Acid-induced responses in hamster chorda tympani and
intracellular pH tracking by taste receptor
cells
Robert E.
Stewart1,
Vijay
Lyall1,
George M.
Feldman1,2,3,
Gerard L.
Heck1, and
John A.
DeSimone1
Departments of 1 Physiology and
2 Medicine, Virginia Commonwealth
University, Richmond 23298; and
3 McGuire Veterans Affairs Medical
Center, Richmond, Virginia 23249
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ABSTRACT |
HCl- and NaCl-induced hamster chorda tympani nerve responses
were recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na+ channel blockers,
amiloride and benzamil, and to methylisobutylamiloride (MIA), an
Na+/H+
exchange blocker. Responses to NaCl were unaffected by MIA but were
suppressed by benzamil. Microfluorometric and imaging techniques were
used to monitor the relationship between external pH
(pHo) and the intracellular pH
(pHi) of fungiform papilla taste
receptor cells (TRCs) following
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading.
TRC pHi responded rapidly and
monotonically to changes in pHo.
This response was unaffected by
Na+ removal or the presence of
amiloride, benzamil, or MIA. The neural records and the data from
isolated TRCs suggest that the principal transduction pathway for acid
taste in hamster is similar to that in rat. This may involve the
monitoring of changes in TRC pHi mediated through amiloride-insensitive
H+ transport across TRC membranes.
This is an example of cell monitoring of environmental pH through pH
tracking, i.e., a linear change in
pHi in response to a change in
pHo, as has been proposed for carotid bodies. In taste, the H+
transport sites may be concentrated on the basolateral membranes of
TRCs and, therefore, are responsive to an attenuated
H+ concentration from diffusion of
acids across the tight junctions.
lingual voltage clamp; microfluorometry; imaging; fungiform
papillae
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INTRODUCTION |
SOURNESS IS THE TASTE quality that humans typically
associate with acidic stimuli (20). The quality and intensity of acidic stimuli vary considerably, depending on
H+ concentration, the accompanying
anion species, and, in the case of "weak" acids, titratable
H+ stored as undissociated acid
(8, 25). Several cellular mechanisms for
H+ transduction in taste receptor
cells (TRCs) of amphibians and mammals have been suggested from results
of neurophysiological, electrophysiological, microfluorometric, and
imaging studies.
Whole cell and loose patch voltage-clamp studies in situ and in
isolated Necturus TRCs have shown that
acidic stimuli depolarize and elicit action potentials from taste cells
by decreasing a resting, outward
K+ conductance, probably by
H+ blockage of a voltage-sensitive
apical K+ channel (14, 15).
Studies on isolated bullfrog TRCs suggest that acids induce a
depolarizing current by increasing an
H+-gated
Ca2+ conductance (22).
Electrophysiological recordings from isolated hamster TRCs and from
intact hamster taste buds in vitro (10, 11) have provided evidence for
H+ transduction by translocation
through amiloride-sensitive, apical Na+ channels. In these studies,
fast current transients elicited from taste buds by both
H+ and
Na+ were inhibited by amiloride
(10), as were voltage-clamping currents in whole cell patch-clamp
studies (11). On the other hand, recordings from the rat chorda tympani
nerve during voltage clamp of the lingual receptive field failed
to demonstrate either voltage or amiloride sensitivity of HCl taste
responses (6). Notably, HCl taste stimuli caused an increase in the
lingual transepithelial resistance in rats and the polarity of the
transepithelial potential reversed sign on application of rinse
solutions or salt stimuli (6). These observations imply that
H+ pass through the paracellular
pathway and are buffered by fixed anionic sites. Therefore,
acid-induced effects on TRCs may not be restricted to the apical cell
membrane.
More recently, measurement of intracellular pH
(pHi) in isolated rat taste bud
fragments (TBFs) by microfluorometry, and in single isolated TRCs by
imaging techniques, showed that changes in external pH
(pHo) induced parallel changes
in TRC pHi (18, 19). The
pHo-induced changes in TRC
pHi were rapid and monotonic and
displayed an average slope close to unity. It was suggested that
changes in TRC pHi may be involved
in acid taste transduction. Therefore, as in amphibians, considerable
diversity in acid transduction mechanisms may exist in mammalian
species.
In epithelial cells (17, 23, 24), including hamster TRCs (11),
H+ can permeate cell membranes via
amiloride-sensitive apical Na+
channels. In the present study, we tested the hypothesis that acid
taste transduction involves H+
flux via amiloride-sensitive apical
Na+ channels in hamster TRCs by
using both in vivo and in vitro approaches. This was accomplished by
recording hamster chorda tympani responses to HCl and NaCl under
current and voltage clamp and in the presence and absence of amiloride
and its analogs. In addition, using microfluorometric and imaging
techniques, we investigated the relationship between pHo and TRC
pHi in isolated hamster TBFs and
single TRCs from fungiform papillae in the presence and absence of
amiloride and its analogs. We predicted that, if apical
H+ flux contributes appreciably to
the stimulation of taste nerves by HCl, taste nerve responses to HCl
should be modulated by voltage clamp of the lingual receptive field and
suppressed by amiloride and its analogs in a manner analogous to that
observed with Na+ salts. In
addition, amiloride and its analogs should significantly affect
pHi and the
pHi vs.
pHo relation observed in isolated
TRCs (18).
We found that HCl caused a concentration-dependent increase in hamster
chorda tympani responses. However, chorda tympani responses to HCl, at
concentrations comparable to those used to obtain NaCl responses, were
virtually insensitive both to voltage clamping and to amiloride or its
analogs. In the subset of isolated
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded hamster TRCs investigated,
pHi showed a strong, linear
dependence on pHo that was
unaffected by amiloride and its analogs. This pH tracking behavior is
similar to that observed in rat TRCs (18) and analogous to that
reported for pH-sensing carotid body type 1 cells (2). This further
suggests that changes in TRC pHi
may be involved generally in H+
detection. Moreover, the data suggest that hamster fungiform papilla
taste cells, like those of rat (6), transduce
H+ stimuli at a site below the
taste bud tight junction.
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MATERIALS AND METHODS |
Recording Chorda Tympani Responses to Acid
Stimuli.
The rinse solution contained 15 mM
KHCO3 and 20 mM KCl (pH 8.3). A
Na+-depleted Krebs-Henseleit
solution (DKH) containing (in mM) 6 KCl, 2 CaCl2, 1.2 MgSO4, 1.3 NaH2PO4,
25 NaHCO3, and 5.6 glucose (pH
7.5) was applied after each stimulus series to maintain a stable
transepithelial potential. The transepithelial potential in the
presence of 150 mM NaCl rarely varied more than ±5 mV during an
experiment. Stimulus series were with 1 and 10 mM HCl and 10 and 100 mM
NaCl.
Nerve preparation and recording.
Neural responses were recorded from the chorda tympani nerves of male
Syrian Golden hamsters (Charles River Laboratories; 70-120 days of
age) during chemical stimulation of the tongue. Hamsters were
anesthetized by brief exposure to ether followed by intraperitoneal
injection of pentobarbital (90 mg/kg). Supplemental pentobarbital (30 mg/kg) was administered as necessary to maintain surgical anesthesia.
Body temperature was maintained at 36-37°C with a circulating
water heating pad. The left chorda tympani was exposed laterally as it
exited the tympanic bulla, as previously described (13). After the
chorda tympani was dissected free from surrounding tissue, it was cut
proximally, desheathed, and placed onto a 32G platinum-iridium wire
electrode. An indifferent electrode was placed in nearby tissue.
Simultaneous epithelial voltage clamping and chorda tympani neural
recordings have been described (31). Stimulus solutions and rinses were
injected (4 ml; 1 ml/s) into a Lucite chamber affixed by vacuum to a
28-mm2 patch of the anterior
dorsal lingual surface. The chamber was fitted with separate Ag-AgCl
electrodes for measurement of current and potential, and reference
electrodes were placed noninvasively on the ventral lingual epithelium.
The current-passing electrode within the chamber served as a virtual
ground, ensuring that only current passing through the stimulated patch
was collected. Neural responses were differentially amplified with a
custom-built, optically coupled isolation amplifier, then full-wave
rectified and integrated with a time constant of 1 s. Integrated neural
responses and voltage and current records were recorded on a Linseis
TYP7045 chart recorder (Princeton Junction, NJ).
Stimuli were applied under zero current clamp, and steady-state
potentials were recorded from the display of the voltage-current clamp
amplifier (Physiologic Instruments VCC600, San Diego, CA). Chorda
tympani responses were then obtained under voltage clamp at +60 and
60 mV relative to the zero current clamp potential recorded for
each stimulus. Each stimulus series was bracketed by application of 150 mM NaCl under zero current clamp. Neural data were
excluded when bracketing responses varied by more than 10%.
Steady-state response magnitudes, the height of each integrated chorda
tympani response 30 s after application of the stimulus, were expressed
relative to the mean 150 mM NaCl response magnitude bracketing a
stimulus.
The protocol for stimulation of the lingual epithelium was as follows.
First, the reference stimulus (denoted
RN) was applied and allowed to
remain on the tongue for 40-50 s. The reference solution was then
rinsed with several applications (typically three) of the
KHCO3-KCl solution (denoted
R0) for a total of at least 60 s. Next, the stimuli of interest were applied as a concentration series
(denoted S1 and
S2), with each allowed to remain
in the chamber for 40-50 s. Individual stimulus applications were
followed by rinsing as described above. After the second stimulus in a series was rinsed, the reference stimulus was reapplied and rinsed. At
this point, DKH was applied, allowed to remain on the tongue for 60 s,
and then rinsed. Finally, the reference stimulus was applied and then
rinsed, and the next concentration series was started. This protocol
can be represented as follows
The voltage-sensitivity index (VSI) was used as a measure of
the relative voltage dependence of chorda tympani responses to various
stimuli (6, 32). The VSI was defined as
where
R(C,
60) is the mean chorda tympani response magnitude at
concentration C and clamp voltage of
60 mV and R(C,+60) is the
mean response at the same concentration and clamp voltage of +60 mV.
Chorda tympani responses were also obtained with conventional recording
techniques (i.e., without voltage clamping) in a separate set of
hamsters. Chorda tympani responses to 100 mM NaCl were obtained in the
absence and presence of 5 µM benzamil, a specific Na+ channel blocker, or 1 µM
methylisobutylamiloride (MIA), a specific Na+/H+
exchange blocker (both from Research Biochemicals International, Natick, MA) (17). In addition, responses to 1 and 10 mM HCl were
obtained in the absence and presence of 50 µM amiloride (Sigma Chemical, St. Louis, MO) or 5 µM benzamil or 1 µM MIA. Responses to
these NaCl and HCl solutions were expressed relative to responses to
300 mM NH4Cl. Solutions containing
amiloride, benzamil, or MIA were made freshly each day from frozen
stock solutions as described in Composition of
Solutions.
pHi Measurements in Isolated TBFs
Preparation of TBFs.
Hamsters were anesthetized with ether and then killed by cervical
dislocation. The tongue was rapidly removed and stored in ice-cold
HEPES-buffered Tyrode solution (pH 7.4) preequilibrated with 100%
O2. Fungiform TBFs were prepared
by the conventional collagenase (Boehringer Mannheim, Indianapolis, IN)
method. The details of the method and the tests used to determine the
viability of TRCs in isolated TBFs were as described previously (18).
Perfusion chamber.
The open perfusion chamber consisted of a standard glass slide onto
which a piece of Silastic sheet (with a
4-cm2 cutout window in the center)
was glued (18). An infusion pump delivered solutions (4 ml/min) into
the chamber through ports located in three sides of the chamber. The
chamber surface was precoated with Cell-Tak (1 µg/cm2; Collaborative Research,
Bedford, MA) to affix the cells. Fluid exchange, measured as BCECF
(Molecular Probes, Eugene, OR)-acid washout from the chamber, had an
initial rapid phase, during which 70% of the dye was cleared from the
chamber in 4 s (n = 5), and a slower
phase, during which another 20% of dye was cleared in 33 s (18). All
tubing was thoroughly washed with distilled water after each
experiment. In addition, a fresh chamber was made for each experiment
by attaching the Silastic sheet to a new glass slide precoated with
Cell-Tak.
pHi measurements.
TBFs were loaded with BCECF by incubation in HEPES-buffered Tyrode
solution (pH 7.4) containing 30 µM BCECF-AM (Molecular Probes) for 1 h in the dark at 4°C. TBFs were then transferred to the
Cell-Tak-coated perfusion chamber and washed 15 min later by perfusion
with 30 ml of HEPES-buffered Tyrode solution. As described earlier
(18), both microfluorometric and imaging techniques were used to
measure pHi in isolated TBFs.
Briefly, the BCECF-loaded TBFs and single isolated TRCs were observed
through a Zeiss ×40 objective (numerical aperture = 0.9).
Excitation light was alternated between 440 and 490 nm using a filter
wheel in the microfluorometry system and a slider in the imaging
system. The dichroic beam splitter and emission filters were 515 and
535 nm, respectively. Filters were obtained from Omega Optical. In the
imaging system, each image was constructed from the average of eight
frames. The pHi was determined
from the ratio of fluorescence emission intensities, F490/F440
ratio, where F is fluorescence, and the signals were calibrated using
high-K+ plus nigericin solutions.
All measurements were made at room temperature (22 ± 1°C).
As in our previous study (18), measurements of
pHi on isolated TBFs were accepted
only if the following criteria were met: 1) the TRCs retained the BCECF
throughout the experiment and 2) the
TBFs gave a satisfactory pH calibration with nigericin-containing, high-K+ calibrating solutions at
the end of the experiment. The reported values of
pHi were averaged over a 30-s
interval following the establishment of a steady state.
Composition of Solutions
Except where otherwise noted, all reagents were obtained from Sigma
Chemical. HEPES-buffered Tyrode solutions contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 sodium pyruvate, 10 glucose, and 10 HEPES; the pH was adjusted to 7.4 with NaOH, unless otherwise mentioned. Na+-free
HEPES solutions contained 140 mM
N-methyl-D-glucamine
(NMDG)-HCl and 10 mM NMDG-pyruvate. In some Tyrode solutions, 30 mM
NaCl was replaced with 30 mM sodium acetate. The
pHi-calibrating solutions contained (in mM) 140 KCl, 4.6 NaCl, 1 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, and
0.01 nigericin. Stock solutions of benzamil were made in distilled
water, whereas stock solutions of BCECF-AM, amiloride, MIA, and
nigericin were made in DMSO (Molecular Probes). Equivalent amounts of
DMSO were added to control solutions. The final concentration of DMSO
in the solutions was <0.02% (vol/vol).
Statistical Analyses
Data are expressed as mean ± SE, unless indicated otherwise.
Potential-dependent differences in chorda tympani response magnitudes were assessed using ANOVA with Student-Newman-Keuls posttests (SNK)
where appropriate. Differences between response magnitudes with and
without Na+ channel and antiport
blockers were determined using paired Student's t-tests. Differences in TRC
pHi were analyzed using both
paired and unpaired Student's
t-tests. The slopes and correlation
coefficients of the least squares regression lines drawn through data
plots of pHi vs.
pHo are represented by
S and
r, respectively.
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RESULTS |
Chorda Tympani Responses to HCl and NaCl Under Voltage Clamp
Figure 1 shows chorda tympani
responses to 10 mM NaCl and to 1 and 10 mM HCl under zero current clamp
and ±60-mV voltage clamp. Chorda tympani responses to 10 mM NaCl
were suppressed under submucosal-positive voltage clamp, whereas those
obtained under submucosal-negative voltage clamp were elevated relative
to responses at zero current clamp. Similar, but more pronounced,
changes in chorda tympani response magnitude under voltage clamp were
observed with 100 mM NaCl. ANOVA with post hoc SNK posttests revealed,
for both 10 mM (Fig. 2) and 100 mM NaCl,
that chorda tympani responses under positive voltage clamp were
significantly suppressed and those under negative voltage clamp were
significantly elevated vs. responses obtained under zero current clamp
[with 10 mM: F(2,12) = 21.60, P < 0.0001; with 100 mM:
F(2,11) = 88.90, P < 0.001; posttest
P values < 0.05]. Figure 1
also shows chorda tympani responses to 1 and 10 mM HCl obtained from
the same hamster. Lingual receptive field voltage perturbations had
little impact on the magnitude of chorda tympani responses to HCl.
Indeed, no significant effect of voltage-clamp condition on chorda
tympani response magnitudes to either 1 or 10 mM HCl was detected
[ANOVA F(2,10)
1.87, P values
0.21] (Fig. 2). This
lack of HCl response voltage sensitivity is illustrated further by
calculations of VSIs for each stimulus (Table
1).

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Fig. 1.
Integrated chorda tympani taste responses to 1 and 10 mM HCl
(middle and
bottom) and to 10 mM NaCl
(top) obtained under zero current
clamp and under +60- and 60-mV voltage clamp. The magnitude of
responses to 10 mM NaCl exhibits marked sensitivity to imposed voltage
perturbations. In contrast, chorda tympani responses to 1 and 10 mM HCl
showed little change in magnitude when transmural potential was clamped
at values either positive or negative to the zero current clamp
potential elicited by these stimuli. Superimposed spikes are caused by
applied current (zero current clamp) or voltage (voltage clamp) pulses
used to monitor transmural resistance.
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Fig. 2.
Mean chorda tympani relative response magnitudes for 1 and 10 mM HCl
and 10 mM NaCl obtained with zero current clamp and with +60- or
60-mV voltage clamp. Responses to either concentration of HCl
showed no significant sensitivity to applied voltage perturbations. In
contrast, responses to 10 mM NaCl were significantly suppressed under
positive voltage clamp (relative to both zero current clamp and
60-mV voltage clamp) and significantly elevated under negative
voltage clamp (relative to both zero current clamp and positive voltage
clamp) (n = 6).
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As expected, positive VSIs for NaCl stimuli were obtained, reflecting
the voltage dependence of Na+
movement into taste cells via apical
Na+ channels. In contrast, VSIs
for HCl stimuli were both close to zero. The ratio of the VSI for 10 mM
NaCl to that for 10 mM HCl is 16.6, whereas the ratio of the VSIs for
100 mM NaCl to 10 mM HCl is 36.8. That is, chorda tympani responses to
10 and 100 mM NaCl are, respectively, 17 and 37 times more sensitive to
voltage perturbation than are those to 10 mM HCl.
Another feature of the hamster HCl taste response is the transmural
potential response to HCl application and rinse. Figure 3 depicts integrated chorda tympani
responses and corresponding potentials evoked under zero current clamp
by 10 mM HCl and 100 mM NaCl. The potential attending application of
100 mM NaCl was reminiscent of those observed in the rat on application
of Na+ stimuli to the lingual
receptive field. Specifically, there was a rapid, inwardly directed
positive change in potential followed by a pseudo-steady-state
potential that paralleled the tonic phase of the chorda tympani
response. After rinse with NaCl, the potential abruptly returned to
resting value after a brief overshoot. In comparison, the positive
potential change in response to application of 10 mM HCl had a somewhat
slower initial ascending phase followed by a broad, slowly rising
component. After rinse with HCl, a sizable, positive-going potential
was observed, followed by a slow return to baseline potential values.
It is notable that the duration of the postrinse "afterpotential"
seemed to be highly correlated temporally with persistent elevated
baseline neural activity that was frequently observed after rinsing HCl
stimuli. These data are consistent with the HCl-induced responses in
rats reported earlier by DeSimone et al. (6).

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Fig. 3.
Integrated chorda tympani (B) and
corresponding evoked transepithelial potential
(A) responses to 10 mM HCl and 100 mM NaCl. Potentials evoked by 10 mM HCl exhibited an initial rising
phase, followed by a broad rising phase. When rinse (R) was applied, a
brief potential reversal was observed, followed by a slow return to
baseline values. This potential reversal and slow relaxation to
baseline corresponded well with large rinse transients and persistent
elevated activity in the neural response on and following rinse of HCl.
In comparison, 100 mM NaCl application was attended by a rapid,
positive change in potential to a pseudo-steady-state value that
paralled the tonic phase of the chorda tympani response. After rinse
with NaCl, transepithelial potential returned abruptly to resting
values. Voltage scale applies only to potential records.
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Sensitivity of Chorda Tympani HCl and NaCl Responses to Amiloride
and Its Analogs
As expected, chorda tympani responses to 100 mM NaCl were significantly
suppressed when this stimulus was dissolved in 5 µM benzamil
(Student's t = 5.92, P < 0.002) (Figs.
4 and 5). In
comparison, 1 µM MIA had no measurable effect on chorda tympani
responses to 100 mM NaCl (Student's t = 1.10, P
0.35). Also, 50 µM
amiloride, 5 µM benzamil, and 1 µM MIA did not affect the magnitude
of chorda tympani responses to 1 or 10 mM HCl
(P values
0.12). However, in three
preparations, considerable (up to 50%) suppression of 10 mM HCl
responses by benzamil was observed, whereas in no cases were responses
to HCl suppressed by amiloride. Suppression by benzamil occurred in
preparations that had unusually large chorda tympani responses to 10 mM
HCl (up to twice the mean relative response magnitude to this
stimulus).

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Fig. 4.
Integrated chorda tympani responses to 100 mM NaCl and 10 mM HCl in the
absence or presence of benzamil. As expected, responses to 100 mM NaCl
were virtually eliminated when the solvent was 5 µM benzamil
hydrochloride. In contrast, responses to 10 mM HCl were essentially
unaffected by 5 µM benzamil hydrochloride.
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Fig. 5.
Influence of amiloride and analogs on mean chorda tympani relative
response magnitudes to HCl and NaCl. No significant effects of
amiloride (50 µM) or benzamil (5 µM) on chorda tympani responses to
1 or 10 mM HCl were observed. In contrast, benzamil significantly
suppressed chorda tympani responses to 100 mM NaCl.
Methylisobutylamiloride (MIA, 1 µM) did not significantly affect
chorda tympani responses to HCl or NaCl.
* P < 0.002; nd, not
determined (n = 5-8).
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Characteristics and Pharmacology of pH Tracking by TRCs
In typical preparations, hamster fungiform papilla TBFs (Fig.
6A) and
individual TRCs were readily identified (Fig.
7A).
Occasionally, some TRCs were rounded. In some taste buds, BCECF was
distributed in most, if not all, cells (18). These cases were normally
selected for further study. In individual TRCs, BCECF fluorescence was intracellular (Fig. 7B). The TBFs
and TRCs retained the dye without significant loss for several hours,
were then calibrated satisfactorily, and were determined to be viable
and functional by criteria described elsewhere (18). As done
previously, microfluorometric measurement of
pHi was carried out exclusively on
a subset of TBFs in which the dye appeared to be distributed in most
cells (18). In single, isolated TRCs,
pHi was measured by imaging (see
Fig. 7).

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Fig. 6.
Acid-induced responses of an isolated taste bud fragment (TBF) loaded
with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF). A: an optical section through
an isolated hamster fungiform papilla TBF. Apical pole of the TBF
(arrow) and several taste receptor cells (TRCs) can be easily
recognized by their spindle shape. Scale bar, 5 µm. TBF was incubated
in HEPES-buffered Tyrode solution containing 30 µM BCECF-AM for 1 h
at 4°C in the dark. TBF was arbitrarily divided in small regions of
interest (ROIs; 3 × 3 µm). B:
effect of step changes in external pH
(pHo; 7.77, 7.39, and 6.78) on
intracellular pH (pHi) in
individual ROIs. Hatched bars represent the slope of the relationship
between pHi and
pHo in individual ROIs. Solid bar
represents the mean slope (S = 1.06 ± 0.05; r = 0.973 ± 0.005).
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Fig. 7.
Pseudocolor ratio images of TRCs loaded with BCECF.
A: an optical section through at least
2 fungiform TRCs. Scale bar, 5 µm.
B: same optical section is shown as
BCECF fluorescence image, excited at the pH-sensitive wavelength, 490 nm. Note that the dye distribution pattern reflects the irregular
shapes of the TRCs. Same optical section is shown as the ratio of BCECF
fluorescence pseudocolor images when excited alternately at 490 and 440 nm at the steady-state pHo of 6.6 (C) and 7.7 (D). Note that a decrease in
pHo induced parallel decreases in
pHi in both the soma and apical
processes.
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Effect of changes in pHo on
pHi.
At pHo of 7.4, the mean resting
TRC pHi was 7.46 ± 0.04 (n = 6). This value compares well with
the resting values of rat TRC pHi
measured under identical conditions (18). Figure
8 shows the effect of step changes in
pHo on TBF
pHi. When the TBF was initially
perfused with HEPES-buffered Tyrode solution at pH 7.42, the cells
maintained a mean resting pHi near
7.4. If pHo was decreased to 6.78 and subsequently raised to 7.87, pHi responded rapidly and
stabilized at values near 6.8 and 8.0, respectively. When pHo was returned to 7.42, pHi promptly returned to a value
close to 7.4. Figure 9 shows the relation
of pHi to
pHo in six TBFs. The
S of the regression lines of
pHo vs.
pHi plotted for individual TBFs
varied between 0.94 and 1.24 and gave a mean value of 1.08 ± 0.04. The mean r was 0.996 ± 0.003. The
maximum rates of change in pHi
(maximum
pHi/min) for different
pHo steps varied between 1.8 and
4.3 and depended on the pHo
gradient (Table 2). The
pHo-induced changes in
pHi were monotonic and stable.
Overall, pHo-induced changes in
hamster fungiform TBF pHi were
very similar to those seen in rat TBFs (18).

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Fig. 8.
Effect of changing pHo on
pHi. A TBF was perfused with
Tyrode solution of pH 7.42, 6.78, and 7.87. Top horizontal bar
represents periods during which the TBF was perfused with solutions of
different pHs.
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Fig. 9.
Relationship between pHo and
pHi. Steady-state relationship
between pHo and
pHi in 5 TBFs under control
conditions ( ). Line of best fit determined by linear regression
(S = 1.08 ± 0.04;
r = 0.996 ± 0.003). Steady-state
relationship between pHo and
pHi in 4 paired TBFs in the
presence of 0.1 mM amiloride ( ; S = 1.10 ± 0.05; r = 0.996 ± 0.003). Steady-state relationship between
pHo and
pHi in 4 paired TBFs in the
absence of external Na+ ( ;
S = 1.05 ± 0.05;
r = 0.996 ± 0.001).
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Effect of amiloride on pH tracking.
Figure 10 shows the effect of 100 µM
amiloride on the pHo-induced
change in TBF pHi. Exposure to
amiloride induced a small, significant decrease in resting TBF
pHi. In four paired experiments in
the presence of amiloride, pHi was
7.42 ± 0.06, a value significantly lower than the
pHi of 7.50 ± 0.06 measured in
its absence (
pHi = 0.08 ± 0.03, P < 0.05, paired).
However, as shown in Fig. 10, the
pHo-induced changes in TBF
pHi were not affected by
amiloride. The relationship between
pHo and
pHi in four paired experiments is
also shown in Fig. 9. The S of the
regression lines of pHo vs.
pHi plots for individual TBFs
varied between 0.99 and 1.24, with a mean of 1.10 ± 0.05 (mean r = 0.996 ± 0.003). As also
shown in Table 2, amiloride did not affect the maximum rates of change in pHi (maximum
pHi/min) for different
pHo steps.

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Fig. 10.
Effect of changing pHo on
pHi in the presence of 0.1 mM
amiloride. A TBF was initially perfused with Tyrode solution of pH
7.48. This was followed by perfusion with solutions of pH 7.48, 6.82, and 7.89 in the continuous presence of 100 µM amiloride. Top
horizontal bar represents periods during which the TBF was perfused
with solutions of different pHs.
|
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Effect of Na+
substitution.
Figure 11 shows the effect of
Na+ removal on
pHo-induced effects on TBF
pHi. Replacement of
Na+ with
NMDG+ induced a small decrease in
resting TBF pHi. In four paired
experiments, the pHi in the
absence of Na+ was 7.38 ± 0.04, a value significantly lower than the
pHi of 7.50 ± 0.06 in control
medium (
pHi = 0.11 ± 0.04, P < 0.05, paired). However,
pHo-induced changes in TBF
pHi were not affected by
Na+ removal (Fig. 11). The
relationship between pHo and
pHi in four paired experiments in
the absence of Na+ is plotted in
Fig. 9. The S of the regression lines
of pHo vs. pHi plotted for individual TBFs
varied between 0.92 and 1.14 and gave a mean of 1.05 ± 0.05 (mean
r = 0.996 ± 0.001). As also shown in Table 2, Na+ removal did not
affect the maximum rates of change in
pHi (maximum
pHi/min) for different
pHo steps.

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Fig. 11.
Effect of changing pHo on
pHi in the absence of external
Na+. A TBF was initially perfused
with Tyrode solution of pH 7.40. This was followed by perfusion with
Na+-free solutions of pH 7.38, 6.68, and 7.79. Top horizontal bar represents periods during which the
TBF was perfused with solutions of different pHs.
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|
In three additional experiments, TBFs were bathed in
Na+-free solutions containing 100 µM amiloride. In the continuous presence of amiloride, the
pHo-induced changes in TRC
pHi were not different from those
observed in TRCs bathed in
Na+-free solutions without
amiloride, as shown in Figs. 9 and 10.
Effect of weak acids.
To determine if the neutral forms of weak acids are membrane permeable
and contribute to changes in pHi,
TBFs were perfused with Tyrode solutions containing 30 mM sodium
acetate at constant pHo. TBFs
responded to sodium acetate solution with a rapid intracellular acidification followed by a spontaneous recovery of
pHi (Fig. 12). When sodium acetate was subsequently
removed, there was a rapid intracellular alkalinization followed by a
recovery phase. Similar changes in
pHi were observed in two
additional TBFs exposed to sodium acetate. These data are consistent
with the effects of weak acids on rat TBFs (18) and suggest that TRCs
behave like many other cells: 1) the
TRC membranes are intact and are able to partition undissociated and
dissociated species of weak acids;
2) BCECF is intracellular and
reflects changes in pHi; and
3) at
iso-pHo TRCs exposed to weak acids
can regulate pHi.

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Fig. 12.
Effect of sodium acetate on TRC
pHi. A TBF initially perfused with
control Tyrode solution was exposed to a similar Tyrode solution
containing 30 mM sodium acetate. Top horizontal bar represents periods
during which the TBF was perfused with solutions with and without
acetate. Broken lines represent the time periods during which the TBFs
were not exposed to excitation light to prevent quenching of the dye.
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|
Changes in TRC pHi Monitored by Imaging
To determine whether pHo has
regional effects on pHi of TBFs
and isolated TRCs, imaging studies were performed. Images of an
isolated fungiform papilla TBF were acquired within the same optical
plane at pHo of 7.77, 7.39, and
6.78. The acquired images were arbitrarily divided into 20 small
regions of interest (ROIs; 3 × 3 µm). Each ROI was individually
calibrated and served as its own control (18). For example, at
pHo of 7.77, the TBF
pHi in individual ROIs varied
between 7.47 and 8.3 (mean = 7.95 ± 0.06). At
pHo of 7.39, the
pHi varied between 7.04 and 7.66 (mean = 7.40 ± 0.033), and, at
pHo of 6.78, the
pHi varied between 6.79 and 6.96 (mean = 6.88 ± 0.01). The relationship between
pHo and mean
pHi in individual ROIs was linear.
Within individual ROIs, the r values
of the regression between pHo and
pHi ranged between 0.942 and 0.999 (mean = 0.973 ± 0.004), whereas S
of the regression lines varied between 0.51 and 1.42 (mean = 1.06 ± 0.05). In five TBFs, the relationship between
pHo and mean
pHi within individual ROIs was
also linear and had mean S values that
varied between 0.73 to 1.22 (mean = 0.96 ± 0.09;
r = 0.992 ± 0.005). This
relationship between pHo and
pHi is similar to that observed
microfluorometrically (Fig. 9). Finally, Fig. 7,
A-D,
shows the pHo-induced changes in
pHi in two fungiform papilla TRCs.
The TRCs responded to a decrease in
pHo with a parallel decrease in
pHi in both the soma and apical
processes.
In fungiform papilla TBFs, the relationship between
pHo vs.
pHi in 20 individual ROIs also
remained linear in the presence of 5 µM benzamil. The
S of the relationship between
pHo and
pHi in individual ROIs varied
between 0.69 and 1.29 (mean S = 1.03 ± 0.04), and the r values varied
between 0.95 and 0.99 (mean r = 0.985 ± 0.003). These values were not significantly different from those obtained under control conditions. In additional, in paired
experiments in three isolated TRCs exposed to 1 µM MIA or 5 µM
benzamil, the mean S of the
relationship between pHi and pHo was 1.04 ± 0.08 (mean
r = 0.98 ± 0.008). These results
are consistent with data obtained microfluorometrically, which
demonstrate no significant effect of amiloride on the relationship
between pHo and
pHi in TBFs (Fig. 9).
 |
DISCUSSION |
Chorda Tympani Response to NaCl
The amiloride-sensitive Na+
channel has been implicated in the transduction of
Na+ salt taste stimuli in every
mammalian species examined (28). Using in vivo receptive field
voltage-clamp methods, Ye et al. (30, 31) demonstrated that the
magnitude of rat chorda tympani responses to
Na+ salt stimuli can be modulated
by alteration of the driving force for
Na+ across the taste cell apical
membrane. The present results extend those findings by showing that
hamster chorda tympani responses to NaCl are also highly sensitive to
voltage perturbations applied to the lingual receptive field (29). We
conclude that the appropriate stimulus dimension for
Na+ taste stimuli in hamster is
the electrochemical concentration, as previously demonstrated in rat
(29, 30).
Chorda Tympani Response to HCl
Protons have been shown to permeate amiloride-sensitive
Na+ channels in epithelial cells
(17, 23, 24), including hamster fungiform papilla TRCs (10, 11). In the
present work, we conducted experiments to probe further the hypothesis
that apical, amiloride-sensitive Na+ channels are a major
transduction site for acid stimuli in hamster fungiform papilla taste
cells. Four specific experimental predictions were tested. First,
hamster chorda tympani responses to HCl should be modulated by applied
voltage perturbations, i.e., responses to HCl should be elevated under
negative voltage clamp and suppressed under positive voltage clamp in a
manner similar to NaCl. Second, chorda tympani responses should be
inhibited by apical Na+ channel
blockers, amiloride and benzamil, in a manner similar to NaCl response
inhibition. Third, resting steady-state TRC
pHi should be sensitive to
amiloride and benzamil. Fourth, if
pHo-induced changes in TRC
pHi have a steep relationship
similar to that observed in rat TRCs (18, 19), then the
S of this relationship should decrease
in the presence of amiloride or benzamil.
Voltage Sensitivity in HCl Chorda Tympani Responses
Chorda tympani recordings show that, in general, HCl responses were not
significantly influenced by voltage perturbation. This was unexpected
given the significant pH-induced currents and conductance increases
observed in whole taste buds (10) and single taste cells (11) in in
vitro electrophysiological recordings. Using 10 mM NaCl, we observed a
highly voltage-sensitive chorda tympani response; however, the chorda
tympani response to 10 mM HCl was voltage insensitive. Given the
conductance measurements reported from whole cell recordings on hamster
taste cells (11) and taste buds in situ (10) and with the use of the
Goldman-Hodgkin-Katz ion flux equation, the expected channel
conductance for the case of a mucosal-side stimulus
consisting of 10 mM H+ relative to
that for a 10 mM Na+ stimulus can
be estimated. The conductance (g) ratio
(gH to
gNa) is
estimated to be at least 25 for intracellular potentials less than
zero, consistent with the reported permeability ratio for the channel
(10). The expected proton influx through the apical amiloride-sensitive
channels should be, therefore, greater than that for
Na+ at the same concentration.
Were this proton traffic to cause taste nerve excitation, the voltage
sensitivity in the chorda tympani response to HCl would be comparable
to that observed with NaCl and, therefore, easily detectable in our
whole nerve recordings, especially in recordings made under variable
lingual voltage-clamp conditions. That this is not so requires some
further consideration.
It is possible that two populations of chorda tympani fibers contribute
to the whole nerve response to acids. One population of axons
innervates taste cells that possess apical, amiloride-sensitive Na+ channels (i.e., N fibers),
whereas the other population innervates taste cells that lack apical
Na+ channels (i.e., H fibers)
(13). It is conceivable, therefore, that in the presence of a dominant
H fiber response component, the N fiber (i.e., voltage and amiloride
sensitive) component of acid responses could be obscured. However, this
is unlikely because, in the hamster chorda tympani, N fibers comprise a
much larger segment of the total fiber population than do H fibers (13). It follows that a larger proportion of the whole nerve response
to acids should be contributed by N fibers than by H fibers. Because
the conductance of protons through the apical, amiloride-sensitive
Na+ channels is expected to be
greater than that of an equal concentration of
Na+, there is little reason to
believe a priori that protons would activate a smaller population of N
fibers than would Na+. On the
other hand, it is possible that voltage sensitivity to protons is
present only in the early transient of the N fiber response, i.e.,
voltage sensitivity is a time-dependent parameter. However, recordings
from whole hamster taste buds (10) and single taste cells (11) in vitro
clearly show pH-induced, amiloride-sensitive currents that were
sustained for tens of seconds. In addition, the present data indicate
that neither voltage perturbations nor pharmacological probes altered
the transient part of the chorda tympani response to HCl (Figs. 1 and
4). The virtual absence of an observable N fiber contribution to the
HCl response, despite the high proton conductance of the apical,
amiloride-sensitive Na+ channels,
suggests that some intervening process limits excitation of N fiber
taste afferents when their associated taste cells are stimulated by
acids. This is consistent with hamster chorda tympani single-unit
studies that show acids in general to be poor N fiber stimuli. Although
in a minority of N fibers HCl is a better stimulus than other acids, in
two-thirds of the N fibers HCl is as poor a stimulus as other acids
(13). It will, nonetheless, be important and revealing to determine if
voltage- and amiloride-sensitive acid responses are detectable in some
single chorda tympani axons and, if so, how such responses differ
quantitatively from those evoked by
Na+ salt stimuli.
Effect of Amiloride Analogs
In general, HCl chorda tympani responses in hamster were not
significantly inhibited by amiloride or benzamil, similar to results
for rat (6). Consistent with these results, responses of neurons in the
hamster nucleus of the solitary tract (NST) to anterior lingual
stimulation with 3 mM HCl were not affected by lingual preadaptation
with 10 µM amiloride (26). It must be noted, however, that
suppression by amiloride of hamster chorda tympani HCl responses has
been reported. However, it was either not statistically significant
(12) or was observed only inconsistently (13). We also noted an aspect
of this inconsistency because in three preparations benzamil (but not
amiloride) suppressed HCl responses.
Hettinger and Frank (13) suggested that some of the inconsistencies in
the suppression by amiloride of single chorda tympani neuron responses
to HCl could be attributed to "old" preparations that had been
stimulated repeatedly and, therefore, to progressive degradation of
Na+ channels. However, in our
three exceptional cases, the preparations were subjected to neither
unusually numerous HCl stimulations nor unusually lengthy recording
durations. Instead, a common feature in these three cases was an
unusually large baseline HCl response. Another aspect regarding
amiloride effects on acid responses is the mode of application of
amiloride during an experiment.
In a recent study (1), acid responses recorded from hamster NST neurons
that were classified as "NaCl-best" were suppressed when acidic
stimuli were dissolved in 10 µM amiloride and applied during the
tonic phase of the response to the stimulus without amiloride. This
finding contrasts with earlier results that showed that preadaptation
with amiloride did not significantly inhibit the response to acid of
such neurons (26). These conflicting results suggest that detection of
a suppressive effect of amiloride depends on the order of stimulus and
drug application. However, Hettinger and Frank (13) noted that, for
NaCl stimuli, brief pretreatment of NaCl-best chorda tympani fibers
with amiloride inhibited responses as completely as addition of the
drug during stimulation. In summary, these results emphasize that there
are variable effects of amiloride and its derivatives on whole nerve, single chorda tympani fibers and single NST unit responses to HCl that
are presently poorly understood. We speculate that the drug and
stimulus actions are governed by physiological state-dependent changes
in the TRC at the channel level and perhaps during adaptation in some
animals. Changes in channel function could be related to individual
differences in degradation and turnover (13) and/or hormonal
regulation of apical Na+ channels
(11, 13).
Studies on the effect of amiloride on taste behavior in hamsters
suggest that this drug may diminish the aversiveness of NaCl taste
(13). However, postingestional effects of amiloride could not be
completely ruled out. These studies have been confirmed and extended to
include the effect of amiloride on acid-related taste behavior in
hamsters (9). The taste aversiveness of pH 2.4 citric acid was
diminished when dissolved in 30 µM amiloride, although overall this
solution was still rejected. The results were obtained in 96-h,
two-bottle preference aversion tests. The lengthy duration of this
testing period, however, makes it difficult to distinguish taste-guided
behaviors, per se, from ingestive behaviors that result from
postabsorptive consequences of amiloride and citric acid intake.
Tracking of pH by Hamster TRCs
Steady-state TRC pHi.
In control solutions (pHo = 7.4),
the mean resting TRC pHi was 7.46 ± 0.04, and, like rat TRCs (18), hamster fungiform TRCs are capable
of regulating pHi when challenged
with weak acids (Fig. 12). Incubation of TBFs in amiloride or in
Na+-free Ringer solutions (Figs.
9-11) induced small, but significant, decreases in steady-state
TRC pHi, suggesting that some
taste cell pH regulatory mechanisms are partially amiloride and
Na+ sensitive. For example, a
decrease in pHi under such
conditions may be attributed to inhibition of an electroneutral,
amiloride-sensitive Na+/H+
exchange in the TRC membranes. Alternatively, the inhibition by
amiloride of apical Na+ channels
or Na+ removal in the absence of
amiloride could cause hyperpolarization of the cell membrane potential
and a consequent potential-dependent decrease in
pHi (17). However, the nature of
the pH regulatory pathways in TRCs is not known.
Relationship between pHi and
pHo in TRCs.
As in earlier work (18), we used two techniques, a photomultiplier tube
(PMT) system and an image acquisition system, to monitor
pHi. The two systems weigh the
data differently. The PMT system yields
pHi values on the basis of the
total amount of light emitted in the area of study, and all the cells
within the area contribute to the
pHi in proportion to their BCECF
content. The image acquisition system yields
pHi values in the spatial domain in which the ROIs are calibrated and analyzed individually. Therefore, PMT studies do not yield information regarding individual cells in the
area limited by the field diaphragm. Figures 8-12
present data from PMT studies and yield data from an average of three to five cells. The variation in
pHi in individual ROIs in TBFs was
investigated by imaging. Figure 6 shows that step changes in
pHo induced a wide distribution in
pHi values in different ROIs in
the TBFs. In 20 ROIs, the S of the
relation between pHo and
pHi varied between 0.51 and 1.42, with a mean value (±SD) of 1.05 ± 0.22. This distribution
demonstrates a heterogeneity in cell pH tracking ability that may
correspond to cells with different functions. The variation in the
pHi response suggests that it is
unlikely that all cells, in which
pHi varies with
pHo, participate in acid-taste
transduction (18). It is possible that acid-induced changes in
pHi modulate taste cell responses to other taste stimuli, such as sugars or alkaloids (7, 27). A unit
change in pHo caused changes in
pHi of ~0.65 pH units in type 1 cells of the carotid body, the primary chemosensors of arterial blood
pH (3, 21). Although a linear relationship between
pHi and
pHo appears to be characteristic
of cells that function as pH sensors (21), it is possible that cells
that are not acid sensors may also have this property.
Effect of amiloride, benzamil, and
Na+ removal on
pHo-induced changes in
pHi.
As shown in Figs. 6-11, microfluorometric and imaging studies
revealed that the association between
pHi and
pHo was insensitive to amiloride,
benzamil, MIA, Na+ removal, or
Na+ removal plus amiloride. These
data suggest that pHo-induced
changes in pHi are largely
independent of both
Na+/H+
exchange and H+ flux via apical,
amiloride-sensitive Na+ channels
in TRC membranes. In this respect, hamster fungiform papilla TRCs are
similar to type 1 cells of the rat carotid body (3). Despite the
presence of pH regulatory mechanisms (including Na+/H+
exchange,
Cl
/HCO
3
exchange, and a
Na+-HCO
3-dependent
mechanism), pHo-induced changes in
type 1 cell pHi were, as in
hamster TRCs, also rapid and monotonic (3, 4). Finally, the present
data (Fig. 8) suggest that hamster fungiform papilla TRCs, like those
in rat (18) and like type 1 cells of the carotid body (3), regulate
pHi in response to an acid load,
if pHo is held constant.
Relationship Between Chorda Tympani Recordings and pH Tracking by
TRCs
Measurements of TRC pHi were
limited to a minimum pH of 6.5 to remain within the dynamic range of
the pH-sensitive dye. On the other hand, solutions of pH greater than
~4.5 do not stimulate gustatory neural responses when applied to the
tongue surface. This creates an apparently large discrepancy between
the pHo tracking properties of
TRCs and the threshold pH required to stimulate a neural response.
However, this discrepancy relies on the assumption that receptor cells
in situ encounter a concentration of protons that is equivalent to that
in the bulk, mucosal stimulus solution (i.e., pH 4.5 or less). However,
as suggested elsewhere (6), the actual
H+ membrane pathways in
acid-sensing cells may lie below the tight junctions. Taste cells
would, therefore, sense lower H+
concentrations than those applied to the lingual surface because the
final concentration of H+ achieved
below the tight junctions by diffusion will be limited by the buffering
power of fixed anionic sites (18). This is supported by the observation
that the pH of the lateral intracellular spaces of Madin-Darby canine
kidney epithelial cell monolayers grown on permeable supports is
minimally sensitive to changes in
pHo (5). Consequently, testing
isolated cells with solutions of higher pH than would normally give a
neural response when applied to the tongue in vivo may be a reasonable
approximation of in situ taste cell function. However, to test this
hypothesis, further studies are needed in which
pHi is measured at lower
pHo values. In addition, it is
important to note that the present studies were done in isolated TBFs
and TRCs exposed to symmetric changes in
pHo. Because TRCs are normally
polarized, a critical question relates to the effects of unilateral
changes in apical pHo on the
pHi of TRCs in intact epithelium
that contains polarized taste buds.
Chorda tympani responses to HCl were insensitive to amiloride and its
analogs. Similarly, in our in vitro studies, amiloride and its analogs
did not affect pHo-induced changes
in TRC pHi. In addition, our data
show that both chorda tympani responses to HCl and pH tracking by TRCs
are independent of
Na+/H+
exchange in TRC membranes. Although the in vitro measurements of
pHi presented here do not
distinguish between H+ flux across
the apical and the basolateral membrane of TRCs, results from chorda
tympani recordings in hamsters (this study) and in rats (6) suggest
that protons that evoke most of the neural response to acids do not
traverse an apical conductance. Instead,
H+, like
K+ (31), may access a basolateral
transduction site following diffusion through taste bud tight
junctions. Indeed, this would account for the insensitivity of the
HCl-evoked chorda tympani response to both voltage perturbation and
amiloride analogs.
It is interesting that, under our experimental conditions, the ability
of TRCs to recover from changes in
pHi is also dependent on
pHo. Both hamster and rat (18)
TRCs recover spontaneously from changes in
pHi caused by exposure of the
cells to weak acids or bases at
iso-pHo. In contrast, when changes
in pHi were caused by alteration
of pHo, TRCs did not spontaneously
recover their pHi but instead
tracked pHo monotonically. Because
TRC pHi was a function of external
HCO
3 concentration at constant
PCO2 (18),
it is suggested that, under physiological conditions, salivary
HCO
3 participates in the maintenance
of steady-state TRC pHi and
neutralizes acid-induced changes in
pHi (27).
Overall, our data lead to the following conclusions.
1) The chorda tympani
responses of hamster to NaCl are, like rat, predominantly mediated
through an amiloride- and voltage-sensitive transduction pathway, most
probably apical membrane epithelial
Na+ channels.
2) The transduction
pathways that yield chorda tympani responses to HCl in hamster are,
like those of rat, predominantly voltage and amiloride
insensitive. 3) Isolated
taste cells in hamster, like those of rat, have cellular mechanisms
that correct perturbations in pHi
when they are made at constant extracellular pH.
4) However, a significant
fraction of cells rapidly changes pHi in proportion to changes made
in pHo, i.e., they track pH. 5) pH tracking may be a component of
the cellular transduction mechanism in
H+ taste sensing, analogous to
H+-sensing mechanisms proposed for
carotid body chemoreceptor cells. This last point should be regarded as
a fresh source of testable hypotheses aimed at elucidating cellular
events in H+ taste sensing.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Alexandre Fabiato for use of the image intensifier and
Drs. Steven Price, David Hill, and Jeanine Stewart for helpful
suggestions. Donald Spragg provided expert technical help with images.
 |
FOOTNOTES |
This work was supported by National Institute of Deafness and Other
Communications Disorders Grants DC-02422 and DC-00122 (J. A. DeSimone)
and by the A. D. Williams Foundation and the Department of Veterans
Affairs (G. M. Feldman). During this work, R. E. Stewart was supported
by National Heart, Lung, and Blood Institute Training Grant HL-07110.
Address for reprint requests and present address of R. E. Stewart:
Dept. of Psychology, Washington and Lee Univ., Lexington, VA 24450.
Received 15 May 1997; accepted in final form 3 April 1998.
 |
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