Chem. Senses 27: 383-394,
2002
© Oxford University Press 2002
Hypoosmotic Stimuli Activate a Chloride Conductance in Rat Taste Cells
Department of Biology, Utah State University, Logan, UT 84322, USA
Correspondence to be sent to: Timothy A. Gilbertson, Department of Biology, 5305 Old Main Hill, Logan, UT 84322-5305, USA. e-mail: tag{at}biology.usu.edu
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The oral cavity is subjected to a wide range of osmotic conditions, yet little is known about how solution osmolarity affects performance of the gustatory system. In order to elucidate the mechanism by which hypoosmotic stimuli affect the peripheral taste system, I have attempted to characterize the effects of hypoosmotic stimuli on individual rat taste receptor cells (TRCs) using whole-cell patch clamp recording. Currents elicited in response to voltage ramps (-90 to +60 mV) were recorded in control saline and in solutions varying only in osmolarity (-30, -60 and -90 mOsm). In roughly two-thirds of cells, hypoosmotic solutions (230 mOsm) caused a 15% increase in cell capacitance and activated a reversible conductance that exhibited marked adaptation in the continued presence of the stimulus. Similar responses could be elicited in taste cells from taste buds in the foliate and vallate papillae, the soft palate, the nasopharynx and the epiglottis. Ion substitution experiments were consistent with the interpretation that the predominant ion carried through these apparent volume- or stretch-activated channels was Cl- under normal conditions. Reversal potentials for the hypoosmotic-induced current closely matched those predicted by the Goldman—Hodgkin—Katz constant field equation for a Cl- conductance. The relative permeability sequence of the hypoosmotic-activated current in TRCs was thiocyanate-
l-
Br- > Cl- F-
isethionate- > gluconate-. Pharmacological
experiments revealed that this Cl- conductance was inhibited by
4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and
5-nitro-3-(3-phenyl-propylamino)benzoic acid (EC50 = 1.3 and 4.6
µM, respectively), but not by CdCl2 (300 µM) nor
GdCl3 (200 µM). I hypothesize that this hypoosmotic-activated
Cl- conductance, which is similar to the well-characterized
swelling-activated Cl- current, may contribute to volume regulation
and could represent the transduction mechanism by which the presence of
hypoosmotic stimuli, including water, may be signaled in taste receptor
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Taste receptor cells (TRCs) respond to a variety of sapid molecules corresponding to the basic human perceptual correlates of salty, sour, sweet, bitter and umami. The molecules that comprise tastants are varied and encompass such chemicals as small ions up to complex organic molecules that are common to many bitter and sweet taste stimuli (Gilbertson and Kinnamon, 1996
). Accordingly, the mechanisms that have been elucidated for
the transduction of taste stimuli are varied and include direct interaction
with ion channels and activation of ionotropic and metabotropic receptors [for
reviews see (Lindemann, 1996;
Gilbertson et al.,
2000)]. TRCs also respond to a variety of compounds outside the
realm of the basic tastes that serve to modulate the response of the receptor
cell to other tastants. A classic example of this phenomenon is the
enhancement of TRC responses to monosodium glutamate by
5'-ribonucleotides (Ninomiya et
al., 1992). Additional factors that have been reported to
modify gustatory responses at the level of the receptor cell include
astringents (Schiffmann et al.,
1992), divalent cations (Myers
et al., 1993), anions
(Gilbertson et al.,
1997a), polyunsaturated fatty acids
(Gilbertson et al.,
1997b; Gilbertson,
1998), other lipids (Schiffman
et al., 1995) and O2 availability
(Singh et al.,
1997).
An additional factor that has received comparatively little attention is
the osmotic status of the taste stimulus. Taste stimuli range from the very
hypoosmotic to those an order of magnitude greater than the osmolarity of the
taste cell interior milieu (Feldman and
Barnett, 1995). Much of our current understanding about the
transduction mechanisms in mammalian TRCs has come from experiments in which
isolated taste buds are recorded from using electrophysiological or calcium
imaging techniques. In these experiments, taste stimuli are typically
presented in an isosmotic solution, a condition not always the case in
vivo. Recently, Lyall and colleagues
(Lyall et al., 1999)
demonstrated that hyperosmotic stimuli enhance rat chorda tympani responses to
NaCl, thereby demonstrating that changes in TRC volume can affect
tastant-induced activity in the peripheral gustatory system.
Higher vertebrates require that intracellular and extracellular osmolarity
be well controlled, and differences between the intracellular and
extracellular osmolarities of >1% can often lead to the activation of
compensatory mechanisms (Wenning,
1999). Most cells when exposed to hypoosmotic stimuli have
mechanisms to compensate for the osmotic difference and increase their cell
volumes in response. Typically, these cells allow the influx of water through
aquaporins (AQPs) (Echevarría and
Ilundáin, 1998; Heymann
and Engel, 1999) followed by a transient or sustained increase in
cell volume. For cells like aortic baroreceptors
(Cunningham et al.,
1995), cardiac myocytes (Hall
et al., 1997;
Köhler et al.,
1998) and central nervous system neurons
(Law, 1996) that often are
exposed to changes in extracellular osmolarity, this increase in cell volume
is sensed by corresponding changes in the activity of mechanosensitive ion
channels. These mechanosensitive channels include those that have been
characterized as stretch-activated, stretch-inactivated and pressure-sensitive
(Morris, 1990), and have ionic
permeabilities that include Cl-, K+ or
Ca2+.
Little is known about how taste cells respond to hypoosmotic changes on
either the apical or basolateral membranes and how this might affect their
ability to respond to taste stimuli. Recently, my laboratory demonstrated the
presence of three varieties of AQPs in taste cells using immunocytochemistry
and reverse transcriptase—polymerase chain reaction
(Kim et al., 1999).
Interestingly, aquaporin-5 (AQP-5) was apparently restricted to the apical
membranes, while two other AQPs, AQP-1 and AQP-2, were present predominantly
on the basolateral membrane. Thus, there are the molecular components present
in TRCs to allow rapid water entry and to compensate for changes in
osmolarity. In the present study, I have investigated the effects of
hypoosmotic stimuli on isolated rat taste buds using whole-cell patch clamp
recording. Consistent with other osmotic-sensitive cells, TRCs responded to
hypoosmotic stimuli by increases in cell volume, as measured by changes in
cell capacitance, and the subsequent activation of stretch-sensitive
Cl- channels. This mechanism may have implications both for the
response to hypoosmotic taste stimuli and in the gustatory response to water
(Cohen et al., 1955;
Ishiko and Sato, 1973;
Sato et al., 1995;
Lindemann, 1996).
Part of these results has appeared in abstract form
(Gilbertson et al.,
2000).
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Materials and methods |
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Taste bud isolation and patch clamp recording
Individual fungiform taste buds were isolated from the tongues of 2- to
5-month-old male Sprague—Dawley rats using techniques described
previously (Béhé et
al., 1990; Doolin and
Gilbertson, 1996). Briefly, tongues were isolated and injected
between the muscle layer and the lingual epithelium with 
1.0 ml of
physiological saline (Tyrode) containing a mixture of collagenase I (0.5
mg/ml; Boeringer Mannheim, Indianapolis, IN), dispase (5 mg/ml; Boeringer
Mannheim) and trypsin inhibitor (1 mg/ml; type I-S; Sigma Chemical Corp., St
Louis, MO). The injected tongue was incubated in a
Ca2+—Mg2+-free Tyrode containing 2 mM glycine,
N, N'-(1,2-ethanediylbis(oxy-2,
1-phenylene))bis(N-(carboxymethyl))-tetrapotassium salt (BAPTA,
Molecular Probes Inc., Eugene, OR) and bubbled with O2 for 25 min
at room temperature. Following incubation, the tongue was washed with saline,
and the epithelium was removed from the underlying muscle layer with forceps
and pinned out in a SylgardTM-lined Petri dish. Individual taste buds
were removed from the epithelium under low magnification (x50) with a
suction pipette (200 µm pore) and plated out into the recording
chamber. In some experiments, taste buds were isolated from the foliate and
vallate papillae (Doolin and Gilbertson,
1996), soft palate, nasopharynx and epiglottis using methods
similar to those used for other non-lingual taste buds
(Gilbertson and Fontenot,
1998). The recording chamber consisted of a Cell-Tak Tissue
Adhesive (Boehringer Mannheim) coated microscope slide fitted with an O-ring.
Though all taste bud types responded to decreases in osmolarity of
extracellular solutions in a qualitatively similar fashion (cf. Figures
1 and
2), the majority of the present
work focused on the response in taste cells from the fungiform papillae. The
criteria used to establish a cell as a taste receptor cell and not an
epithelial cell was the same as used in previous work
(Gilbertson et al.
1993; Gilbertson and Fontenot,
1998). Once in the recording chamber, cells were perfused with
extracellular solution (Tyrode) containing (in mM): 140 NaCl, 5 KCl, 1
CaCl2, 1 MgCl2, 10
N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid
(HEPES), 10 glucose and 10 Na+ pyruvate (310 mOsm). The pH was
adjusted to 7.4 with NaOH. In the initial experiments, a standard
intracellular (pipette) solution was used that contained (in mM): 140 KCl, 1
CaCl2, 2 MgCl2, 10 HEPES, 11 ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA) and 3 ATP. The pH was adjusted to 7.2 with KOH (310 mOsm). In most
experiments, however, a low K+ intracellular solution was used, in
which 90 mM CsCl and 100 mM mannitol were substituted for the KCl and the pH
was adjusted to 7.2 with CsOH (310 mOsm). This CsCl intracellular
solution helped to eliminate most of the voltage-activated outward
K+ current, which facilitated the analysis of the
hypoosmotic-induced current and set the Cl- equilibrium potential
(ECl) to near zero. Nonetheless, the results obtained were
equivalent regardless of the intracellular solution used.
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) and at -90 mV (O) versus
time from the peak of the response at time 0. The nearly superimposed solid
and dotted lines are the best fits with single exponential functions to the
-90 mV data and the +60 mV datasets, respectively. The time constants for the
adaptation (
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Voltage-activated currents were recorded from individual taste receptor
cells (TRCs) maintained in the taste bud by using the whole-cell patch clamp
configuration. Patch pipettes were pulled to a resistance of 5-10 M
when filled with intracellular solution. Series resistance and cell
capacitance were compensated optimally before the recording. The holding
potential in all experiments was -80 mV. In most cases, ramp protocols from
-90 mV to +60 mV (480 ms duration, 0.31 V/s) were used to generate
instantaneous current—voltage (I—V) relationships in the
various solutions. Command potentials were delivered and current data were
recorded with pCLAMP software (versions 7/8) interfaced to an AxoPatch 200A or
200B amplifier with a Digidata 1200A A/D board (Axon Instruments, Foster City,
CA). Data were collected at 10 kHz and filtered online at 2 kHz. No records
were leak subtracted in the present study.
Solutions
To elicit hypoosmotic responses in TRCs, solutions were prepared that varied only in osmolarity from the control solution. A control solution was prepared that contained 100 mM mannitol, and hypoosmotic solutions were made by decreasing the total mannitol concentration (to 10, 40 or 70 mM; Table 1). Thus, the ionic components of all solutions were kept constant. The control solution contained 90 mM NaCl and substitution for the Cl- with Na salts containing other anions (Table 1) was used in experiments designed to determine the relative permeability of the hypoosmotic-induced current. Junction potentials were determined empirically for all solutions and reversal potentials were corrected for junction potentials prior to analysis. The osmolarity of all solutions used in the present study was measured with a vapor pressure osmometer (Model 5500, Wescor, Logan, UT) and adjusted, if necessary, with water or mannitol.
The pharmacology of the hypoosmotic-induced current was examined using a
number of Cl- channel blockers known to target volume- or
stretch-activated conductances (Quasthoff,
1994; Xu et al.,
1997; Hume et al.,
2000;
Pérez-Samartín et
al., 2000), including DIDS
(4,4'-diiso-thiocyanatostilbene-2,2'-disulfonic acid),
CdCl2, GdCl3 and NPPB
[5-nitro-3-(3-phenylpropylamino)benzoic acid]. These inhibitors were added to
both the control (310 mOsm) and hypoosmotic (230 mOsm) solutions, and applied
by bath perfusion. The effectiveness of these inhibitors was determined by
measuring the net hypoosmotic-induced current in the absence and presence of
the inhibitors at the peak of the response at +60 mV. This potential was
chosen to eliminate the complication of the strong voltage-dependence of
Cl- channel inhibition by DIDS
(Xu et al., 1997)
(Figure 5A). Relative
inhibition was calculated as the ratio of the peak current in the presence of
the inhibitor over the current in the absence of the inhibitor.
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Data analysis
To isolate the hypoosmotic-induced current, the control traces (e.g.
I—V curves) that immediately preceded the experimental
condition were subtracted from those in the presence of hypoosmotic stimuli.
The traces selected to reflect the experimental condition were those that were
collected during the peak of the response to the hypoosmotic stimuli. Data
were collected every 30-60 s during the control, experimental and recovery
conditions. In general, the peak of the response to hypoosmotic stimulation
occurred 1-3 min after solution change. This peak value was determined
empirically for each cell used for the analysis. Because the responses to
hypoosmotic stimuli adapted significantly (see
Figure 1B) and I could not be
sure the properties of the adapted response were equivalent to those prior to
adaptation, I was careful to choose responses that represented the peak of the
current response for subsequent analysis.
Statistical analysis was performed using SPSS software (v. 7.5, SPSS Inc.,
Chicago, IL) with the level of significance (
) set at 0.05 in all
cases. One-way analyses of variance (ANOVAs) were conducted to determine the
differences among the magnitude of current densities induced by hypoosmotic
stimuli in the various taste bud types and to determine the significant
differences in the relative perme-abilities for the anions carrying the
hypoosmotic-induced current. Specific details are given in the text.
The Institutional Animal Care and Use Committees of Pennington Biomedical Research Center and Utah State University approved all procedures involving animals.
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Results |
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Effects of hypoosmotic stimuli on taste receptor cells
To examine the effects of hypoosmotic stimuli on taste receptor cells, I used whole-cell patch clamping on taste buds isolated from the tongue and oral cavity in 2- to 5-month-old male Sprague—Dawley rats. Control currents were elicited in an isotonic saline (310 mOsm) in response to voltage ramps from -90 to +60 mV (0.31 V/s) and displayed a typically outwardly rectifying waveform when a high K+ intracellular solution was used (Figure 1A). Changing to a hypoosmotic (230 mOsm) extracellular solution (Table 1) led to a pronounced increase in conductance in response to the voltage ramp in roughly two-thirds of fungiform taste cells (61.1%, n = 144 cells). This change was evident within a few seconds following solution change and continued to increase in magnitude for 1 to several min (see below). The remaining cells showed little or no response to the hypoosmotic stimulus. To isolate the net hypoosmotic-induced current in taste cells (IHYPO-T) the control I—V curve was subtracted from that generated in the presence of hypoosmotic stimuli (Figure 1A). Examination of IHYPO-T showed that it had a weakly outwardly rectifying waveform with a reversal potential near 0 mV with equimolar Cl- on the two sides of the membrane.
As shown in Figure 1A, the
magnitude of IHYPO-T increased with decreasing
extracellular osmolarity. Concomitant with the increase in whole-cell
conductance, hypoosmotic stimuli also lead to an increase in whole-cell
membrane capacitance, which reflects a change in membrane surface area
(Thiele et al.,
1998). In normal saline the mean capacitance of fungiform taste
receptor cells in the present study was 9.7 ± 0.5 pF (n = 42
cells); when placed into hypoosmotic saline the capacitance increased
significantly to 11.1 ± 0.6 pF (n = 42 cells, P <
0.0001, Student's t-test), an increase of 14.9%. The change in
membrane capacitance was correlated with the presence of the
hypoosmotic-induced current. Cells that showed no significant
IHYPO-T upon stimulation with hypoosmotic stimuli also did
not have a significant change in their membrane capacitance (data not
shown).
Though the initial response to the hypoosmotic stimulus was rapid (within a
few seconds after complete solution change), the magnitude of
IHYPO-T did not reach a maximum until 1-3 min after
changing solutions from the control to hypoosmotic solutions (the time for
complete solution change in the chamber used was <5 s). Following its
maximal activation, IHYPO-T began to decay despite the
continued presence of the hypoosmotic stimulus.
Figure 1B shows the decay of
IHYPO-T as a function of time following the peak of the
response. Within 5 min, IHYPO-T decayed to 20%
of its maximum value. There was little or no voltage dependence to this decay
since time constants (
) measured at +60 (+60 = 162 s)
were not significantly different from those measured at -90 mV
(-90 = 155 s). Adaptation was also seen in the measurements of
membrane capacitance. As IHYPO-T adapted to near
prestimulus levels, the change in capacitance associated with the hypoosmotic
stimulus also returned close to control levels in most cells (data not
shown).
Hypoosmotic stimuli affect many types of taste buds in the oral cavity
In addition to investigating hypoosmotic responses of fungiform taste receptor cells, I also examined the effects of hypoosmotic stimuli on taste cells maintained in isolated taste buds from the foliate and vallate papillae of the tongue and from the soft palate, nasopharynx and epiglottis. In all taste bud types, hypoosmotic stimuli activated a conductance in a subset of cells that looked qualitatively similar to that seen in the fungiform taste receptor cells (Figure 2A-E). Recordings were taken from too few of the other classes of taste buds in the present study to make a detailed analysis meaningful. Nonetheless, there was a limited degree of variability among the various taste bud types in terms of percent responsive cells (fungiform: 61.1%; vallate: 62.5%; foliate: 72.7%; palate: 56.3%; nasopharynx: 41.7%; epiglottis: 85.7%). The current density (pA/pF) of IHYPO-T showed no significant differences across taste bud types (Figure 2F; P = 0.51, F = 0.865, df = 5).
IHYPO-T is a Cl- current
As shown in Figures 1 and
2, the reversal potential for
IHYPO-T was close to zero with equimolar Cl- on
both sides of the membrane in all cell types examined. Because of this and
because complete replacement of extracellular NaCl with
N-methyl-D-glucamine Cl did not appreciably alter the response to
hypoosmotic stimuli (data not shown), it was hypothesized that
IHYPO-T might be carried largely by Cl- ions.
To test the involvement of Cl- in the hypoosmotic-induced response,
Na gluconate was substituted for NaCl either partially (60 mM Na gluconate; 30
mM NaCl) or completely (90 mM Na gluconate; 0 mM NaCl) in the hypoosmotic
solution listed in Table 1.
Consistent with Cl- contributing to IHYPO-T,
changes in extracellular Cl- activity (concentration x
activity coefficient) led to changes in the reversal potential of
IHYPO-T that were closely predicted by the Nernst equation
for a pure Cl- conductance (ECl = RT/F
ln[Cl]i/[Cl]o; Figure
3). Taking into account the contribution of the relative
permeability for gluconate determined empirically (see below and
Table 2) I applied the
Goldman—Hodgkin—Katz (GHK) equation in the following form:
 | (1) |
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) of NaCl (99
mM:
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Anion permeability of the hypoosmotic-induced conductance
To examine the anion permeability of IHYPO-T,
extracellular NaCl was replaced with sodium salts containing various anions in
both the control and hypoosmotic (230 mOsm) solutions
(Table 1). Reversal potentials
of IHYPO-T were measured at the peak of the response (i.e.
before the onset of adaptation) to the hypoosmotic stimulus during voltage
ramps from -90 to +60 mV. For each cell included in this analysis, reversal
potentials were measured in Cl--containing extracellular solution
and from 2-3 other solutions that had equimolar replacement of Cl-
with one of the following anions: thiocyanate (SCN-),
I-, Br-, F-, isethionate- or
gluconate-. Figure 4
shows typical I—V curves generated in the presence of
Cl- and four other solutions in which the Cl- was
replaced with SCN-, I-, Br- and
isethionate-. All reversal potentials were corrected for errors due
to pipette junction potentials. Mean reversal potentials obtained in the
various anionic solutions are listed in
Table 2. Assuming that
IHYPO-T was carried solely by anions and that the
intracellular Cl- concentration did not change significantly during
the hypoosmotic response, the relative permeabilities for the replacement
anions (Px/PCl) were calculated from
the averaged reversal potentials (Erev) by the GHK
equation (equation 1). The relative permeabilities for the various anions
through the hypoosmotic-activated channel are listed in
Table 2. The statistical
comparison among the relative permeabilities for the various anionic solutions
was performed using Tukey's HSD post-hoc test following a one-way ANOVA.
Table 3 lists the levels of
significance obtained in this analysis (SPSS 7.5, SPSS Inc., Chicago, IL) and
reveals a permeability sequence for IHYPO-T as follows:
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Effects of Cl- channel inhibitors on hypoosmotic-induced currents
The effects of several Cl- channel inhibitors and blockers of
stretch-activated Cl- channels on IHYPO-T were
examined. Both GdCl3 (200 µM) and CdCl2 (300 µM),
two known inhibitors of stretch-activated Cl- channels
(Quasthoff, 1994;
Pérez-Samartín et
al., 2000), had no significant effect on hypoosmotic-induced
currents in fungiform TRCs (Figure
5A,B). Two other Cl- channel inhibitors significantly
blocked currents though the hypoosmotic-activated channels. DIDS (200 µM)
and NPPB (100 µM) inhibited >90% of IHYPO-T measured
at +60 mV (Figure 5B).
Consistent with other reports (Xu et
al., 1997), the inhibition by DIDS was significantly voltage
dependent. While 200 µM DIDS inhibited 96 ± 2.1% of
IHYPO-T at +60 mV, it only inhibited 28 ± 11% of
the same current measured at -90 mV (n = 18). NPPB showed no similar
voltage dependence, inhibiting roughly equal proportions of
IHYPO-T at all potentials. Concentration-response
functions were generated for DIDS (0.02-200 µM) and NPPB (0.01-100 µM)
for their ability to inhibit IHYPO-T measured at its
maximal activation at +60 mV. These inhibitors blocked
IHYPO-T in a concentration-dependent manner, and best fits
to the data with a logistic function (Origin 6.1, OriginLab Corp.,
Northampton, MA) showed that the EC50 for DIDS was 1.3 µM and
for NPPB, 4.6 µM (Figure
5C).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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During feeding, the peripheral gustatory response reflects not only the response to the individual tastants (i.e. salty, sour, bitter, sweet, umami) that may be contained in food, but also the context in which it is presented. Such factors as ionic composition of the saliva, the presence of molecules that may act as taste modulators or those with additional sensory cues (e.g. texture, temperature) may all affect the response generated in the TRCs and carried forward to the gustatory afferents. In the present study, I have investigated the effects of hypoosmotic stimuli on the activity of taste receptor cells using whole-cell patch clamp recording. By altering only the osmotic composition of the stimuli through changes in mannitol concentration and keeping all ionic constituents constant, I have attempted to isolate the response attributable to decreasing osmolarity of the extracellular solution. In roughly two-thirds of cells examined, hypoosmotic stimuli (230-280 mOsm) caused an increase in the membrane capacitance of the TRC and an increase in whole-cell conductance that showed marked adaptation over several minutes of exposure. The increase in conductance was attributable to the activation of a DIDS- and NPPB-sensitive Cl- current whose magnitude was inversely correlated with solution osmolarity (Figure 1A, inset). These data, coupled with those of Lyall et al. (Lyall et al., 1999
) that demonstrated effects of hyperosmotic stimuli on TRC
gustatory responses, are consistent with the interpretation that the osmotic
properties of taste stimuli may have profound effects on peripheral gustatory
responses. The present study in particular demonstrates that increases in cell
volume directly lead to activation of volume- or stretch-activated
Cl- channels that may contribute to the well-known gustatory
response to water (Zotterman,
1956; Shingai,
1980). Nature of the hypoosmotic response in taste receptor cells
Using whole-cell patch clamp recording, the perfusion of stimuli that
varied only in their osmolarity led to significant changes in the
current-voltage relationship generated in response to a voltage ramp from -90
to +60 mV. This hypoosmotic response was evident as an increase in the slope
of the I-V curve (Figure
1A). The net response (IHYPO-T) exhibited a
moderate outwardly rectifying conductance (Figures
1A,
2 and
4). Though the ability to
respond to changes in solution osmolarity is a feature of many cells types,
not all the TRCs in the present study responded to decreases in osmolarity.
Approximately one-third of TRCs could be classified as non-responsive; these
cells showed neither the changes in conductance nor the change in membrane
capacitance (surface area) that characterized the responsive TRCs. Our
preliminary study that looked at the distribution of AQP-1, -2 and -5 in rat
TRCs using antibodies directed against these water channels
(Kim et al., 1999)
has shown that 50% of TRCs contain these AQP molecules. Though in the
present study I have not attempted to determine a correlation between AQP
expression and the hypoosmotic responses, one explanation for the
non-responsive TRCs may be that they lack a highly permeable route for water
entry needed to mount a fast (<1 min) response to hypoosmotic stimuli.
The responses to hypoosmotic solutions were also characterized by marked
adaptation despite the continued presence of the stimulus
(Figure 1B). This adaptation
was evident by both the adaptation of IHYPO-T and the
change in membrane surface area. Adaptation of changes in membrane capacitance
and IHYPO-T suggests that TRCs are capable of recovering
from prolonged changes in the osmolarity of the extracellular milieu. A
similar time course for adaptation of water responses in the frog
(Andersson and Zotterman, 1950;
Nomura and Ishizaki, 1972) and
rat (Shingai, 1980) has been
reported. Adaptation of hypoosmotic responses is common in a variety of cell
types that function as osmometers (Bourque
and Oliet, 1997; van der Wijk
et al., 2000) and it may be a mechanism to protect the
cells from cytoskeletal damage during prolonged hypoosmotic conditions. This
response, termed the regulatory volume decrease (RVD), is a typical feature of
most cell types that display these types of swelling-activated Cl-
currents (ICl,swell) and follows a similar time course to
that shown in Figure 1B [for a
review see (Hume et al.,
2000)]. TRC responses to hyperosmotic solutions, on the other
hand, did not show a similar type of adaptation
(Lyall et al.,
1999).
Ion substitution experiments revealed that the outwardly rectifying
IHYPO-T was a Cl- current since changes in
Cl- concentration produced changes in the reversal potential for
IHYPO-T that were closely predicted by the GHK equation
(Figure 3). Because I could not
be sure that the process of regulatory volume decrease did not begin before
the maximal activation of IHYPO-T, I have chosen to assume
that the Cl- concentration inside the cell did not appreciably
change during osmotic-induced swelling. If the increase in surface area did
lead to a significant reduction in intracellular Cl- concentration and I have
overestimated [Cl-]in, then one would predict a slight
(a few millivolts) shift in ECl toward a more negative
value. Nonetheless, the relative permeability sequence for the various anions,
which is an important defining characteristic of IHYPO-T,
would remain the same. I determined the permeability sequence for
IHYPO-T in the present study was SCN-
I- Br- > Cl- F-
isethionate- > gluconate-, which follows the relative
permeability for the fairly ubiquitous ICl,swell
(Jentsch et al.,
1999). Similarities between IHYPO-T described
in the present study and the well-described ICl,swell
extend into the pharmacology of this current. Both the stilbene derivative
DIDS and the Cl- channel inhibitor NPPB inhibited
IHYPO-T in a manner consistent with these compounds'
ability to inhibit ICl,swell
(Hume et al., 2000).
Thus, from all indications, the hypoosmotic-activated current in TRCs appears
qualitatively similar to the ICl,swell present in a
variety of cell types.
The molecular identity of ICl,swell remains unclear.
There is controversy about which, if any, of the identified ClC gene family of
Cl- channels (Jentsch et
al., 1999) mediates the swelling-activated conductance. One
of the potential candidates for ICl,swell remains ClC-3.
When expressed, this current shows outward rectification, an I-
> Cl- > F- permeability sequence and sensitivity
to stilbene derivatives (Duan et
al., 1997; Shimada et
al., 2000). However, experiments using heterologous
expression of ClC-3 are complicated first, by the fact that this channel is
expressed to some degree in most cells types, including those used for
heterologous expression, such as Xenopus oocytes, and Chinese hamster
ovary and NIH/3T3 cell lines (Li et
al., 2000; Stobrawa
et al., 2001); and second by the fact that other
laboratories have been unable to reproduce these expression data
(Li et al., 2000).
Further support for the involvement of ClC-3 in ICl,swell
has come from a recent study by Duan and colleagues
(Duan et al., 2001)
that used antibodies against ClC-3 to inhibit the native
ICl,swell in guinea pig cardiac myocytes and in
heterologously expressed ClC-3 in Xenopus oocytes, suggesting that
ClC-3 was an important contributor to this current. Despite this evidence,
ClC-3 knockouts exhibit normal ICl,swell responses in both
hepatocytes and pancreatic acinar cells, two cells that express substantial
ICl,swell in wild-type animals
(Stobrawa et al.,
2001). This study concluded that ClC-3 is an intracellular channel
similar to the closely related ClC-4 and -5 channels, whose properties vary
dramatically from the native ICl,swell
(Friedrich et al.,
1999). Clearly, the molecular nature of the channel mediating
ICl,swell and IHYPO-T remains an open
question (Clapham, 2001).
At least one member of the ClC family of chloride channels has been
identified in taste receptor cells. Despite the fact that ClC-3 remains the
only viable candidate of the ClC family for mediating
ICl,swell, it is not apparently found in mammalian TRCs.
Miyamoto and colleagues (Miyamoto et
al., 2001) failed to identify ClC-3 protein using
immunocytochemical techniques, but did locate ClC-2 protein on the basolateral
membranes of both taste cells and epithelial cells in the lingual epithelium
of C57BL/6 mice. They suggested that ClC-2 channels might contribute to the
inwardly rectifying current mediating KCl responses in TRCs. ClC-2, however,
is unlikely to be involved in generating the IHYPO-T
reported in the present study. ClC-2 currents are inwardly rectifying, have a
Cl- > Br- > I- permeability sequence,
and are inhibited by millimolar concentrations of Cd2+
(Okada et al., 1998;
Wills and Fong, 2001),
features distinct from IHYPO-T in rat TRCs.
Implications of IHYPO-T in taste receptor cells
The identification of an ICl,swell-like conductance in
rat taste receptor cells, which I have termed IHYPO-T to
represent the hypoosmotic-activated current in taste cells, implies that
mammalian TRCs are capable of responding electrophysiologically to decreases
in extracellular solution tonicity. In addition, the changes in both membrane
capacitance (surface area) (Thiele et
al., 1998) and IHYPO-T returned to near
control levels despite continual perfusion of the hypoosmotic stimulus. This
result is consistent with the interpretation that TRCs are capable of RVD.
Though in taste cells the mechanism of RVD has not been investigated, it is
likely to involve the efflux of solutes during hypoosmotic stimulation, as is
the case in other cell types (Bond et
al., 1998; Deleuze et
al., 1998; Song et
al., 1998).
In the present study, stimuli were applied to the entire surface of the
taste cells and not restricted to their apical membranes, as is the case
in vivo. As such, no conclusions can easily be drawn about whether
these responses reflect those that are attributable to taste responses or
merely compensatory responses during osmotic changes as might occur in the
interstitial fluid surrounding the taste bud. Nonetheless, there are several
parallels between this and other studies that are suggestive of the fact that
this conductance may also be important for gustatory processing. First,
previous work from my laboratory has shown that TRCs from the fungiform
papillae contain apically localized AQP-5 water channels
(Kim et al., 1999)
that would provide a route for rapid water entry during such hypoosmotic
stimulation. Thus, irrespective of the cellular localization of these putative
stretch-activated channels that underlie IHYPO-T, water
entry through these apical channels could lead to activation of this
Cl- conductance.
Secondly, in many cell types there exists a basally active Cl-
conductance that has all the hallmarks of both IHYPO-T and
ICl,swell, including enhancement by swelling, outward
rectification, I- > Br- > Cl-
permeability sequence and stilbene derivative sensitivity
(Duan and Nattel, 1994;
Duan et al., 1997).
It has been hypothesized that this current, termed ICl,b,
is actually the same current as ICl,swell
(Hume et al., 2000),
and that this conductance may be partially active under normal, isotonic
conditions. Interestingly, a recent report implicated a NPPB-sensitive
Cl- conductance in the transduction of sour tastants
(Miyamoto et al.,
1998). This conductance was inhibited by NPPB applied
basolaterally in the same concentration range
(Miyamoto et al.,
1998) as the NPPB block of IHYPO-T
(Figure 5), and, like
IHYPO-T, was outwardly rectifying and
Gd3+-insensitive. Thus, if IHYPO-T represents a
constitutively active current in TRCs, then this may be one of the targets for
modulation by sour tastants (e.g. acids). Additionally,
ICl,b has been shown to be inhibited by hyperosmotic
stimuli (Duan et al.,
1995), suggesting that it may contribute not only to hypoosmotic
responses, but also to hyperosmotic responses, and may, in this way,
contribute to the hyperosmotic responses reported by Lyall et al.
(Lyall et al., 1999).
Further studies aimed at elucidating the contribution of
IHYPO-T to sour taste transduction and hyperosmotic taste
responses appear warranted.
A third potential role of this conductance in gustatory processing may be
in its contribution to `water' taste
(Zotterman, 1956;
Storey and Johnson, 1975;
Shingai, 1980). The gustatory
response to water has been demonstrated in a variety of species [for a review
see (Lindemann, 1996)]. In cat
(Cohen et al., 1955),
pig (Zotterman, 1956) and frog
(Andersson and Zotterman, 1950)
tongue, afferent nerve fibers were shown to respond to orally applied
hypoosmotic (low ionic strength) stimuli by increasing their firing rates.
These responses were sensitive to mucosal Cl- concentrations,
suggesting that this anion played a role in the water response. This
dependence of the water response on mucosal Cl- was supported by
more recent studies in the frog tongue that demonstrated that increasing the
mucosal Cl- inhibited the water response
(Okada et al., 1993).
If IHYPO-T is involved in this response, increasing
mucosal (extracellular) Cl- concentrations would be predicted to
reduce net efflux of Cl- and hence decrease the net inward current,
provided that these channels could sense mucosal ion concentrations (i.e. were
apically localized). Moreover, the water response in the frog was sensitive to
stilbene derivatives like DIDS (Okada
et al., 1993). In the rabbit, water responses were
similarly inhibited by extracellular Br-, I- and
Cl- ions (Shingai,
1977), all of which were markedly permeant through the
hypoosmotic-activated channels in the present study. Taken together, there is
a good correlation between the properties known for the water response in
taste cells and the properties of IHYPO-T described in the
current study.
An inherent property of the water response in the oral cavity is that the
most vigorous responses occur in the laryngeal water receptors and, by
comparison, water responses originating in the lingual taste buds are quite
modest (Shingai and Shimada,
1976; Smith and Hanimori,
1991). I have shown that all the taste receptor cells investigated
in this study respond in a similar fashion to hypoosmotic stimuli
(Figure 2), suggesting a common
mechanism underlying the generation of IHYPO-T. Though a
slightly greater percentage of hypoosmotic-responsive cells was found in the
area of the epiglottis (85.7% of cells responded versus 61.1% in the fungiform
taste buds), there was no significant difference in the density of
IHYPO-T in various areas of the oral cavity
(Figure 2F), arguing against
higher levels of expression of hypoosmotic-activated channels mediating the
differences in water responsiveness between the tongue and the back of the
oral cavity. It is possible that local differences in ion concentrations,
either intracellularly or extracellularly, or differences in cellular
expression (apical versus basolateral) of the channels underlying
IHYPO-T may play a role in generating this difference.
|
Acknowledgments |
|---|
I wish to acknowledge the expert technical assistance of Nikki D. Siears and Huai Zhang, and their substantial contributions to this study. I also wish to thank Insook Kim and Lidong Liu, for their contributions to the study and their helpful discussions, and Christine Foley, for comments on an earlier version of this manuscript. This work was supported in part by research grants DC02507 and DK55809 to from the National Institutes of Health.
|
References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Accepted February 13, 2002