Decrease in rat taste receptor cell intracellular pH is the
proximate stimulus in sour taste transduction
Vijay
Lyall1,
Rammy I.
Alam1,
Duy Q.
Phan1,
Glenn L.
Ereso1,
Tam-Hao T.
Phan1,
Shahbaz A.
Malik1,
Marshall H.
Montrose2,
Shaoyou
Chu2,
Gerard L.
Heck1,
George M.
Feldman1,3, and
John A.
DeSimone1
1 Department of Physiology, Virginia Commonwealth
University, Richmond 23298-0551, 3 McGuire Veterans Affairs
Medical Center, Richmond, Virginia 23249; and 2 Department of
Physiology and Biophysics, Indiana University School of Medicine,
Indianapolis, Indiana 46202-5120
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ABSTRACT |
Taste receptor cells (TRCs)
respond to acid stimulation, initiating perception of sour taste.
Paradoxically, the pH of weak acidic stimuli correlates poorly with the
perception of their sourness. A fundamental issue surrounding sour
taste reception is the identity of the sour stimulus. We tested the
hypothesis that acids induce sour taste perception by penetrating
plasma membranes as H+ ions or as undissociated molecules
and decreasing the intracellular pH (pHi) of TRCs. Our data
suggest that taste nerve responses to weak acids (acetic acid and
CO2) are independent of stimulus pH but strongly correlate
with the intracellular acidification of polarized TRCs. Taste nerve
responses to CO2 were voltage sensitive and were blocked
with MK-417, a specific blocker of carbonic anhydrase. Strong acids
(HCl) decrease pHi in a subset of TRCs that contain a
pathway for H+ entry. Both the apical membrane and the
paracellular shunt pathway restrict H+ entry such that a
large decrease in apical pH is translated into a relatively small
change in TRC pHi within the physiological range. We
conclude that a decrease in TRC pHi is the proximate stimulus in rat sour taste transduction.
hydrogen ion conductance; intracellular signaling; chorda tympani; acid stimulation
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INTRODUCTION |
SOURNESS is a
primary taste sensation (25). Stimuli evoking a sour
sensation yield dissociable H+ ions. It is natural to
assume that taste receptor cells (TRCs) are extracellular pH
(pHo) detectors, and that sourness should, therefore, be a
graded function of stimulus pH. However, this is not generally true.
The poor correlation between sourness and stimulus pH has been amply
demonstrated in both human (1, 12, 17, 21, 24, 29) and
animal (2, 3, 27) studies. At the same pH, acetic acid is
a more potent sour stimulus than HCl (12, 27, 29).
Moreover, the sourness of acetic acid (and other weak acids) is
essentially the same as that of a buffer consisting of the acid plus
its conjugate base, even though the latter has a higher pH (2,
12). It has been speculated that weak acids might induce sour
taste perception by penetrating plasma membranes as undissociated
molecules and decreasing intracellular pH (pHi) upon
dissociating within the TRCs (13, 27, 35). We have
previously demonstrated that isolated rat (23) and hamster (34) TRCs respond to changes in pHo with
parallel changes in pHi. This suggests that changes in TRC
pHi may indeed be the appropriate stimulus variable for
sour taste transduction. If so, it would explain the paradoxical
correlation between stimulus pH and sourness. However, before a firm
conclusion can be drawn regarding the role of pHi in acid
sensing, measurements of TRC pHi must be made in an intact
taste bud maintained with its natural polarity, and a direct
correlation must be established between changes in pHi of
polarized TRCs and the taste nerve responses to acids. Finally, pharmacological agents that specifically inhibit the normal
acid-induced decrease in pHi should also inhibit the
acid-induced neural response. We have made these measurements, and the
results demonstrate unequivocally that changes in pHi in
TRCs represent the proximate sour taste stimulus in rat.
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MATERIALS AND METHODS |
Chorda tympani recording.
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 chorda
tympani (CT) nerve was exposed laterally as it exited the tympanic
bulla (9, 34, 39) and placed onto a 32-gauge 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 with the use of a band-pass filter
with cut-off frequencies of 40 Hz-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 Linseis TYP7045 chart recorder and
also captured on disk with Labview software and were analyzed off-line
(40). 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 or voltage clamp (39,
40). 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 acetic acid, citric acid, or HCl
solutions at pH 3.0 or with solutions containing 10 mM of each of the
above acids. To measure the CT response to dissolved CO2,
we stimulated the lingual surface with a solution containing 72 mM
KHCO3 buffered to pH 7.4 with a 10% CO2-90%
O2 mixture. In these experiments the rinse solutions
contained 72 mM KCl buffered to pH 7.4 with 10 mM HEPES. To evaluate
the role of carbonic anhydrase in dissolved-CO2-induced CT
responses, we studied the effect of topical application of 50 mM MK-417
(Merck, Rahway, NJ), a cell-permeable blocker of carbonic anhydrase
(11), on CT responses. In some experiments the CT
responses were recorded at the slower perfusion rate of 1 ml/min to
obtain flow rates comparable to those required in pHi
measurements in isolated single fungiform papillae.
pHi measurements.
Rats were anesthetized with methoxyflurane 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 HEPES, pH 7.4. The lingual epithelium was isolated by collagenase
treatment (23, 34). A small piece of the anterior lingual
epithelium containing a single fungiform papilla (Fig.
1, a-d) was mounted in a
special microscopy chamber (7). The tissue was
intermittently perfused with Ringer solution containing 25 µM of the
pH-sensitive fluoroprobe
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-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 camera (Imago, TILL Photonics Applied Scientific
Instrumentation, Eugene, OR) attached to an image intensifier (model
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 (diameter 2-3 µm2) were chosen in which the
changes in fluorescence intensity ratio (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 pHi were calibrated by bilateral perfusion of
high-K+ calibrating solutions between pH 6.5 and 8.0 containing 10 µM nigericin (23, 34). All experiments
were performed at room temperature (22 ± 1°C).

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Fig. 1.
Microscopy chamber. a: mounting of an isolated piece of
lingual epithelium (7) containing a single fungiform
papilla in the microscopy chamber. Images (b-d)
represent transmitted and fluorescent images of the taste bud when
viewed from the basolateral side (bar, 10 µm) at ×10 and ×40
magnification. Individual taste receptor cells (TRCs) can be observed
in the taste bud region (c). The fluorescence image of the
same taste bud excited at 490 nm is shown (d). The dye is
specifically loaded in TRCs and is excluded from the surrounding
supporting cells.
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Stimulus solutions.
Stimulus solutions were without HEPES and contained HCl, acetic acid,
citric acid, and tartaric acid at the concentrations given in the text.
Stimulus solution containing 8.9 mM acetic acid (pH 3.2) was titrated
to pH 6.0 with 155 mM potassium acetate (155 mM potassium acetate
replaced 150 mM NaCl and 5 mM KCl in the solution).
Bicarbonate-buffered solution contained (in mM) 78 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 72 NaHCO3 and was bubbled with a 10% CO2-90%
O2 mixture (pH 7.4). In some experiments both HEPES- and
bicarbonate-buffered solutions contained either 5 or 50 mM MK-417, a
membrane-permeable blocker of carbonic anhydrases (11).
Data analysis.
Results are presented as means ± SE of the number of ROIs in the
taste bud. Student's t-test was employed to analyze the
differences between sets of data.
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RESULTS |
Effect of acid stimulation on CT nerve activity.
The lingual application of acid stimuli increases CT nerve activity
(Fig. 2). At pH 3.0 (Fig. 2A),
acetic acid gave a larger CT response than either citric acid or HCl,
indicating that acetic acid is a stronger sour taste stimulus than
citric acid and HCl (2, 12, 24, 27). In contrast, when
each acid was applied at a concentration of 10 mM, all three acids
produced CT responses of similar magnitude even though the pH values of
the acid solutions varied between 2.0 and 3.4 (Fig. 2B).
Thus CT responses are not graded functions of the lingual surface pH
(pHo) (2, 12, 24, 27).

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Fig. 2.
Effect of acid stimuli on left chorda tympani (CT) nerve
activity. The integrated CT responses were monitored in anesthetized
rats while the anterior lingual surface was suffused with a rinse
solution (R; 10 mM KCl) and with solutions containing acetic acid,
citric acid, or hydrochloric acid (HCl) at pH 3.0 (A) or
with solutions containing 10 mM acids (B).
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Effect of acid stimulation on TRC pHi.
Acidic stimuli were applied to the apical side while changes were
monitored in TRC pHi in situ from the basolateral side. Acidic stimuli decreased TRC pHi (Fig.
3, A-C). Acetic acid at pH 3.1 induced greater decreases in TRC pHi than citric
acid, tartaric acid (Fig. 3A), and HCl (Fig. 3B).
In contrast, the changes in pHi produced by individual
acids at 10 mM concentration were of similar magnitude (Fig.
3C). In each case, therefore, the decrease in
pHi correlates with the respective taste neural responses, while pHo does not.

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Fig. 3.
Acid-induced decrease in intracellular pH
(pHi) of polarized TRCs. TRC pHi was monitored
while the tissue was perfused on both sides with control solution (R;
pH 7.4) and during perfusion of the lingual surface with solutions
containing acids at pH 3.1 (A and B) or acids at
10 mM concentration (C). A: a decrease in
extracellular pH (pHo) from 7.4 to 3.1 ( pHo = 4.3) with acetic acid, citric acid, and
tartaric acid decreased the mean pHi in a taste bud by
0.34, 0.16 and 0.13, respectively [n = 3 regions of
interest (ROIs)]. The ratio pHi/ pHo for
these 3 acids was 0.08, 0.04, and 0.03, respectively. B: in
another taste bud, acetic acid and HCl at pH 3.1 decreased mean
pHi by 0.37 and 0.27, respectively (n = 5 ROIs). The pHi/ pHo values for these 2 acids were 0.086 and 0.063, respectively. C: when the acids
were all present at 10 mM concentration, the mean change in
pHi for acetic acid, citric acid, and HCl was 0.23, 0.21, and 0.24, respectively (n = 4 ROIs). Similar results
were obtained in 2 other experiments.
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At pH 3.0, the acetic acid concentration is 58.3 mM. Of this, 57.3 mM
is undissociated acid. Citric acid, a much stronger acid, has a
concentration of 2.2 mM, of which 1.19 mM is undissociated. The
corresponding parameters for tartaric acid are similar to those for
citric acid. At pH 3.0, acetic acid induced a greater decrease in TRC
pHi (Fig. 3A) and a greater CT response (Fig. 2A) for two reasons: 1) it is present at higher
concentration than either citric acid or tartaric acid, and
2) a much higher proportion of the acetic acid is present as
the membrane-permeable undissociated form (13). At 10 mM
concentration, acetic acid has a pH of 3.4. Of this, 9.59 mM is
undissociated, while 10 mM citric acid has a pH of 2.6 and 7.49 mM is
undissociated. The concentrations of the undissociated forms of acetic
acid and citric acid in the stimulus solutions are comparable and,
therefore, induce comparable changes in pHi (Fig.
3C) and CT responses (Fig. 2B) for both acids.
Within the normal range of pHi (Fig. 3, A and
C), diffusing citric acid delivers three equivalents of
H+ ions to the TRCs. This suggests that the intrinsic
permeability of acetic acid is about three times that of citric acid.
Effect of pHo on acetic acid responses.
If the permeability of the undissociated form of a weak acid is the
critical factor in determining its intensity as a stimulus, the changes
in pHi and the CT response should both be independent of
pHo. To test this hypothesis, we obtained the CT responses to unbuffered acetic acid (pH ~3) and to the same concentration of
acetic acid buffered at pH 6 with potassium acetate. At a given concentration, the buffered (pH 6.0) and unbuffered acetic acid (pH
3.0) gave similar responses (Fig.
4A). In parallel experiments, perfusing solutions containing 8.9 mM acetic acid plus 150 mM potassium
acetate (pH 5.9) and 8.9 mM acetic acid alone (pH 3.2) across the
apical side of a single fungiform papilla induced changes in TRC
pHi of similar magnitude (Fig. 4B). Thus the CT
response and the changes in TRC pHi are independent of
pHo and the acetate concentration. Both the CT response and
the changes in TRC pHi were found to be independent of
apical Na+ and were unaffected by 1 mM
-cyano-4-hydroxycinnamate (4), a blocker of
monocarboxylate transporter I (unpublished observations). The data
suggest that undissociated acetic acid molecules enter TRCs by passive
diffusion across the apical membranes (13).

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Fig. 4.
Effect of pHo on acetic acid-induced changes
in CT responses and TRC pHi. A: CT responses
were recorded while the lingual surface was perfused with solutions
containing 10 (pH 3.40), 30 (pH 3.15), and 50 mM (pH 3.05) acetic acid
(AA) or with solutions containing 10, 30, and 50 mM acetic acid
titrated to pH 6.0 with 175, 525, and 875 mM potassium acetate (KA),
respectively. The CT responses increased with acetic acid concentration
and were independent of pHo. The solutions containing 175 and 525 mM potassium acetate (pH > 9.0) alone did not produce
significant changes in CT activity. At 875 mM potassium acetate, a
small CT response was noted. Similar results were obtained in 2 other
experiments. B: TRC pHi was monitored while the
tissue was perfused on both sides with control solution (pH 7.4) and
when the lingual surface was perfused with an unbuffered solution
containing 155 mM potassium acetate (with KA replacing all NaCl and KCl
in the Ringer solution) at pH 8.1. The mean TRC pHi in the
presence of 155 mM potassium acetate solution was taken as the baseline
pHi. In the second step, perfusing a solution of 155 mM
potassium acetate buffered to pH 5.9 with 8.9 mM acetic acid produced a
reversible decrease in TRC pHi from baseline. In the third
step, perfusing a solution containing 8.9 mM acetic acid alone (pH 3.2)
produced a decrease in TRC pHi similar to that observed in
the presence of 155 mM potassium acetate plus 8.9 mM acetic acid (pH
5.9). Values are means ± SE of 3 ROIs in the taste bud.
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Effect of CO2 on CT nerve activity.
Initially the lingual surface was suffused with a rinse solution
containing 72 mM KCl buffered to pH 7.4 with 10 mM HEPES, and the CT
activity was taken as the baseline activity. Suffusing the lingual
surface with a solution containing 72 mM KHCO3 buffered to
pH 7.4 with 10% CO2-90% O2 produced
reversible increases in CT activity (Fig.
5A). Cell membranes are freely
permeable to dissolved CO2, and its conversion to
H2CO3 catalyzed by intracellular carbonic
anhydrase (8, 16) represents penetration of acid equivalents across TRC membranes (23, 34). This was tested directly by recording CT responses in the presence of MK-417, a
specific blocker of carbonic anhydrases (11). We applied
50 mM MK-417 topically on the lingual surface in the rinse solution for
15 min. This inhibited the dissolved-CO2-induced CT
responses by ~50% (Fig. 5A, middle). Upon
washout, the effects of MK-417 were completely reversible (Fig.
5A, right). In a similar study (20),
topical application of MK-927, another membrane-permeable carbonic
anhydrase inhibitor, to the rat tongue inhibited CT responses to
carbonated water by 62%.

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Fig. 5.
CO2-induced changes in CT activity.
A: CT responses were recorded while the lingual surface was
suffused with rinse solution (R = 72 mM KCl solution buffered to
pH 7.4 with HEPES) or with 72 mM KHCO3 solution buffered to
pH 7.4 with a 10% CO2-90% O2 gas mixture
(left). The lingual surface was treated with rinse solution
containing 50 mM MK-417 for 15 min, and the CT responses were recorded
(middle). The lingual surface was then rinsed with the rinse
solution without the drug for 15 min, and CT responses were recorded
again (right). B: responses to 72 mM
KHCO3 solution buffered to pH 7.4 with a 10%
CO2-90% O2 gas mixture were recorded under
open-circuit conditions [i.e., under zero current clamp (0 CC)], at
60 mV, and at +60 mV of voltage clamp.
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Voltage dependence of CO2 induced CT nerve activity.
Data summarized in Fig. 5B indicate that unlike CT responses
to HCl (9, 34) and acetic acid (data not shown), the CT responses induced by dissolved CO2 are voltage sensitive.
Compared with open-circuit conditions (i.e., under 0 current clamp),
the CT responses were enhanced at
60 mV and suppressed at +60 mV, similar to voltage-clamp effects on responses to NaCl (39,
40).
Effect of CO2 on TRC pHi.
The generation of intracellular H+ ion was confirmed
directly by the observed decrease in TRC pHi produced by
perfusing the lingual surface with a solution containing 72 mM
NaHCO3 buffered to pH 7.4 with 10% CO2-90%
O2 (Fig. 6A). The
changes in TRC pHi were significantly attenuated in the
presence of 5 mM MK-417 (Fig. 6B). In the presence of 50 mM
MK-417, dissolved CO2 induced a maximum decrease in
pHi of 0.05 ± 0.008 pH unit. These data suggest a
direct role of carbonic anhydrase in CO2-induced sour taste transduction (20). Dissolved CO2 entry across
the apical membrane of TRCs is independent of pHo and the
HCO
concentration in the stimulus solution. The fact
that this acidic stimulus can be presented at an alkaline
pHo is further evidence that the proximate acidic stimulus
is the change in pHi and not pHo. Fully
dissociated strong acids, such as HCl (Fig. 1), HNO3, and
H2SO4, are also potent stimuli of the taste
nerves (2, 3, 9, 24, 30) and decrease TRC pHi
(Fig. 3, B and C), suggesting that H+
ions also gain entry into TRCs but, in all probability, not by diffusing through the membrane lipid bilayer like dissolved
CO2 and the neutral forms of weak acids.

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Fig. 6.
CO2-induced changes in TRC pHi.
A: TRC pHi was monitored while the tissue was
perfused on both sides with control solution buffered to pH 7.4 with
HEPES and when the lingual surface was perfused with the solutions
buffered to pH 7.4 with 72 mM NaHCO3 plus 10%
CO2-90% O2 (72 mM NaHCO3 replaced
72 mM NaCl in the solution). B: TRC pHi was
monitored while the tissue was perfused on both sides with control
solution (HEPES) and when the lingual surface was perfused with the
solutions buffered to pH 7.4 with 72 mM NaHCO3 plus 10%
CO2-90% O2
(HCO /CO2) in the presence
( ) and absence ( ) of 5 mM MK-417.
Values are means ± SE of 7 ROIs in the taste bud.
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Temporal relation between CT activity and TRC pHi.
Consistent with our previous studies (9, 34, 39, 40), CT
nerve activity was monitored in vivo at normal physiological temperature while the lingual surface was suffused with stimulating solutions at the rate of 1 ml/s. In contrast, our in vitro
pHi measurements were made in a small microscopy chamber at
room temperature where the maximum rates of perfusion were 1 ml/min. It
is desirable to control for these differences in the rate of stimulus
application when comparing the CT recordings with the changes in TRC
pHi. It was not possible, for technical reasons, to
increase the flow rate in vitro beyond 1 ml/min. Therefore, we made
some CT recordings with stimulus and rinse applied at 1 ml/min.
Consistent with previous studies (10, 31), the phasic
parts of the CT responses were strongly influenced by the flow rate
(Fig. 7); however, the magnitude of the
maximum CT response to either dissolved CO2 (Fig.
7A) or HCl at pH 3.0 (Fig. 7B) was not affected
by the flow rate. At the flow rate of 1 ml/min both acid stimuli,
dissolved CO2 (Fig. 8,
A and B) and HCl (Fig. 8, C and
D) induced CT-response profiles that were similar to the
pHi changes observed in vitro. The data show that under the
additional constraint of comparable flow rates, the CT nerve response
and the changes in TRC pHi are temporally well correlated.

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Fig. 7.
Effect of flow rate on CT responses. A: CT
responses were recorded while the lingual surface was suffused with
rinse solution (R = 72 mM KCl solution buffered to pH 7.4 with
HEPES) or with 72 mM KHCO3 solution buffered to pH 7.4 with
a 10% CO2-90% O2 gas mixture at the rate of 1 ml/s or 1 ml/min. B: CT responses were recorded while the
lingual surface was suffused with rinse solution (R = 10 mM KCl)
or with HCl solution at pH 3.0 at the rate of 1 ml/s or 1 ml/min.
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Fig. 8.
Temporal relation between CT activity and TRC
pHi. The temporal relation between the CT responses
(A) and the TRC pHi changes (B) to
dissolved CO2 is shown when both the rinse and stimulus
were applied at the rate of 1 ml/min. The temporal relation between the
CT responses (C) and the TRC pHi changes
(D) to HCl stimulation (pH 3.0) is shown when both the rinse
and stimulus were applied at the rate of 1 ml/min.
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Regional differences in HCl- and acetic acid-induced changes in pH
within a taste bud.
No CT responses were observed with HCl at pH 5.0 (data not shown). A
decrease in pHo from 7.4 to 5.0 induced an average decrease in TRC pHi of 0.11 ± 0.003 pH unit in a taste bud
(n = 12). We compared the regional changes in TRC
pHi in three taste buds exposed to apical acetic acid or
HCl at pH 3.0. All 58 ROIs in taste buds responded with a decrease in
pHi >0.11 pH unit. The mean decrease in pHi
produced by acetic acid (
pHi = 0.40 ± 0.01)
was significantly greater than that produced by HCl
(
pHi = 0.29 ± 0.01; P < 0.001; paired). In 56 ROIs, acetic acid produced a greater decrease in pHi than HCl did. The changes in pHi varied
widely within ROIs. After HCl treatment (Fig.
9A), 35 of 58 ROIs responded
with a decrease in pHi between 0.2 and 0.299 pH unit, and
20 of 58 ROIs (34.5%) responded with a decrease in pHi
>0.3 pH unit. It is likely that TRCs in the ROIs that respond with the
greatest decrease in pHi participate most in sour
transduction. In contrast, after acetic acid treatment, 5 of 58 ROIs
responded with a decrease in pHi between 0.2 and 0.299 pH
unit, and 53 of 58 ROIs (91.4%) changed pHi >0.3 pH unit
(Fig. 9B). The data suggest that HCl-induced CT responses
are elicited by a subpopulation of TRCs contained in different ROIs
within the taste bud that contain either an H+ entry
mechanism in their apical membrane or in their basolateral membrane
accessible via paracellular shunts. In contrast for acetic acid, the
influx of acid equivalents into TRCs is augmented by a significant flow
of unionized acetic acid. This would account for the significantly
greater decrease in TRC pHi, consistent with the greater CT
response.

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Fig. 9.
TRC pHi changes in different ROIs in the
taste buds. Three tissues were exposed to apical acetic acid
(A) or HCl (B) at pH 3.1, and changes in TRC
pHi were monitored in 58 ROIs in the taste buds. For each
acid, a histogram shows the number of ROIs that fall within a given
pHi interval.
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DISCUSSION |
Our results show that weak acids enter rat TRCs from the apical
side as neutral molecules (acetic acid or dissolved CO2). H+ ions (HCl) also gain access to TRCs, but the pathways
involved have not yet been fully elucidated. Irrespective of the mode
of acid entry, however, the data indicate that the consequent decrease in TRC pHi serves as the proximate stimulus in sour taste transduction.
pHi as the proximate stimulus in acid detection.
The hypothesis that a decrease in TRC pHi is the proximate
stimulus in rat sour taste transduction is borne out by our studies with acetic acid stimuli at pH 6.0 and dissolved CO2 as an
acid stimulus. Even at pH 6.0, a pHo that is significantly
above the threshold pH that gives a detectable CT response, acetic acid was as good a stimulus as at pH 3.0. Acetic acid responses were completely independent of pHo and K-acetate concentration.
Our data indicate that the permeability of the undissociated acetic acid is the critical factor in determining its intensity as a stimulus
(13, 27). Inside the cell, acetic acid is dissociated into
H+ and acetate ions. Thus a decrease in TRC pHi
is the proximate stimulus for acetic acid-induced increase in CT
responses at pH 6.0.
At constant pHo, dissolved CO2 decreased TRC
pHi and increased CT responses. A membrane-permeable
carbonic anhydrase blocker, MK-417, attenuated CT responses and
inhibited changes in TRC pHi. It is interesting to note
that unlike CT responses to HCl (9, 34) and acetic acid
(data not shown), the CO2 responses were voltage sensitive.
Inside the cell, the dissolved CO2 is converted to
H2CO3 in a reaction catalyzed by intracellular
carbonic anhydrases. The H2CO3 yields free
H+ and HCO
ions. It is likely that
HCO
ions exit TRCs via conductive pathways and/or
ion exchangers in the cell membranes (unpublished observations).
Because CO2 permeability across cell membranes is not
voltage sensitive, voltage changes most likely affect CT responses
indirectly by modulating the HCO
flux across TRC
membranes. Because the hydration of CO2 leads to the
formation of H+ and HCO
in
stoichiometrically equal proportions, alteration in the concentration
of the latter under voltage-clamp conditions will produce immediate
changes in pHi. If a decrease in pHi is the
necessary precursor to a sour taste response, the time course of the CT
response ought to follow that of the pHi when the sour
stimulus is applied at a given fixed rate. The optimal means of making
this determination would be to measure both pHi and the
neural response in the same system simultaneously. For technical
reasons, this was not possible. However, when we obtained the
pHi profiles and the CT response for both HCl and
CO2 in separate experiments but at the same stimulus flow
rate, their time courses were quite comparable (cf. Fig. 8). This
result is consistent with the conclusion that the change in
pHi is the actual rate-limiting stimulus in acid taste transduction.
Similar studies in other mammalian pH-sensors have also identified
pHi as the proximate stimulus in acid detection. A decrease in pHi induced by elevated levels of CO2
depolarized pH-sensing neurons (28, 30, 38) and increased
action potential frequency. In cultured ventrolateral medullary
neurons [37] during NH4Cl pulses a decrease
in pHi led to increased action potential frequency. A
decrease in pHi induced by blocking the neuron membrane
Na+/H+ exchanger resulted in increased cell
excitability (38). Acid detection by carotid body type I
cells also depends on a decrease in pHi (5,
6). It appears, therefore, that mammalian pH sensory cells have
the common property of responding to changes in pHi as the
proximate stimulus in acid detection.
Proton interactions with TRCs.
Our data indicate that H+ ions rapidly enter TRCs. Within
the taste bud, a subpopulation of TRCs appears to contain an
H+ entry mechanism either in the apical membranes or in the
basolateral membranes made accessible via paracellular shunts. During
acid stimulation, this subset of TRCs responds with greater changes in
pHi and most likely participates in CT responses elicited
by strong, fully dissociated mineral acids. However, the exact nature of the H+ entry mechanism remains unknown principally
because the effect of H+ ions on TRC membrane conductances
varies considerably among species (9, 22, 26, 33).
H+ entry through apical amiloride-sensitive epithelial
Na+ channels (ENaC) has been suggested as a possible
mechanism for acid transduction (14, 22, 33). However,
HCl-induced CT responses are not affected by apical Na+,
amiloride, or changes in the transepithelial potential of the receptive
field under stimulation (9, 34). Similarly, HCl-induced changes in TRC pHi were unaffected by apical
Na+, amiloride, or changes in basolateral K+
concentration (unpublished observations). In mouse TRCs, acid responses
were insensitive to amiloride but were blocked by
5-nitro-2-(3-phenylpropyl-amino)benzoic acid, a Cl
channel blocker (26). Both the mammalian brain
Na+ channel (BNC1) (36) and the HCN family of
channels (32) have been shown to be present in rat vallate
TRCs. However, recent evidence suggests that BNC1 is most
likely not involved in sour taste transduction (19).
Alternatively, H+ ions may pass through paracellular shunts
to reach TRC basolateral membrane sites (9). These sites
could belong to the HCN channel family.
pHi regulation and pH tracking in TRCs.
The acid-induced changes in TRC pHi were sustained. A
sustained change in pHi is a common feature of acid-sensing
cells (30, 37, 38). This, however, does not imply that
TRCs lack the ability to regulate pHi. We have previously
(23, 34) shown that at constant pHo, isolated
TRCs recover from intracellular acid loading. It is likely that during
acid stimulation, the inhibition of pH recovery mechanisms contributes
to a sustained decrease in pHi (Ref. 30 and
unpublished observations). This ensures that for a given acid stimulus,
the induced pHi changes will be maximal and graded.
In contrast to the apical membrane, the relationship between
pHi and pHo across the basolateral membrane
gave a slope of 0.69 (unpublished observations). A similar relationship
between pHi and pHo was observed in isolated
TRCs (23, 34). This suggests that the apical membrane of
TRCs is significantly less conductive to H+ ions relative
to the basolateral membrane and acts as a filter for H+
ions. In concert with the paracellular shunt pathway (9,
34), apical cell membranes of TRCs help to regulate
H+ concentration, thereby protecting the sensory apparatus
from hyperacidic conditions. Consequently, a large change in apical pHo is translated into a relatively small change in
pHi. This ensures that variations in TRC pHi
remain within the physiological range during the rigors of acid
stimulation (5, 30, 38).
Role of pHi in transduction.
In the case of rat acid-sensitive taste receptors, transduction steps
subsequent to the decrease in pHi are still unknown. In
carotid body type I cells, decrease in pHi leads to a
conductance increase, membrane depolarization, and an increase in
intracellular Ca2+ concentration (5). In the
acid-sensors in the locus coeruleus, the proximate stimulus is still a
decrease in intracellular pH, but in this case depolarization results
from proton-induced closure of K+ channels
(28). Divergence in the subsequent transduction steps in
these cases suggests the possibility of alternate mechanisms leading to
depolarization in taste cells. To some extent, one should anticipate
this given the diversity of mechanisms that have been proposed for sour
taste transduction in various species. For example, in the case of
Necturus, a decrease in apical pHo depolarizes
taste cells by closing apical K+ channels
(18). Our preliminary data (unpublished observations) suggest that TRC pHi is regulated via intracellular second
messengers (e.g., intracellular Ca2+ concentration and
cAMP). This suggests that, similar to other sensory cells, sustained
changes in TRC pHi during acid transduction must involve
activation of intracellular signaling mechanisms that result in
depolarization of TRC receptor potential and release of
neurotransmitter. This is consistent with the studies of Gilbertson et
al. (15) where the contribution of cAMP to the development of acid-responses is also indicated.
 |
ACKNOWLEDGEMENTS |
We thank Victoria Bickel for assistance with stimulation chamber
fabrication, Dr. Niley Desai for assistance with imaging, and Dr.
Steven Price for helpful suggestions.
 |
FOOTNOTES |
This work was supported by National Institute of Deafness and other
Communications Disorders Grants DC-02422 and DC-00122 and by the
Department of Veterans Affairs. During this study, V. Lyall was
partially supported by the Veterans Affairs Merit Review.
Address for reprint requests and other correspondence: V. Lyall, Dept. of Physiology, Virginia Commonwealth Univ., Sanger Hall
3002, 1101 E. Marshall St., Richmond, VA 23298-0551 (E-mail: vlyall{at}hsc.vcu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 January 2001; accepted in final form 24 April 2001.
 |
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