Effects of osmolarity on taste receptor cell size and
function
Vijay
Lyall1,
Gerard L.
Heck1,
John A.
DeSimone1, and
George M.
Feldman1,2,3
1 Department of Physiology and
2 Department of Medicine, Virginia
Commonwealth University, Richmond 23298; and 3 McGuire
Veterans Affairs Medical Center, Richmond, Virginia 23249
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ABSTRACT |
Osmotic effects on
salt taste were studied by recording from the rat chorda tympani (CT)
nerve and by measuring changes in cell volume of isolated rat fungiform
taste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO did
not induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions also
contained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO.
The enhanced CT responses to NaCl were reversed when the saccharides
were removed and were completely blocked by addition of 100 µM
amiloride to the stimulus solution. Exposure of TRCs to hyperosmotic
solutions of mannitol or cellobiose induced a rapid and sustained
decrease in cell volume that was completely reversible, whereas
exposure to hypertonic urea or DMSO did not induce sustained reductions
in cell volume. These data suggest that the osmolyte-induced increase
in the CT response to NaCl involves a sustained decrease in TRC volume
and the activation of amiloride-sensitive apical
Na+ channels.
chorda tympani; cell volume; calcein; fluorescence imaging; salt
taste
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INTRODUCTION |
TASTE RECEPTOR CELLS (TRCs) provide for sensory
transduction of chemical stimuli normally found in foods and initiate
quality coding in the gustatory neuraxis (32). Taste quality, a key factor in the decision to accept or reject a substance as a meal, is a
complex entity that includes significant influences from environmental
and cultural, as well as physiological, factors (30). Taste quality
perception may also be influenced by factors of long-term duration,
such as metabolic or hormonal status, that often reflect individual
nutritional and health factors (28). In the taste periphery, variations
in the physicochemical properties of taste stimuli, including mixtures,
are probable sources of acute and widely experienced variations in
taste intensity and quality. Stimulus properties such as viscosity (7)
or tonicity (10) do not activate the peripheral taste organs directly,
but they may exert a modulatory effect on the response of TRCs.
In the case of tonicity, TRCs are exposed to osmolalities ranging from
nearly zero to >2,500 mosmol/kg, all under physiological conditions
(10). TRCs must be robust, as evidenced by their ability to transduce
stimulus quality and intensity under extremes that would incapacitate
many cells. On exposure to anisotonic conditions, most cells initially
behave as osmometers and alter their volumes according to the tonicity
of the extracellular compartment. However, some cells do not
demonstrate short-term recovery from volume perturbations (26, 36),
whereas many other cell types are capable of actively restoring their
volumes, despite continuous hypotonic and hypertonic challenge (17).
Little is known regarding the reaction of individual TRCs to rapid
changes in osmotic pressure and possible mechanisms of cell volume
recovery. In most cells, recovery from volume perturbations involves
the activation of a variety of solute transport mechanisms. Given that
many of these solutes are normal stimuli for TRCs, one might reasonably
expect osmotic changes in the oral cavity fluid to have consequences for the encoding of taste sensation. This would suggest mechanisms in
polarized epithelial cells permitting stimulus-induced changes in one
cell membrane to be communicated to the contralateral membrane via
changes in cell volume, intracellular ion activities, and membrane
voltages (36).
Although not polarized epithelial cells, supraoptic neurons are
well-studied examples of osmoreceptors that serve as cell volume
transducers (6). Although TRCs are not osmoreceptors per se, studies
have shown that active solute transport and arterial-venous exchange of
solutes along the length of cat fungiform papillae exposed to isotonic
fluids result in the papilla tips becoming hypertonic (15). Thus TRCs
may experience changing osmotic pressure gradients depending on the
permeability properties of various solutes and their effects on cell
metabolism and papillary blood flow. We have investigated the effects
of osmotic pressure on taste at the level of the sensory afferents by
recording from the rat chorda tympani (CT) and on the cellular level by
measuring changes in cell volume of isolated rat fungiform TRCs with
use of imaging techniques. Because taste stimuli have their own
intrinsic osmotic pressures, we have employed conditions that separate
the osmotic pressure variables from those relating to taste stimulus intensity. Correlation of the electrophysiological results with the
cell volume measurements indicates that a sustained decrease in TRC
volume is the necessary precursor to the enhancement of the taste
neural response to isotonic NaCl by certain osmolytes.
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MATERIALS AND METHODS |
Recording CT Responses
Nerve preparation and recording.
Female Sprague-Dawley rats (150-200 g) were anesthetized by brief
exposure to ether followed by injection of pentobarbital sodium (60 mg/kg ip). Supplemental pentobarbital sodium (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 was exposed laterally as it exited the
tympanic bulla, as previously described (38). After the CT was
dissected free from surrounding tissue, it was cut proximally, desheathed, 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 and recorded on a modified Toshiba DX-900 videocassette recorder. For display, responses were filtered using a
band-pass filter with cutoff 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. The voltage output of the integrator is a
measure of the neural response (the number of individual nerve fibers
firing at a given time) and is proportional to the number of spikes per
second (4). Integrated neural responses and current and voltage records
were recorded on a chart recorder (model TYP7045, Linseis, Princeton
Junction, NJ). For display, the integrated neural records were plotted
in scaled arbitrary chart units relative to baseline in rinse
solutions. An upward pen excursion corresponds to an increase in
magnitude of the integrated neural response (i.e., increased spike
frequency) at a given point in time.
Stimulation chamber and in situ transepithelial potential
recording.
Solutions were injected (3 ml, 1 ml/s) into a Lucite chamber affixed by
vacuum to a 28-mm2 patch of
anterior dorsal lingual surface. The chamber was fitted with a Ag-AgCl
electrode for current passing and a salt-bridge electrode for measuring
the in situ lingual transepithelial potential (Vis).
Corresponding reference electrodes were placed noninvasively on the
ventral lingual epithelium. The
Vis and applied
currents were measured and programmed, respectively, using a
voltage-current-clamp amplifier (model VCC600, Physiologic Instruments,
San Diego, CA). All experiments were performed while the lingual
epithelium was maintained under zero current-clamp mode, and all
voltages were referenced to the musocal side. The current-passing
electrode within the chamber served as a virtual ground, ensuring that
only current passing through the stimulated patch was collected. A periodic (15-s) bidirectional constant-current pulse (4 µA) was generated across the lingual receptive field contained in the stimulation chamber. The current also perturbed the steady-state Vis, and this
yielded a measure of the relative changes in in situ tissue resistance
(Ris). For
comparison purposes the data are presented as relative changes in
Ris and
Vis with respect to their values in the rinse solution rather than their absolute values. This is because it is necessary to place the voltage and current-passing reference electrodes noninvasively along the ventral lingual surface rather than embed them in the muscle close to the
dorsal surface (16). This avoids injury and inflammation, which is
essential in preserving normal peripheral taste sensory function.
Sublingual electrode placement adds another series resistance, due to
muscle and connective tissue, that varies among animals because of
variations in muscle thickness and the position of the reference
electrodes. However, on stimulating the tongue with taste stimuli, the
added series resistance does not change with solution composition, so
measured changes in
Vis and
Ris in vivo correlate well with changes in transepithelial potential and
resistance, respectively, as determined from previous studies (34, 38, 39).
The time course of lingual potentials, programmed currents, and
integrated CT responses were also captured on disk during an experiment
by use of Labview software and then analyzed off-line in a manner
similar to that previously described (38). The numerical value of an
integrated CT response was obtained as the area under the integrated CT
response curve for a time interval of 75 s from the onset of
chemically evoked neural activity. The area under the integrated CT
response curve under control conditions was normalized to 100%, and
the increase or decrease in the area under experimental conditions was
expressed relative to control.
The protocol for stimulation of the lingual epithelium was as follows:
Before the experiment was started, the responses to three reference
stimuli were tested. The reference solution was injected into the
chamber and allowed to remain on the tongue for 40-60 s. The
reference solution was then rinsed with several applications of rinse
solution. Next, the stimulus series with solutions of different
osmolarities containing mannitol, cellobiose, urea, or DMSO was applied
to the tongue. At the end of this series the three reference stimuli
were reapplied and rinsed. The data from the stimulus series with
various osmolarities were accepted if the nerve responses to three
reference stimuli did not differ by >10% before and after the
stimulus series.
Solutions.
The reference solutions were 0.3 M NaCl, 0.3 M KCl, and 0.3 M
NH4Cl, and the rinse solution
between the application of each reference solution was 15 mM
KHCO3-15 mM KCl (38). Thereafter, all solutions that superfused the tongue contained 20 mM KCl. Additional solutes varied according to the protocol under
investigation. The solutes included NaCl, mannitol, cellobiose, urea,
and DMSO at the concentrations noted. In a given experiment, rinse
solutions included 20 mM KCl and various concentrations of mannitol,
cellobiose, urea, or DMSO to control the nominal solution osmotic
pressure but always excluded the taste stimulus, NaCl. Stimulus
solutions always contained 20 mM KCl and 150 mM NaCl and various
concentrations of mannitol, cellobiose, urea, or DMSO to adjust the
osmotic pressure. In some experiments the rinse solutions and
all stimulus solutions contained 100 µM amiloride (Sigma Chemical,
St. Louis, MO).
Cell Volume Measurements in Isolated TRCs
Preparation of TRCs.
Female Sprague-Dawley rats weighing 150-200 g were anesthetized
with methoxyflurane and then killed by cervical dislocation. The
tongues were rapidly removed and stored in ice-cold HEPES-buffered solution (pH 7.4) preequilibrated with 100%
O2. The lingual epithelium was
isolated by injection of collagenase (Boehringer-Mannheim, Indianapolis, IN) and incubation in a
Ca2+-free solution (3). Then taste
bud fragments (TBFs) and TRCs were prepared from the fungiform
papillae, as described previously (19, 34).
Perfusion chamber.
The open perfusion chamber consisted of a standard glass slide onto
which a piece of silicone rubber sheet with a
4-cm2 cutout window in the center
was glued (19). Cells were affixed to the slide with Cell-Tak (1 µg/cm2; Collaborative Research,
Bedford, MA), and a fresh chamber was prepared for each experiment. The
chamber was perfused at 4 ml/min.
Measurement of cell dimensions.
After an initial wash perfusion for 15 min with HEPES-buffered
solution, TRCs were visualized through a ×40 objective (Zeiss; 0.9 numerical aperture) with a Zeiss Axioskop. Transmitted images were
acquired with a video camera (model ITC 510, Ikagami) and digitized at
10-s intervals with a software-controlled frame grabber board (Digidata
2000 Image Lightning Board and Imaging Workbench, Axon Instruments,
Foster City, CA). Changes in length of the cell major and minor axes
were measured using Transform (Fortner Research, Sterling, VA). With
the assumption that the TRC body has the shape of an ellipsoid, the TRC
volume (V) was calculated using the following formula: V = 4
a3/3
.
This formula is based on the following relations:
S = 4
a2{(1/2a)ln[(1 + a)/(1
a)]}, where
S is surface area,
a =
and
is the ratio
a/c, where
a is one-half the minor axis length and c is one-half the major axis length.
Measurement of calcein fluorescence.
In some experiments, relative changes in cell volume were monitored
using the fluoroprobe calcein, because the calcein fluorescence varies
inversely with its concentration (36, 37). TRCs in the perfusion
chamber were loaded with calcein in its AM form (25 µM) at 4°C
overnight. Before the experiment was started, the cells were superfused
with room temperature control solution for 30 min. The imaging setup,
described above, was used with the addition of an image intensifier
(Videoscope, Washington, DC), an epifluorescent light source (TILL
Photonics Polychrome II, Applied Scientific Instrumentation, Eugene,
OR), a 515-nm dichroic beam splitter (Omega Optical), and a 535-nm
emission filter (20-nm band pass, Omega Optical). The cells,
illuminated with 490-nm light, were imaged at 10-s intervals, and 16 frames were averaged. Small regions of interest (~5
µm2) in cells were chosen in
which fluorescence was monitored. Photobleaching of calcein was <5%
(see Figs. 11 and 12).
In separate experiments, TBFs and TRCs were imaged with a confocal
laser scanning imaging system (LSM 410 or LSM 510, Carl Zeiss,
Heidelberg, Germany). The excitation light was 488 nm, and the light
emitted above 515 nm was measured. To evaluate the relationship between
calcein fluorescence and cell size, TRCs were exposed to hypertonic
NaCl. Images were obtained at 20-s intervals, and in each image cell
size and mean calcein fluorescence intensity were measured. In four
TRCs the mean changes in calcein fluorescence were linearly related to
changes in cell size with a slope of 0.58. A similar linear
relationship between calcein fluorescence intensity and cell size has
been reported in gallbladder epithelial cells (36) and in rat
hepatocytes (37).
Solutions.
HEPES-buffered control solutions (pH 7.4) contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 sodium pyruvate, 10 glucose, and 10 HEPES. The hypotonic solution was NaCl free. The
solution osmolarity was increased by the addition of mannitol,
cellobiose, urea, DMSO, or NaCl.
Statistical Analyses
Values are means ± SE; n
represents the number of animals from which CT recordings were made in
the group. In in vitro experiments n
represents the number of TRCs in an experiment. Statistical significance was assessed with the paired Student's
t-test, and significance was achieved
when two-tailed P < 0.05.
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RESULTS |
In Vivo Studies
Effects of osmolarity on CT activity.
To verify that D-mannitol does
not evoke a neural response in the rat CT, the tongue was superfused
first with a solution that approximated normal saliva, i.e., 20 mM KCl,
and then with a solution containing 300 mM mannitol + 20 mM KCl. As
shown in Fig.
1A,
apart from a mechanical rinse artifact (rapid transient upward
deflection in the baseline neural record), no significant chemically
evoked neural response occurred. In the bottom
trace, exposure of the tongue to mannitol solution also
caused a small positive increase in
Vis (referenced
to the mucosal side) and an increase in
Ris (i.e.,
increase in the amplitude of the transient voltage excursions in
response to the periodic bipolar current pulses). The observed changes
in Ris cannot, of
course, be attributed to intrinsic properties of the added
nonelectrolyte osmolyte. Similar changes in
Ris and
Vis were observed
when cellobiose (which also did not evoke a CT response) was used
rather than mannitol (data not shown). The data from both saccharides
were, therefore, pooled and analyzed. In 10 animals the tongues were
initially superfused with a rinse solution containing 20 mM KCl. On
increase in the osmolarity of the rinse solutions with 300 mM mannitol (n = 7) or cellobiose
(n = 3),
Ris increased by
53.3 ± 17.1% (P < 0.025). These
changes were accompanied by an increase in
Vis by 4.7 ± 1.4 mV (P < 0.01). In seven
additional animals, increasing the saccharide concentration from 300 to
600 mM increased
Ris further by
23.0 ± 3.4% (Fig. 1B).


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Fig. 1.
A: effect of mannitol on chorda
tympani (CT) response (top trace)
and lingual in situ transepithelial potential
(Vis) and
resistance (Ris;
bottom trace) in vivo. CT responses
are measured as voltage output of a spike integrator and displayed
graphically in arbitrary chart units. Periodic vertical deflections in
neural record from baseline are transient responses to a periodic
(15-s) bipolar 4-µA constant-current pulse across lingual receptive
field contained in stimulation chamber. Current also perturbed
steady-state Vis,
and this yielded a measure of relative changes in
Ris.
B: relative increase in
Ris (mean ± SE) in several animals when saccharide concentration was increased from
0 to 300 or from 300 to 600 mM. Solid bars, paired differences between
data sets.
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In parallel experiments the tongue was first superfused with 20 mM KCl,
which was then replaced by another solution containing 20 mM KCl + 600 mM urea (n = 3) or DMSO
(n = 3). As was the case with mannitol
(cf. Fig. 1A), no significant
chemically evoked neural responses occurred with urea or DMSO.
Application of urea or DMSO did not significantly alter
Vis and
Ris (data not shown).
In the next series of experiments we investigated the effect of
mannitol on the neural response to stimulation with 150 mM NaCl. The
first stimulation series was performed under near-isosmotic conditions.
The tongue was superfused with a solution containing 20 mM KCl + 300 mM mannitol for several minutes and then stimulated with a
solution in which 300 mM mannitol was replaced with 150 mM NaCl (an
isosmotic change). As shown in Fig.
2A (also
see Figs. 3-5),
the 150 mM NaCl stimulus solution induced an increase in the CT
response (top trace). These changes
were accompanied by a small decrease in
Vis
(bottom trace) and
Ris. After
several minutes the salt stimulus was added once again, and after an
infusion artifact the CT response assumed its original time course. In the third step the salt stimulus was replaced by a hypertonic salt
stimulus containing 150 mM NaCl + 20 mM KCl + 300 mM mannitol. The CT
record shows a further increase in activity (upward deflection) that
follows a new time course of elevated neural activity compared with
that seen in the absence of mannitol. On rinsing the hypertonic salt
stimulus with the solution containing 20 mM KCl + 300 mM mannitol, the
original baseline was reachieved and
Vis and
Ris returned to
values close to their baseline. Similar results were obtained when the
solutions contained cellobiose in place of mannitol (data not shown).


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Fig. 2.
A: effect of hyperosmolarity on CT
response to 150 mM NaCl (top trace)
and Vis and
Ris
(bottom trace) in vivo. CT responses
are measured as voltage output of a spike integrator and displayed
graphically in arbitrary chart units. Tongue was treated with an
isosmotic rinse solution containing 20 mM KCl + 300 mM mannitol, and CT
response was taken as baseline. Perfusion of tongue with a salt
stimulus with same osmolarity (300 mM mannitol replaced with 150 mM
NaCl) represents neural response to 150 mM NaCl. In next step,
perfusion with a hypertonic salt stimulus containing 20 mM KCl + 150 mM
NaCl + 300 mM mannitol further increased CT response. In final step, on
exposure of tongue to rinse solution, CT response declined to baseline
value. B: summary of data from several
such experiments represented as percent change in CT response
calculated from changes in relative area under response curve. Percent
increase in area in presence of 150 mM NaCl + 300 mM saccharides is
expressed relative to that of 150 mM NaCl alone. Values are means ± SE of number of animals in parentheses. Solid bars, paired differences
in CT responses between sets of data (see text for details).
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Fig. 3.
A: effect of hyposmolarity on CT
response to 150 mM NaCl (top trace)
and Vis and
Ris
(bottom trace) in vivo. CT responses
are measured as voltage output of a spike integrator and displayed
graphically in arbitrary chart units. Tongue was initially treated with
a hypertonic rinse solution containing 20 mM KCl + 600 mM mannitol, and
CT response was taken as baseline. Perfusion of tongue with a salt
stimulus with same osmolarity as rinse solution (300 mM mannitol
replaced with 150 mM NaCl) represents neural response to 150 mM NaCl
under hypertonic conditions. In next step, perfusion with an isotonic
salt stimulus containing 20 mM KCl + 150 mM NaCl decreased CT response.
In final step, on exposure of tongue to rinse solution, CT response
declined to baseline value. B: summary
of data from several such experiments represented as percent change in
CT response calculated from changes in relative area under response
curve. Percent decrease in area in presence of 150 mM NaCl alone is
expressed relative to that of 150 mM NaCl + 300 mM saccharide. Values
are means ± SE of number of animals in parentheses. Solid bars,
paired differences in CT responses between sets of data (see text for
details).
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Fig. 4.
Effect of urea on CT response to 150 mM NaCl (top
trace) and
Vis and
Ris
(bottom trace) in vivo. Tongue was
treated with an isosmotic rinse solution containing 20 mM KCl + 300 mM
urea, and CT response was taken as baseline. Superfusion of tongue with
a salt stimulus with same osmolarity as rinse solution (300 mM urea
replaced with 150 mM NaCl) represents neural response to 150 mM NaCl.
In next step, superfusion with a hypertonic salt stimulus containing 20 mM KCl + 150 mM NaCl + 600 mM urea had no effect on neural response to
150 mM NaCl. In final step, on exposure of tongue to rinse solution, CT
response declined to baseline value.
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Fig. 5.
Effect of hyperosmolarity on CT response to 150 mM NaCl
(top trace) and
Vis and
Ris
(bottom trace) in vivo in absence
(A) and presence
(B) of amiloride. In
A, responses are similar to those in
Fig. 2A. In
B, tongue was treated with an
isosmotic rinse solution containing 20 µM KCl + 300 mM mannitol + 100 µM amiloride, and CT response was taken as baseline. Perfusion of
tongue with a salt stimulus with same osmolarity (300 mM mannitol
replaced with 150 mM NaCl) induced no significant CT activity above
baseline. In next step, perfusion with a hypertonic salt stimulus
containing 20 mM KCl + 150 mM NaCl + 300 mM mannitol + 100 µM
amiloride also induced no further increase in CT response above
baseline. In final step, on exposure of tongue to rinse solution, there
was no significant decline in CT response.
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The CT responses from several animals are summarized in Fig.
2B. The integrated CT responses were
calculated as the area under the response curve in the presence of 150 mM NaCl and in the presence of 150 mM NaCl + 300 mM mannitol or
cellobiose. The percent change in area in the presence of 150 mM
NaCl + 300 mM saccharides is expressed relative to that in
the presence of 150 mM NaCl alone. The data show that superfusing the
tongue with a second salt stimulus (150 mM NaCl + 20 mM KCl) does not
change the steady-state CT response (bars
1 and 2). However,
on superfusion, a similar salt stimulus containing 300 mM saccharide
(bar 3) induced an increase in CT
response by 49.0 ± 17.9% (solid bar;
P < 0.025, n = 8). Although during a second
stimulation with hypertonic stimulus solution the CT response decreased
(bar 4), it was still 26.7 ± 5.2% greater (P < 0.005) than in
the absence of saccharides.
Changing from a solution with 300 mM mannitol to a stimulus solution
containing 150 mM NaCl caused an electronegative change in
Vis by 8.8 ± 1.8 mV (P < 0.001). The changes in
Vis were in the
opposite direction from those observed when saccharides were administered in the absence of NaCl (Fig.
1A). The apical lingual surface
was initially superfused with rinse solutions containing 20 mM KCl + 300 mM mannitol. On superfusion of the tongue surface with a stimulus
solution containing 150 mM NaCl,
Ris decreased by
47.6 ± 12.8% (n = 8) relative to
the rinse solution. However, on superfusion, the salt stimulus
containing, in addition, 300 mM mannitol did not change
Ris
(
Ris = 2.1 ± 2.1%, P > 0.05, n = 8) or
Vis.
To investigate whether the lowering of tonicity of the salt stimulus
would attenuate the nerve response, the same experiment was done under
hypertonic salt stimulus. The tongue was rinsed with a rinse solution
containing 20 mM KCl + 600 mM mannitol for several minutes and then
stimulated with hypertonic salt solution containing 20 mM KCl + 150 mM
NaCl + 300 mM mannitol (no change in tonicity, because the rinse and
the salt stimulus have an osmotic pressure of ~640 mosM). As shown in
Fig. 3A, there was an increase in the
CT response (top trace) due to NaCl
that maintained its time course during a second exposure to the
hypertonic salt stimulus. These changes were accompanied by a small
decrease in Vis
(bottom trace) and a small decrease
in Ris. This was
replaced by an isosmotic salt stimulus without mannitol. It caused a
significant decrease in neural activity, but in this case the decrease
in neural activity was more protracted in time. Similar responses were
obtained with cellobiose (data not shown). The data from several
animals are presented in Fig. 3B, in
which the relative CT response is expressed as the area under the CT
response curve in the presence and absence of saccharides. The data
show that superfusing the tongue with a second salt stimulus (150 mM
NaCl + 20 mM KCl + 300 mM saccharide) does not change the steady-state
CT response (bars 1 and
2). However, superfusing a similar
salt stimulus without the saccharide (bar 3) induced a decrease in CT response by 17.9 ± 3.2% (solid bar; P < 0.005, n = 8). Subsequently, a second
stimulation with the isotonic salt stimulus (bar
4) caused the CT response to decrease further and was
43.6 ± 9.7% lower (solid bar;
P < 0.005) than stimulation with the hypertonic stimulus.
As observed in the previous experiment, changing from a rinse solution
with 600 mM mannitol to a stimulus solution containing 150 mM NaCl + 300 mM mannitol caused an electronegative change in the transepithelial
potential and a decrease in
Ris by 49.6 ± 5.5% (n = 9) relative to the rinse
solution (data not shown). The changes in
Vis were in the
opposite direction from those observed when saccharides were
administered in the absence of NaCl (Fig. 1). However, superfusing the
salt stimulus containing 150 mM NaCl alone induced a further decrease
in Ris by 3.9 ± 1.5% (P < 0.05, n = 9) without a change in
Vis.
When the lingual epithelium was rinsed with solutions containing
mannitol, a subsequent salt stimulus in which 150 mM NaCl replaced an
equivalent amount of mannitol decreased
Ris but caused an
electronegative shift in
Vis. However, we
observed that when the lingual epithelium was rinsed with rinse
solutions without the saccharides, a subsequent NaCl stimulus always
induced a positive shift in
Vis (39). In
tongues rinsed with 15 mM KCl + 15 mM KHCO3, a subsequent exposure to
300 mM NaCl decreased
Ris by 58.3 ± 4.5% (P < 0.001) and increased
Vis by 9.2 ± 1.6 mV (P < 0.001, n = 10). Similarly, in tongues rinsed
with 10 mM KHCO3, a subsequent exposure to 100 mM NaCl induced a decrease in
Ris by 25.9 ± 5.6% (P < 0.025), with a positive
shift in Vis by
6.2 ± 2.1 mV (P > 0.05, n = 5). However, in additional
experiments, when tongues exposed to 10 mM NaCl were subsequently
treated with 100 mM NaCl, Ris decreased by
33.5 ± 2.9% (P < 0.005) and
Vis increased by 8.6 ± 1.4 mV (P < 0.025, n = 5). These data suggest that
NaCl-induced changes in
Vis depend on the
rinse solution composition. This effect is certainly not restricted to
NaCl, since we observed that, in the same five rats, when 10 mM NaCl
was replaced with 300 mM cellobiose,
Ris increased by
9.5 ± 2.1% (P < 0.025) and Vis decreased by
2.6 ± 0.5 mV (P < 0.025). This
change in Vis is
opposite from that observed with mannitol (Fig.
1A) or cellobiose when the rinse
solution was 20 mM KCl. In contrast to this, in the presence of 150 mM
NaCl (Figs. 2A and
3A), addition or removal of the
saccharides had only minimal effects on
Ris and
Vis.
In the above experiments, altering the osmolarity of the NaCl stimulus
solution with cellobiose or mannitol modulated the CT response to 150 mM NaCl. Because most cells respond to changes in external osmolarity
with changes in cell volume (17), we hypothesized that mannitol and
cellobiose modulate the CT response to NaCl via changes in TRC volume.
To investigate this possibility, in the next set of experiments we
altered the osmolarity of the NaCl stimulus solution with urea and
DMSO, both of which have been shown to have high permeability across
cell membranes (14, 24) and are expected to induce no significant
changes in TRC volume. The tongue was rinsed with 20 mM KCl + 300 mM
urea for several minutes and then stimulated with a solution in which
300 mM urea was replaced with 150 mM NaCl (an isosmotic change, since both solutions have an osmotic pressure of ~340 mosM). As shown in
Fig. 4, there was an increase in CT response (top
trace) and a relative decrease in
Ris and
Vis
(bottom trace). After several minutes the salt stimulus was added once again. After an infusion artifact, the response assumed its original time course. In the third
step the salt stimulus was replaced by a hypertonic salt stimulus
containing 150 mM NaCl + 20 mM KCl + 600 mM urea. There was no change
in the neural activity or its time course compared with that seen in
the absence of urea. On rinsing the hypertonic salt stimulus with the
rinse solution, the original baseline was reachieved. When the infusion
solutions contained 600 mM DMSO in place of urea, once again there was
no change in the neural activity or its time course compared with that
in the absence of DMSO (cf. Fig. 4). In additional experiments the
steady-state CT response in the presence of a hypertonic salt stimulus
containing 150 mM NaCl + 20 mM KCl + 300 mM DMSO (or urea) did not show
any change in neural activity or its time course compared with the response in the presence of 150 mM NaCl + 20 mM KCl. In eight such
experiments when the salt stimulus contained 300 mM urea (n = 4) or DMSO
(n = 4), the integrated CT responses
(cf. Figs. 2B and
3B) were increased by only 3.4 ± 2.9% (P > 0.05) compared with the
steady-state CT activity in the presence of the salt stimulus alone.
The CT nerve responses to NaCl are inhibited by the application of
amiloride (32), suggesting the presence of apical amiloride-sensitive Na+ channels on TRCs innervated by
the CT. To investigate whether apical amiloride-sensitive
Na+ channels in TRCs may modulate
osmotically induced changes in NaCl responses, CT responses were
monitored in stimulus solutions without and with amiloride. As shown in
the experiment in Fig. 5A, when
amiloride was absent the CT responses to 150 mM NaCl and 150 mM NaCl + 300 mM mannitol were similar to those shown previously in Fig.
2A. In the second part of the
experiment the same sequential protocol was used, but 100 µM
amiloride was added to the stimulus solutions. As shown in Fig.
5B, amiloride completely blocked the
CT responses to 150 mM NaCl and to 150 mM NaCl + 300 mM mannitol.
Similar amiloride inhibition of the CT responses was observed in two
additional animals (data not shown). These data suggest that the
amiloride-sensitive Na+ channels
present in the apical membranes of TRCs are involved in osmotically
induced modulation of CT responses to NaCl.
In summary, during stimulation with 150 mM NaCl, changing directly to a
solution containing 150 mM NaCl, but with 300 mM saccharide, caused an
increase in the CT response. In contrast, during stimulation with 150 mM NaCl + 300 mM saccharide, changing directly to a solution containing
150 mM NaCl, but without the saccharide, caused a decrease in the CT
response. On the other hand, urea and DMSO had no significant effect on
the steady-state CT responses to 150 mM NaCl. On the basis of the
differences in the permeability of saccharides vs. DMSO and urea across
cell membranes (14, 17, 24), these results suggest that taste responses
to NaCl may be modulated in part by osmotically induced changes in TRC
volume. This hypothesis was tested directly by measuring changes in TRC
volume with mannitol, cellobiose, urea, and DMSO.
In Vitro Studies
Effect of osmolarity on TRC volume.
Figure 6 shows three-dimensional
reconstructed images of a TBF and isolated TRCs perfused in control
solution without mannitol (A and
C) and after a 1-min perfusion with
a similar solution containing 600 mM mannitol
(B and
D). The images demonstrate that TRCs
shrink in hypertonic solutions and that the cell shrinkage occurs in
major and minor axes. Also, the images demonstrate that some TRCs are
rounded and that they too respond to changes in osmolarity.

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Fig. 6.
Isolated taste bud fragments (A) and
isolated taste receptor cells (TRCs,
C) were loaded with a fluorescent
agent and perfused in HEPES-buffered control solution
(A and
C). Confocal laser microscopy was
used to obtain multiple images of TRCs in
z-axis, and then images were
reassembled into these views of isolated TRCs. TRCs were perfused for 1 min with a similar solution containing 600 mM mannitol
(B and
D) and imaged again. TRCs shrink in
hypertonic media. Decrease in cell size reflects a decrease in
dimensions of major and minor axes. Some TRCs are rounded and also
respond to osmolarity. Bar = 10 µm.
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To evaluate the time course of the nontastant saccharide-induced
changes in cell volume, TRCs were initially perfused with hypotonic
NaCl-free solution, then they were superfused with a similar solution
containing mannitol or cellobiose. Figure 7
shows a representative experiment in which solution tonicity was varied with cellobiose. Although 300 mM cellobiose decreased cell volume by
17.8 ± 5.6% (P < 0.05, n = 5), further raising the
concentration to 600 mM induced an additional decrease in cell volume
by 19.9 ± 3.4% (P < 0.01). The
total decrement in cell volume in 600 mM cellobiose was 37.7 ± 6.6%, and as shown in Fig. 8, the addition of 600 mM mannitol decreased cell volume by 34.3 ± 6.1%
(P < 0.001, n = 11). To assess whether mannitol
had the same effect on cell volume when
Na+ was in the bathing solution,
300 mM mannitol was added to a solution containing 150 mM
Na+. In 9 TRCs (Fig.
9), addition of mannitol induced a 22.6 ± 6.3% (P < 0.01)
decrease in cell volume. In these experiments the decrements in volume
were complete within 30 s of solution change. Similar results were
obtained with cellobiose (data not shown).

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Fig. 7.
Effect of solution osmolarity on TRC volume. TRCs were initially
perfused with NaCl-free solution. As indicated by horizontal bar, cells
were perfused with NaCl-free solution containing, in addition, 300 or
600 mM cellobiose. Percent changes in TRC volume are shown relative to
TRC volume in presence of 300 mM cellobiose. In 5 TRCs, 300 and 600 mM
cellobiose decreased cell volume by 17.8 ± 5.6%
(P < 0.05) and 37.7 ± 6.6%
(P < 0.01), respectively.
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Fig. 8.
Effect of mannitol on TRC volume. TRCs were initially perfused with
NaCl-free solution. As indicated by horizontal bar, perfusion solution
was switched to NaCl-free solution containing, in addition, 600 mM
mannitol. Values represent paired differences in individual TRC volume
(means ± SE; n = 11) between 2 sets of data. Presence of 600 mM mannitol decreased TRC volume by 34.3 ± 6.1% (P < 0.01). On
reperfusion of TRCs with solution without mannitol, TRC volume
increased rapidly, transiently exceeding starting volume
(P < 0.05) and then relaxing to
initial value within 30 s.
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Fig. 9.
Effect of mannitol on TRC volume in presence of NaCl. TRCs were
initially perfused with control solution (containing 140 mM NaCl). As
indicated by horizontal bar, solution was switched to a similar
solution containing, in addition, 300 mM mannitol. Values represent
paired differences in individual TRC volume (means ± SE;
n = 9) between 2 sets of data.
Addition of mannitol decreased TRC volume by 22.6 ± 6.3%
(P < 0.01) in 30 s.
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These data suggest that TRCs behave as osmometers. The changes in cell
volume are rapid and completely reversible (cf. Fig. 8). Although no
spontaneous volume compensation was observed when solution tonicity was
increased, on reducing tonicity from 600 mM mannitol to zero mannitol,
TRC volume increased rapidly (Fig. 8), transiently exceeding the
starting volume (i.e., volume overshoot) and then relaxing to the
initial volume within 30 s. These data suggest that TRCs are capable of
regulatory volume decrease (17). However, it is important to note that
in these experiments the determination of cell volume occurred at 10-s
intervals, thus limiting the detection of more rapid events.
Because volume changes were similar with mannitol or cellobiose (Figs.
7 and 8) and were not affected by the presence and absence of NaCl
(Figs. 7 and 9), the data were pooled to analyze the distribution of
volume responses to increasing osmolarity. As shown in Fig.
10A,
changing from a 340 to a 640 mosM solution reversibly decreased TRC
volume by 26.8 ± 3.5% (P < 0.001, n = 42). The decrements in cell
volume were grouped into the following volume decrease categories:
0-9, 10-19, 20-29, 30-39, 40-49, and >50%.
As shown in Fig. 10B, the cell number
(n), corresponding to an observed
relative decrease in volume, follows a Poisson distribution
[f = (42e
µµx)/x!,
where x = 0, 1, 2, 3, 4, 5,..., are
the volume decrease categories chosen above]. The mean, µ = 2.35, was found from µ = 42/ln(4), where 42 and 4 are the total
number of cells and the number of cells observed in the zeroth
category, respectively. This corresponded to a mean decrease in TRC
volume of 23.5%, a value close to the experimental value of 26.8%.
The distribution of volume decreases suggests that TRCs may form a
heterogeneous population with respect to water and solute permeability.


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Fig. 10.
A: changes in cell volume in 42 TRCs
that were initially bathed in an isotonic solution (solution with or
without NaCl + 300 mM saccharide) and then perfused with a hypertonic
solution (solution with 140 NaCl + 300 mM saccharide or NaCl-free
solution + 600 mM saccharide). Values are means ± SE. Solid
bar, paired differences between 2 data sets.
B: number of TRCs that responded to a
decrease in volume of 0-9, 10-19, 20-29, 30-39,
40-49, and >50%. Cell number corresponding to an observed value
of shrinkage follows a Poisson distribution
[f = (42e µµx)/x!,
where x = 0, 1, 2, 3, 4, 5,... discrete states representing above 6 categories in which TRCs were
arbitrarily divided with a mean value of 2.35 (corresponding to a mean
decrease in TRC volume of 23.5%)]. Values in parentheses above
bars represent theoretically predicted values and are close to those
observed experimentally.
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We also monitored relative changes in TRC volume using calcein, a
fluorescent compound. Because the calcein fluorescence self-quenches as
its concentration increases, intracellular calcein fluorescence varies
with cell volume and serves as a convenient marker for changes in cell
volume (36, 37). Figure
11A
shows a representative experiment in which seven TRCs were initially
bathed in isotonic solution containing 150 mM
Na+; after an increase in mannitol
concentration from 0 to 600 mM, the mean calcein fluorescence decreased
in all seven TRCs. The maximum decrease in fluorescence was complete
within 30 s after the change in perfusion solution and was sustained in
the presence of mannitol. On return to the initial control 150 mM
Na+ solution, the calcein
fluorescence recovered to near its resting value, demonstrating the
reversibility of the saccharide-induced volume changes. In 19 TRCs
investigated (including the 7 TRCs shown in Fig.
11A), the mean decrease in calcein
fluorescence intensity was 19.5 ± 2.9%. These data indicate that
relative changes in calcein fluorescence reflect changes in TRC volume
and follow the same time course observed in Figs. 7-9.


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Fig. 11.
Effect of solution osmolarity on relative changes in TRC volume
measured with calcein. A: 7 TRCs were
initially perfused with control solution (containing 140 mM NaCl). At
time period indicated by horizontal bar, cells were perfused with a
similar solution containing, in addition, 600 mM mannitol. At each time
period, percent change in calcein fluorescence intensity of individual
TRC was calculated relative to baseline fluorescence in control
solution. Error bars, SE. Mannitol induced a rapid reversible decrease
(P < 0.01) in calcein fluorescence
intensity, and this decrease was sustained as long as cells were
superfused with saccharide. B: 4 TRCs
were initially perfused with control solution (containing 140 mM NaCl).
As indicated by horizontal bar, cells were superfused with a similar
solution containing, in addition, 360 mM NaCl (total NaCl = 500 mM).
An increase in NaCl induced a rapid decrease
(P < 0.01) in calcein fluorescence
that was followed by a spontaneous increase in fluorescence toward its
baseline value. On superfusion of cells with control solution, calcein
fluorescence rapidly returned to a value close to its original baseline
value.
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To determine whether changing osmolarity with NaCl affects TRC volume
differently from the changes induced by saccharides, the NaCl
concentration was increased from 140 to 500 mM. As shown in Fig.
11B, increasing NaCl concentration
induced a rapid decrease in calcein fluorescence in four TRCs,
indicating a rapid reduction in cell volume. Unlike the effect of the
saccharides, the fluorescence spontaneously but slowly increased with
time, indicating a spontaneous recovery of cell volume. On superfusion
of TRCs with control solution (140 mM NaCl), the calcein fluorescence
recovered to near its resting value. In 12 TRCs (including the 4 TRCs
shown in Fig. 11B), immediately
after the increase in NaCl concentration the calcein fluorescence
intensity decreased to 54.6 ± 7.0% of its baseline value. However,
in all cells, calcein fluorescence intensity increased spontaneously
with time to 69.8 ± 5.6% of its baseline value (
calcein
fluorescence = 15.2 ± 1.7%, paired difference, P < 0.001). Thus it appears that
TRCs demonstrate spontaneous regulatory volume increase in the presence
of the salt but not in the presence of saccharides. Although such
differences in solute-induced changes in cell volume have been observed
in some cell types (17), the mechanisms involved in regulatory volume
decrease and increase in TRCs are not known.
To evaluate whether urea or DMSO affects cell volume differently from
NaCl or saccharides, calcein fluorescence was monitored as osmolarity
was increased by urea or DMSO. As shown in Fig. 12A, on
exposure to 600 mM urea, there was a rapid decrease in calcein
fluorescence in all seven TRCs that was followed by a spontaneous
recovery of fluorescence to near its resting value. In 19 TRCs
investigated (including the 7 TRCs shown in Fig.
12A), the mean maximum transient
decrease in calcein fluorescence intensity was 7.9 ± 0.4%
(P < 0.001). In all 19 TRCs the
calcein fluorescence intensity increased spontaneously and stabilized
to a new steady-state value, which was 4.0 ± 0.5%
(P < 0.001) above its control value. Although urea had transient effects on TRC volume, it had no sustained effects on TRC volume, which is similar to the lack of sustained effects of urea on cells derived from rabbit thick ascending limb of
Henle's loop (14). As shown in Fig.
12B, exposure to 600 mM DMSO did not
alter calcein fluorescence in three TRCs. In nine TRCs (including the 3 TRCs shown in Fig. 12B), the mean
increase in calcein fluorescence intensity in the presence of DMSO was 2.1 ± 0.7%. These data are consistent with the notion that DMSO and urea (14, 24) exhibit a significant permeability across cell
membranes and thus induce only transient or minimal changes in TRC
volume.


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Fig. 12.
Effect of solution osmolarity on relative changes in TRC volume
measured with calcein. A: 7 TRCs were
initially superfused with control solution (containing 140 mM NaCl). As
indicated by horizontal bar, cells were superfused with a similar
solution containing, in addition, 600 mM urea. Urea induced a rapid
transient decrease in calcein fluorescence that was followed by a rapid
spontaneous increase in fluorescence that stabilized to a value
slightly above its baseline value in control solution.
B: 3 TRCs were initially perfused with
control solution (containing 140 mM NaCl). At time period indicated by
horizontal bar, cells were superfused with a similar solution
containing, in addition, 600 mM DMSO. At each time period, percent
change in calcein fluorescence intensity of individual TRC was
calculated relative to baseline fluorescence in control solution. Error
bars, SE. DMSO induced only minor changes in overall fluorescence
intensity.
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DISCUSSION |
Taste responses arising from a meal normally result from mixtures of
chemicals released from foods during chewing and swallowing. Such
mixtures of taste stimuli often evoke taste responses that are not
easily interpretable in terms of the responses of their isolated
components. These mixture interactions may arise in quality and
intensity and may have their origin in the taste periphery and/or in
higher gustatory centers. Mixture suppression is said to occur when the
intensity of the response of a mixture is less than that of the sum of
its components presented individually. A well-studied example of
mixture suppression, arising at the taste cell level, is the
suppression of rat CT responses to
Na+ salts when presented together
with potassium benzoate (23, 29) or potassium gluconate (33). On the
other hand, mixture enhancement occurs when the intensity of the
response of a mixture is greater than that of the sum of its components
presented individually. The mixtures of mannitol or cellobiose with
NaCl, used in the present study, fall into the latter category.
Although numerous accounts of mixture interaction are to be found in
the literature, the mechanisms underlying it remain, in most cases,
poorly understood. In this study we present evidence that substances,
at hyperosmotic concentrations, that produce sustained TRC shrinkage
(mannitol or cellobiose) enhanced the rat CT response to NaCl when
presented in mixture with NaCl. The reductions in TRC volume were not
only sustained, but they also varied with the concentration of
saccharide, suggesting that the mechanism of TRC shrinkage is osmotic
and that mannitol and cellobiose have high reflection coefficients
(perhaps approaching unity, i.e., nearly TRC membrane-impermeable
solutes). This is in contrast to the effects of hyperosmotic
concentrations of urea and DMSO, which did not sustain a decrease in
TRC volume and had no effect on the CT response to isosmotic NaCl. In
their ability to produce no more than transient TRC shrinkage, it may
be concluded that urea and DMSO have low reflection coefficients; i.e.,
they readily enter TRCs. These results suggest that the capacity of
hyperosmotic agents to cause enhancement in the CT response to NaCl is,
therefore, more a function of their ability to sustain TRC shrinkage
and, therefore, not the result of cellular processes critically
dependent on the permeation of the agents across TRC membranes. On this basis, it seems reasonable to propose that changes in cell volume can
directly affect the sensory function of TRCs.
An important consideration in such a proposal is, of course, the
relative time courses of CT response enhancement and TRC shrinkage
after solution composition changes involving the saccharides. From our
measurements the time course of TRC shrinkage seems sufficiently fast
to be considered a possible precursor of an enhanced sensory nerve
response. What may appear to be differences between the time courses in
the data between the two procedures are accounted for by the slower
image acquisition rate (1 image every 10 s) in the cell volume studies
compared with CT nerve records (integrator time constant of 1 s). Also,
limits on the fluid exchange characteristics of the perfusion chamber
impose delays in the attainment of steady-state cell volume values that
do not exist under in vivo conditions (19, 34). The comparable time
courses of cell shrinkage and changes in neural activity, therefore,
further support the conclusion that the mixture enhancement in the NaCl
response due to mannitol or cellobiose is a consequence of osmotically
induced changes in TRC volume. Although it is also possible that
transient changes in TRC volume also induce changes in CT responses,
our data suggest that sustained changes in TRC volume are more
effective than transient changes. For example, the transient changes in
TRC volume in response to urea (Fig.
12A) were significant; however, as
shown in Fig. 4, a similar concentration of urea did not produce
transient changes in the steady-state CT responses to 150 mM NaCl.
Hence, it is tempting to speculate that transient changes in volume are
significantly attenuated in vivo (see below) and occur on a faster time
scale than shown in Fig. 12A. If the
volume decrease transients in vivo were small and of very short
duration, the CT nerve response transients might not be observed
because of the small volume change and the fact that an integrator time
constant of 1 s precludes the resolution of faster neural transients.
It is important to note that the mechanisms that link TRC volume
changes to TRC sensory activity, including their sensitivities and time
resolutions, are unknown.
Further evidence in support of osmotically induced cell volume change
is indicated by the increased resistance of the anterior lingual
receptive field when hypotonic KCl rinse solution was replaced by 300 mM mannitol (cf. Fig. 1). It is known that increasing the osmotic
pressure of the mucosal bathing solution of leaky ion-transporting
epithelia in vitro results in increased transepithelial resistance.
Studies on frog gallbladder showed that the resistance increased by
~40% when mucosal tonicity was increased by addition of 200 mM
sucrose (5). This is comparable to the 53% increase in resistance we
observed in rat tongue in vivo with 300 mM cellobiose or mannitol. In
frog gallbladder, ultrastructural studies revealed that the increase in
resistance was related to the collapse of the lateral intercellular
spaces and the shrinkage of the epithelial cells (5). Similar studies
with Necturus gallbladder epithelium mounted in an Ussing-type chamber showed that increasing the apical solution osmolarity induced a small positive increase in
transepithelial voltage as well as an increase in tissue resistance
(36). The increase in transepithelial voltage is comparable to our
observation of an increase in
Vis of ~5 mV in
the lingual epithelium. From studies of CT responses to NaCl under
lingual voltage clamp (38), it is unlikely that changes in
Vis of such small
magnitude will significantly influence TRC membrane voltages and,
therefore, neural responses. However, the changes observed in the in
situ lingual electrical parameters are nonetheless similar to changes observed in vitro in other epithelia under osmotic stress. On this
basis, one might anticipate TRC volume to be affected by exposure to
anisotonic conditions in situ. As seen in Figs. 6-12, TRC volume
decreased with increasing osmotic pressure in a reversible manner. In
recent preliminary observations, using immunocytochemical techniques
and RT-PCR, Gilbertson et al. (12) identified aquaporins (AQP1, AQP2,
and AQP5), which are molecules involved in water flow across cell
membranes, in rat and hamster TRCs. In addition, they observed that
changes in external osmolarity induced voltage-activated currents in
TRCs: hypertonic solutions increased inward currents, whereas hypotonic
solutions increased outward currents. In preliminary studies from our
laboratory (20), changes in TRC volume were associated with changes in
intracellular pH and activation of a large anion pathway across TRC
membranes. It is quite likely that the above mechanisms are involved in
TRC volume regulation.
Cell volume changes are central to the osmoreceptor function of
supraoptic neurons, resulting in the release of vasopressin (6). There
are, in addition, afferent neural pathways originating in the
oropharyngeal/laryngeal mucosa and terminating on neurons in the
hypothalamus that release vasopressin (1). This release depends on the
molarity of NaCl in the stimulus and is amiloride sensitive. The
receptors in the mouth for hyperosmotically evoked thirst have not been
identified, but the observations suggest that NaCl-induced changes in
receptor cell volume could play a role.
In the present study, all the osmotic agents were nonelectrolytes, and
the critical cell transport parameter distinguishing those that
produced mixture enhancement from those that did not was their
respective reflection coefficients. In cases involving nonisosmotic
electrolyte solutions, the relationship between TRC volume and
excitation of the taste nerves is, however, far from a simple one, as
can be seen from the variety of dilution or water responses reported in
single taste fibers of various species (9, 31, 40) and psychophysically
in humans (2). It would appear that TRCs in these cases are not
functioning as simple osmometers, because dilution response often
depends specifically on the solute present. In the cat CT (9) and
rabbit superior laryngeal nerve (31), observations of the dilution or
water response depend on the anion present in the solution applied to
the receptive field. Increasing
Cl
concentration inhibits
the response from single water units, whereas increasing sulfate
activates it. If modulation of a
Cl
channel is an important
part of the water response, as suggested (31), the key osmotic effect
may be in recovery from osmotic swelling, not the initial swelling
itself. The Cl
channel
involved could be related to the swelling-activated anion channel,
which is affected by the transmembrane
Cl
gradient, the presence
of foreign anions in the extracellular medium, and anion channel
blockers (35). Evidence for this was obtained by Okada et al. (25), who
found that increasing Cl
concentration and applying the
Cl
channel blockers
4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid
and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
inhibited the water response in frog taste nerves and the
depolarization potential in TRCs resulting from the application of
water to frog tongue. Recent studies in rat TRCs suggest that an anion
channel (12) and a pathway for the exit of large anions (20) may be activated during cell swelling.
Osmotically induced changes in cell volume can exert mechanical stress
on membranes and channel proteins (8). In this regard, it is
interesting to note that rabbit laryngeal water-sensitive single units
are also mechanosensitive (31). In amphibians it has been observed that
cell swelling activates epithelial
Na+ channels and cell shrinkage
suppresses them (11). The cloned rENaC (epithelial
Na+ channel), isolated from the
rat colon, was responsive to changes in external osmotic pressure when
expressed in Xenopus oocytes. However,
in this case, oocyte swelling decreased the channel activity, whereas
use of mannitol to shrink oocytes increased the activity of rENaC (18).
In our studies, amiloride effectively blocked CT responses to 150 mM
NaCl and the subsequent increase in the CT response to 150 mM NaCl + 300 mM mannitol (Fig. 5). These data suggest that an osmotically
induced increase in amiloride-sensitive Na+ channel activity in the apical
membrane of TRCs is the basis of the taste mixture enhancement reported
here. An increase in apical Na+
channel activity and the accompanying decrease in cell volume in
hypertonic solutions should result in an increase in intracellular Na+ activity in TRCs, where the
salt concentration of the applied stimulus remains constant. These
changes in turn could influence cell potentials or other factors
affecting receptor excitability. However, this hypothesis remains to be
tested explicitly.
If a high reflection coefficient is sufficient to account for the
mixture enhancement potential of mannitol and cellobiose, then it is
likely that other nonelectrolytes with this property will have similar
effects on taste responses. Support for this view emerges in the
extensive literature on the taste effects of polycose, a mixture of
short-chain polysaccharides with a mean molecular weight of 1,000 (27).
Polycose, which is highly preferred by rats, gives a strong neural
response in single units of the nucleus tractus solitarius
at mean concentrations of 100-200 mM (13). Surprisingly, the units
most stimulated are those that respond best to salts and acids and not
those most sensitive to sucrose. In a subsequent study, Rehnberg et al.
(27) tested undialyzed and dialyzed polycose on the CT response of
hamsters. They found that removing the ionic contaminants from the
polycose eliminated the CT response. However, the concentration of
ionic contaminants could not alone account for the response; i.e., the presence of saccharide seems necessary to amplify the ionic response. Our results suggest that this may also be accomplished through osmotic
shrinkage of the salt-sensitive receptor cells. Accordingly, the
effects of polycose and other nonelectrolytes of high reflection coefficient merit further investigation along the lines presented here.
In the in vitro experiments, isolated TRCs are exposed to solutions on
apical and basolateral membranes. However, it should be emphasized that
TRCs are structurally polarized columnar epithelial cells with an
apical projection of microvilli above the tight junctions and a smooth
basolateral membrane below. The lingual epithelium actively transports
Na+ from the mucosa to the
submucosa, an indication that structural polarity has functional
consequence (22). In a polarized preparation of lingual epithelium, we
have observed in preliminary studies that a unilateral increase in NaCl
concentration on the apical side induced a decrease in TRC volume,
although a similar change in NaCl concentration on the basolateral side
alone caused a significantly greater decrease in TRC volume. These
unpublished observations suggest that the changes in TRC volume do
occur in the intact epithelium and are significantly attenuated when
the tissue is exposed unilaterally to hypertonic solutions from the
apical side.
In summary, we observed that osmotically effective substances that
produce sustained TRC shrinkage (mannitol or cellobiose) enhanced the
rat CT response to NaCl when presented in mixture with NaCl. In
contrast, osmotically ineffective substances that did not produce
sustained TRC shrinkage (urea and DMSO) had no effect on the CT
response to NaCl. These results indicate that changes in TRC volume
directly affect the sensory function of TRCs.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Thomas U. L. Biber for expertise in conducting
experiments on the confocal laser scanning microscope, Janet K. Taylor
for technical assistance in obtaining the neural recordings, and
Victoria A. Walton for help in image analysis.
 |
FOOTNOTES |
This work was supported by National Institute on Deafness and Other
Communication Disorders Grants DC-00122 and DC-02422 (J. A. DeSimone), the Department of Veterans Affairs (G. M. Feldman), the A. D. Williams Foundation (V. Lyall), and the Jeffress Memorial Trust (J. A. DeSimone).
A preliminary report has appeared as an abstract (21).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. Lyall, Dept.
of Physiology, Virginia Commonwealth University, Sanger Hall 3002, 1101 E. Marshall St., Richmond, VA 23298-0551 (E-mail:Lyall{at}vcu.org).
Received 24 February 1999; accepted in final form 9 June 1999.
 |
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