the other hand, in a binary mixture of sweet and sour stimuli, the sweet stimulus may lead directly to an increase in cell cAMP. Again, assuming a bimodal TRC, the effect would be to enhance the rate of entry of H⁺ ions into the TRC across the cell apical membranes (increased cAMP) and decrease the rate at which the cell NHEs could mitigate the stimulus-induced drop in pH_i (again due to increased cAMP). The perceived intensity of sweet response would be suppressed as a result of the fact that cAMP, produced in connection with sweet taste transduction, would also be available to enhance the sour response. Further studies are required, however, before a role for second messengers in mixture interactions can be firmly established.

The main conclusions of the paper and the proposed mechanism for sour taste activation and adaptation for strong acids are summarized in a schematic (Fig. 13). Our data indicate that cAMP enhances the sour taste of strong acids by activating a Ca²⁺- and amiloride-insensitive apical H⁺ conductance and inhibiting pH_i recovery by blocking NHE activity in TRCs. An increase in [Ca²⁺]_i stimulates pH_i recovery by activating NHE activity, which increases sensory adaptation to acids.

View larger version (26K): [in this window] [in a new window]   Fig. 13. Proposed mechanism for sour taste enhancement and adaptation in rat TRCs. Cyclic AMP activates an amiloride-insensitive and Ca2+-insensitive apical H+ pathway and inhibits Na+-H+ exchange activity in TRCs. Both these effects result in enhanced CT responses to lingual stimulation with HCl. The fact that post-cAMP CT responses are enhanced by hyperpolarization and attenuated by depolarization of the apical membrane potential suggest that the apical H+ pathway is a cAMP-activated H+ channel. An increase in TRC [Ca2+]i activates Na+-H+ exchange activity, which results in accelerated recovery of TRC pHi from intracellular acidification. During acid stimulation the rapid recovery from intracellular acidification results in rapid neural adaptation to HCl stimulation.