Department of Anatomy and Neurobiology, Colorado State University, Fort Collins 80523; and Rocky Mountain Taste and Smell Center, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Lin, Weihong and
Sue C. Kinnamon.
Physiological Evidence for Ionotropic and Metabotropic Glutamate
Receptors in Rat Taste Cells.
J. Neurophysiol. 82: 2061-2069, 1999.
Monosodium glutamate (MSG)
elicits a unique taste in humans called umami. Recent molecular studies
suggest that glutamate receptors similar to those in brain are present
in taste cells, but their precise role in taste transduction remains to
be elucidated. We used giga-seal whole cell recording to examine the
effects of MSG and glutamate receptor agonists on membrane properties of taste cells from rat fungiform papillae. MSG (1 mM) induced three
subsets of responses in cells voltage-clamped at 80 mV: a decrease in
holding current (subset I), an increase in holding current (subset II),
and a biphasic response consisting of an increase, followed by a
decrease in holding current (subset III). Most subset II glutamate
responses were mimicked by the ionotropic glutamate receptor (iGluR)
agonist N-methyl-D-aspartate (NMDA). The
current was potentiated by glycine and was suppressed by the NMDA
receptor antagonist D(
)-2-amino-5-phosphonopentanoic acid (AP5). The group III metabotropic glutamate receptor (mGluR) agonist L-2-amino-4-phosphonobutyric acid (L-AP4)
usually mimicked the subset I glutamate response. This hyperpolarizing
response was suppressed by the mGluR antagonist
(RS)-
-cyclopropyl-4-phosphonophenylglycine (CPPG) and by
8-bromo-cAMP, suggesting a role for cAMP in the transduction pathway.
In a small subset of taste cells, L-AP4 elicited an
increase in holding current, resulting in taste cell depolarization under current clamp. Taken together, our results suggest
that NMDA-like receptors and at least two types of group III mGluRs are
present in taste receptor cells, and these may be coactivated by MSG.
Further studies are required to determine which receptors are located
on the apical membrane and how they contribute to the umami taste.
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INTRODUCTION |
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Monosodium glutamate (MSG) is a natural component
of many foods, including seafood, meats, milk and their by-products,
mushrooms, and some vegetables. Both naturally occurring and purified
MSG have been used to enhance the flavor of foods and increase food palatability. The taste induced by MSG is called "umami," a
Japanese term meaning delicious or savory (Ikeda 1909).
It is believed that the appetitive taste of MSG and other amino acids
reflects the requirement of protein in the diet of most animals.
Several studies have characterized the properties of umami taste (for
review, Bellisle 1999). As a potent taste stimulus, MSG
alters the activity of afferent nerve fibers and central gustatory neurons (Adachi and Aoyama 1991
; Hellekant and
Ninomiya 1991
; Hellekant et al.
1997
; Nakamura and Norgren
1993
; Ninomiya et al. 1991
; Plata-Salaman
et al. 1992
). In general, it is believed that glutamate is the
primary stimulus for the umami taste (Schiffman and Gill
1987
; Yamaguchi 1987
, 1991
;
Yamaguchi and Kimizuka 1979
).
Several studies have provided evidence that glutamate receptors similar
to those in brain may be involved in the transduction of umami taste.
Ligands of brain glutamate receptors, including aspartate and
ibotenate, are potent taste stimuli; these compounds induce responses
in the chorda tympani nerve (Faurion 1991) and elicit an
umami taste in humans (for review, Maga 1983
). Recent studies suggest that group III metabotropic glutamate receptors (mGluRs) may play a key role in this process. Molecular studies have
shown that mGluR4 is specifically expressed in taste cells and not the
surrounding epithelium (Chaudhari et al. 1996
). In addition, behavioral studies show that the group III mGluR agonist L-2-amino-4-phosphonobutyric acid (L-AP4)
generalizes to the taste of glutamate (Chaudhari et al.
1996
). Further, physiological studies have shown that both MSG
and L-AP4 elicit conductance changes (Bigiani et al.
1997
) and alter intracellular Ca2+ levels
(Hayashi et al. 1996
) in taste cells, providing further evidence for a role of these receptors in the transduction process.
Yet, other investigators have provided evidence for the presence of
ionotropic glutamate receptors (iGluRs) in taste cells. These data have
come from bilayer studies, in which taste epithelial membranes were
incorporated (Brand et al. 1991; Teeter et al. 1992
). MSG and N-methyl-D-aspartate
(NMDA) both activated a cation conductance in the bilayers, suggesting
that the receptor may be an NMDA receptor. In addition,
Ca2+ imaging studies have shown increases in intracellular
Ca2+ in response to NMDA in isolated taste cells
(Hayashi et al. 1996
).
Using the whole cell patch-clamp technique and pharmacological agents,
we examined the following questions in the present study.
1) Are both NMDA and mGluRs present in taste cells of
fungiform papillae? 2) What intracellular signaling
pathways are involved? 3) Which ions are involved in
responses to NMDA and L-AP4? Preliminary accounts of this
work have been published in abstract form (Lin et al.
1996; Lin and Kinnamon 1996
).
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METHODS |
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Isolation of taste buds
Four to 12-wk-old male Sprague-Dawley rats were used. Taste buds
were freshly isolated from fungiform papillae with a method adapted
from Béhé et al. (1990). Briefly, rats were
killed with CO2, and the tongue was dissected and
placed into cold Tyrode's solution. Approximately 0.3-0.8 ml of an
enzyme mixture containing 3 mg dispase, 0.7 mg collagenase B
(Boehringer Mannheim, Indianapolis, IN), and 1 mg trypsin inhibitor
(type I-S; Sigma Chemical, St. Louis, MO) in 1.0 ml of Tyrode's was
injected beneath the lingual epithelium of the tongue. The tongue was
then incubated in Ca2+ and
Mg2+-free oxygenated Tyrode's for 30 min, or
until the epithelium could be gently separated from the underlying
muscle and connective tissue. The stripped lingual epithelium was
pinned serosal side up in a silicone elastomer (Sylgard)-covered Petri
dish and incubated in Ca2+ and
Mg2+-free Tyrode's for 20 min. Taste buds were
removed by gentle suction with a glass pipette and plated onto Cell-Tak
(Collaborative Research, Bedford, MA)-coated glass slide chambers. The
chambers were formed by affixing a Sylgard ring (2-mm wall thickness
with an opening diameter of 1.5 cm) to the Cell-Tak-coated slide.
Solutions and chemicals
Normal Tyrode's was used as a standard bath solution,
containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES), 10 glucose, and 10 sodium pyruvate (pH 7.4 with
NaOH). The Ca2+- and
Mg2+-free Tyrode's for isolating taste buds
contained 2 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA; Molecular Probes, Eugene, OR). The 70 mM
Na+ or Na+-free Tyrode's
was obtained by replacing Na+ with equimolar
N-methyl-D-glutamine (NMDG). The pH was adjusted to 7.4 with HCl. For Ba2+-Tyrode's, NaCl was
replaced by 100 mM BaCl2 and 50 mM NMDG. Low Cl Tyrode's contained 140 mM
Na+ gluconate instead of NaCl. Bath solutions
were gravity-fed into the 0.5-ml recording chamber. The standard
intracellular pipette solution contained (in mM) 140 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 11 EGTA, 1 ATP, and 0.4 GTP (pH 7.2 with KOH). Low
Cl
pipette solution contained 130 mM K
gluconate and 10 mM KCl in place of 140 mM KCl.
MSG, guanosine 5'-o-(2)-thiodiphosphate (trilithium salt,
GDP--S) and 8-bromo adenosine 3'5'-cyclic monophosphate
(8-bromo-cAMP) were from Sigma Chemical. The agonists and antagonists
of glutamate receptors were obtained from Tocris Cookson (Ballwin, MO);
these included NMDA, MK-801,
D(
)-2-amino-5-phosphonopentanoic acid (AP5),
L-AP4, (R,S)-
-methyl-4-phosphonophenylglycine
(MPPG), (RS)-
-cyclopropyl-4-phosphonophenylglycine (CPPG), and
(s)-
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA).
Solutions were gravity-fed into the recording chamber; solution
exchange was complete in <10 s. Most of the chemicals used were bath
applied except GDP-
-S, which was included in the pipette solution.
Patch-clamp recordings
The whole cell patch-clamp technique was used (Hamill et
al. 1981). The steady-state holding current was recorded at
80 mV, except as noted. In some experiments, changes in membrane
potential were monitored under current-clamp conditions; responses were monitored at resting potential. The glass pipettes for recording were
pulled from microhematocrit capillary tubes (Scientific Products, McGaw
Park, IL) with a two-stage vertical puller (model PB-7; Narishige,
Tokyo) or a Flaming/Brown micropipette puller (model P-97; Sutter
Instrument, Novato, CA). Pipette resistance was 3-6 M
when filled
with normal pipette solution and 4-8 M
when filled with the low
Cl
pipette solution. Membrane currents were
low-pass filtered at 2 kHz and recorded with an Axopatch patch-clamp
amplifier (model 200B, Axon Instruments, Foster City, CA).
Voltage-activated Na+ and
K+ currents were generated by applying
depolarizing voltage steps from a holding potential of
80 mV; these
were used to distinguish taste cells from nonsensory epithelial cells.
Hyperpolarizing voltage pulses (20 mV) were used to monitor membrane
conductance during whole cell recording. All voltage commands were
generated by an Indec laboratory computer system (Sunnyvale, CA).
Steady membrane currents were recorded on a strip chart recorder
(Linear) as well as on videotape using a VCR (JVC) and analyzed subsequently.
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RESULTS |
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Responses to glutamate
Taste cells from isolated taste buds were voltage clamped at 80
mV, and holding current and membrane conductance were monitored in
response to bath application of MSG (1 mM). Responses were arbitrarily
grouped into three subsets: a decrease in holding current and membrane
conductance (subset I), an increase in holding current (subset II), and
a biphasic response, i.e., an increase, followed by a decrease in
holding current (subset III). A total of 108 cells of the 185 tested
responded to MSG. Of these, 40 cells exhibited subset I responses, 38 cells subset II responses, and 30 cells subset III responses (Fig.
1). In general, it took longer for a
subset I response to reach peak amplitude than a subset II response.
The time taken to reach half of the response amplitude
(T1/2 value) for subset I and II
responses are 22.5 ± 1.1 and 2.7 ± 0.5 s, respectively
(mean ± SE, n = 16, P < 0.001). There were no consistent differences
in membrane resistance, capacitance, or expression of voltage-gated
currents in these glutamate responding cells that could correlated with
the type of glutamate response. We considered that responses to MSG
were induced primarily by glutamate, because the addition of 1 mM MSG
has a negligible effect on bath Na+ concentration. These
results are consistent with a previous study of vallate taste cells
that showed both increases and decreases of holding current in response
to MSG (Bigiani et al. 1997
).
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Metabotropic glutamate receptors in brain are coupled to
G-protein-mediated intracellular pathways; activation of receptors in
group III mGluRs inhibits the activity of adenylate cyclase and
decreases the intracellular cAMP level (for review, Pin and Duvoisin 1995). To determine whether both iGluRs and mGluRs are expressed in fungiform taste cells, we added a nonhydrolyzable GDP
analogue, GDP-
-S (0.2 mM) to the recording pipette to inhibit G
protein-mediated pathways. With GDP-
-S in the pipette solution, subset I responses were abolished after 15-30 min of whole cell recording (n = 4). In addition, subset III
responses were converted to subset II responses (n = 4; Fig. 2A) while subset
II responses usually remained unchanged (n = 9).
Because many group III mGluRs decrease cAMP in brain (Pin and
Duvoisin 1995
), we examined whether intracellular cAMP pathways
are involved in taste cell responses to glutamate. Bath application of
a membrane-permeable cAMP analogue, 8-bromo-cAMP (1 mM) suppressed
subset I responses (7 of 10 cells; Fig. 2B) and the
outward component of the subset III response (n = 3), while having no significant effect on subset II responses (n = 6). The results indicate that both iGluRs and
mGluRs may be present in fungiform taste cells and that subset I
responses may be coupled to G protein-mediated intracellular cAMP
pathways. On the basis of the results above, we utilized specific
glutamate receptor agonists and antagonists in further experiments to
identify possible subtypes of glutamate receptors in taste cells.
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Responses to NMDA
Both glutamate and NMDA activate NMDA receptors in brain; glycine
is usually required as a co-agonist for full activation of the receptor
channel. Extracellular Mg2+ blocks the channel at
negative membrane potentials, resulting in voltage-dependent activation
of the channel. The channel is permeable to Na+,
Ca2+, and K+ (for review,
Collingridge and Watkins 1994). We examined whether NMDA
receptors are present in fungiform taste cells and whether the
receptors are similar to those in brain. Bath application of NMDA (1 mM) alone increased holding current in ~45% of cells tested when
cells were voltage clamped at
80 mV (n = 111). The response generally mimicked subset II responses to glutamate. In the
presence of 10 µM glycine, the amplitude of NMDA-sensitive currents
increased from 5.2 ± 1.1 pA to 7.7 ± 1.0 pA
(n = 10, Fig.
3A). The above experiments
suggest that glycine is a co-factor for the taste cell NMDA receptor;
thus it was added to all solutions in which NMDA was applied. Glycine
(10 µM) applied alone usually did not change holding current.
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Next, we tested blockage by extracellular Mg2+.
Because membrane depolarization removes the Mg2+
block for brain NMDA receptors, we examined the NMDA-sensitive current
at two holding potentials: 80 and
40 mV. Only 4 of 11 cells tested
showed greater current amplitudes at
40 mV than at
80 mV, whereas
in most cells, the peak currents induced at
80 mV were bigger than
those at
40 mV (8.6 ± 0.8 pA and 5.9 ± 0.6 pA,
respectively, n = 7, t < 0.05).
Similar results were obtained from experiments conducted in
extracellular Mg2+-free solution, in which only
three of nine cells showed an increase in the peak current. However,
when cells were bathed in Mg2+-free solution, the
NMDA-induced current appeared to desensitize slower in six of nine
cells tested. In contrast, when cells were bathed in 10 mM
Mg2+ solution, the current desensitized much
faster, although the peak current did not decrease (n = 3). These data suggest that extracellular Mg2+
normally does not block the receptor channel, but once it opens, Mg2+ can partially occlude the channel (Fig.
3B). These results are consistent with the effect of MK-801,
an open channel blocker. MK-801 blocked the current partially (data not
shown), whereas 50 µM AP5, a specific antagonist of the channel,
suppressed most of the current (Fig. 3C; n = 4). In addition, we tested the effect of cAMP on the NMDA-induced
current. Similar to subset II responses to glutamate, 8-bromo-cAMP did
not suppress the NMDA-sensitive current (n = 3, data
not shown).
To determine the reversal potential of the NMDA-sensitive current, we recorded the current at different holding potentials (Fig. 4, A and B). Unlike the NMDA-sensitive current in the brain, the current in taste cells did not reverse at 0 mV, but reversed at potentials considerably more positive than 0 mV (n = 7). Due to the instability of recording at positive potentials, we could not obtain the actual reversal potential of the NMDA-sensitive current. Our results, however, suggest that the reversal potential is near the equilibrium potentials of Na+ and Ca2+. To test whether Na+ and Ca2+ both contribute to the receptor current, we replaced bath Na+ with the impermeant cation NMDG. The peak current induced by NMDA was reduced in 70 mM Na+ solution (from 6.42 ± 2.1 pA to 3.6 ± 1.1 pA; n = 3). However, the current was not totally eliminated, even in Na+-free solution. When 100 mM NMDG was replaced with 100 mM Ba2+ in the bath solution, the current was partially recovered (Fig. 4C), suggesting that both Na+ and divalent cations carry the NMDA-sensitive current.
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Finally, we examined the effect of NMDA on membrane potentials under
current-clamp configuration. When cells were clamped with the standard
pipette solution containing 140 mM KCl and bathed in the normal
Tyrode's solution, resting membrane potentials ranged from 20 to
70 mV. Only a few cells had resting potentials more negative than
50 mV, and some of these fired action potentials spontaneously.
Spontaneously active cells usually had relatively large voltage-gated
Na+ currents (peak current ~2,000 pA) and small
voltage-gated K+ currents. NMDA depolarized these
cells and increased the frequency of action potentials (Fig.
5A, n = 3). In
cells that were not spontaneously active at rest, NMDA caused membrane
depolarization, but action potentials were not always elicited (Fig.
5B; n = 6). In the particular cell shown in
Fig. 5B, replacing normal bath solution to a
Mg2+-free Tyrode's dramatically increased
responses to NMDA. Depolarizations induced by NMDA were often followed
by a small hyperpolarization during wash out of NMDA. Similar wash
effects were often observed with subset II and subset III glutamate
responses under voltage clamp.
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Responses to L-AP4
L-AP4 is a specific agonist for the group III
mGluRs (for review, Thomsen 1997). Previous studies
showed that mGluR4 is expressed in vallate taste cells and
L-AP4 mimics responses to glutamate both physiologically
(Bigiani et al. 1997
) and behaviorally (Chaudhari et al. 1996
). Thus mGluR4 also may be expressed in taste cells of fungiform papillae. Bath application of L-AP4 (10-20
µM) decreased the holding current in 30 of 93 cells tested, mimicking
the subset I response to glutamate. The presence of 8-bromo-cAMP (1 mM)
suppressed the response (Fig.
6A). In addition, concomitant
with this subset of response, L-AP4 induced either a
hyperpolarization or had no effect on membrane potentials for most
cells tested in current-clamp mode. In a few cells, L-AP4
decreased the firing rate of spontaneous action potentials. The mGluR4
antagonists MPPG 500 µM and CPPG 100 µM suppressed responses to
L-AP4 (n = 4 and 3, respectively; Fig.
6B). To determine the reversal potential of this
L-AP4-sensitive current, we recorded the responses at
different holding potentials. When the pipette solution contained 140 mM KCl, the current reversed about 0 mV (n = 2, data
not shown). However, when the pipette solution contained 130 mM K
gluconate and 10 mM KCl the current reversed about
40 mV, a potential
near the equilibrium potential of Cl
(Fig.
6C), suggesting that L-AP4 may suppress a
Cl
conductance. To further examine the role of
Cl
, we conducted experiments in low
Cl
(9 mM) Tyrode's. Under these conditions,
the L-AP4 response was virtually eliminated. This result
was unexpected, because the driving force for
Cl
should be larger under these conditions. It
is possible that extracellular Cl
is involved
in the channel gating, as has been shown in other systems (Pusch
et al. 1995
). Further experiments are required to address this
issue thoroughly.
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In general, the results above are consistent with previous studies on
effects of L-AP4 in vallate taste cells (Bigiani et al. 1997). However, we recorded an increase in
holding current in response to L-AP4 in 16 of 93 cells
(Fig. 7A). Unlike the effect of L-AP4 described above, the amplitude of this response
increased with increasing concentrations of L-AP4 ranging
from 10 µM to 1 mM in the bath. These data are consistent with the
dual effects of L-AP4 reported previously in vallate taste
cells. Although most L-AP4-sensitive taste cells showed a
decrease in intracellular Ca2+, a few cells
showed increases in intracellular Ca2+ in
response to L-AP4 (Hayashi et al. 1996
). The
inward currents elicited by L-AP4 were not significantly
suppressed by the metabotropic antagonist CPPG (100 µM;
n = 2). In addition, this subset of L-AP4 response usually was accompanied by membrane depolarization
(n = 3, Fig. 7A), and replacement of
extracellular Na+ with NMDG eliminated the
response (Fig. 7A). To examine this response further, we
examined the effect of 8-bromo-cAMP on the L-AP4 response.
Curiously, bath perfusion of 8-bromo-cAMP (1 mM) alone caused a
decrease in holding current and membrane conductance (n = 7; Fig. 7B). In the presence of 8-bromo-cAMP, the
L-AP4-induced inward current was transiently potentiated
in four of the seven cells (Fig. 7B). In these cells,
L-AP4 appeared to transiently remove the block of the
conductance. This conductance appears to have similar properties to the
direct cyclic nucleotide conductance recently described in frog taste
cells (Kolesnikov and Margolskee 1995
). Further
experiments will be required to examine this hypothesis.
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To determine whether both mGluR4 and the NMDA receptor channels are located in the same taste cells, we applied glutamate, L-AP4, and NMDA sequentially to several taste cells. Data were pooled from all subsets of glutamate responses and are shown in Fig. 8. In a total of 32 cells that responded to glutamate, 11 cells (34%) responded to both L-AP4 and NMDA, 8 cells (25%) responded to NMDA only, 9 cells (28%) responded to L-AP4 only, and 4 cells (13%) responded to neither NMDA nor L-AP4. The lack of response to both NMDA and L-AP4 suggests that other subsets of glutamate receptors may be expressed in taste cells. Therefore we also tested some taste cells for responses to AMPA, a specific agonist of the AMPA receptor. Bath application of AMPA (100 µM) induced responses similar to the subset II response of glutamate, i.e., an increase in holding current in a small subset of cells. Further study is needed to determine whether AMPA receptors play any role in taste transduction.
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DISCUSSION |
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In this study we present electrophysiological evidence that NMDA
receptors and at least two types of group III metabotropic glutamate
receptors are expressed in rat fungiform taste cells. These findings,
coupled with molecular, biochemical, and behavioral studies
(Chaudhari et al. 1996) suggest that these receptors may play an important role in the transduction of umami taste.
Responses to glutamate
In our study, glutamate induced three different subsets of
responses. Because these responses were pooled from all taste cells tested, we could not rule out the possibility that the multiple responses occur selectively in different cell types. However, we did
obtain results from single taste cells that responded to both NMDA and
L-AP4, suggesting that both receptors are often present in
the same cells. We suggest that subset III responses represent compound
responses that involve co-activation of NMDA and mGluR4 receptors.
Several observations are consistent with this interpretation. First,
cells exhibiting subset III responses usually responded to both NMDA
and L-AP4, suggesting that both types of receptors are
present in these cells. Second, the time to half-maximal activation was
considerably longer for responses elicited by L-AP4 than
for those elicited by NMDA; thus the inward current should precede the
outward current. Finally, GDP--S and 8-bromo-cAMP suppressed only
the glutamate-activated outward current in cells expressing subset III
responses. These data are consistent with other studies showing both
increases and decreases in intracellular Ca2+ or
holding currents in response to glutamate (Bigiani et al. 1997
; Hayashi et al. 1996
). The data presented
in this study showed that the percentage of cells responding
biphasically (28%, Fig. 1B) is lower than the
percentage of cells that respond to both L-AP4 and NMDA
(34%, Fig. 8). This difference could be due to the fact that some
responses to L-AP4 involved increases in holding current.
Taste cells could express receptors for this novel response of
L-AP4 in addition to NMDA receptors. Such a combination
would not likely result in a biphasic response to glutamate.
Responses to NMDA
In vallate taste cells from mice, NMDA increased the membrane
conductance (Teeter et al. 1992) and increased
intracellular Ca2+ levels (Hayashi et al.
1996
). Our data are in agreement with these studies. Our data
on fungiform taste receptor cells differ somewhat from those of
Bigiani et al. (1997)
, who showed that only a small
fraction of vallate taste cells in rats showed increases in holding
current in response to glutamate. In our study, most glutamate-sensitive taste cells exhibited some inward current in
response to glutamate, and many also responded to NMDA. It appears that
there may be differences in glutamate transduction in different species
and even among papillae of the same species.
Our study extended the findings of previous investigators to show that
NMDA receptors in taste cells share many properties with brain NMDA
receptors, such as co-activation with glycine and suppression by AP5
(for review, Collingridge and Watkins 1994). However,
the blocking effect of extracellular Mg2+ on the
NMDA-sensitive current is somewhat less effective in taste cells than
in brain. We observed blockage by Mg2+ in some
cells, whereas in most of the cells tested, the amplitude of the peak
current was not reduced in the presence of Mg2+,
although the desensitization of the channel was much faster. The lack
of Mg2+ block may allow these channels to
participate in the transduction of glutamate in vivo.
Responses to L-AP4
Most of the responses to L-AP4 involved a decrease in
holding current and membrane conductance, concomitant with membrane hyperpolarization in some cells. These results are consistent with
previous findings in taste cells of vallate and foliate papillae (Bigiani et al. 1997; Hayashi et al.
1996
). We showed further that the response is suppressed by
cAMP and by the metabotropic antagonist CPPG, supporting the idea that
mGluR4 is expressed in rat taste cells (Chaudhari et al.
1996
). Suppression by cAMP is to be expected, because
activation of mGluR4 decreases intracellular cAMP levels in brain
(Pin and Duvoisin 1995
). In addition, MSG decreases cAMP
levels in tissue from rat vallate and foliate papillae (Zhou and
Chaudhari 1997
). One difference between our results on
fungiform taste cells and those of Bigiani et al. (1997)
on foliate and vallate taste cells is the conductance that is modulated by L-AP4. In vallate and foliate taste cells, a cation
conductance is closed by L-AP4 stimulation, whereas in
fungiform taste cells, a Cl
conductance is
closed. Although 8-bromo-cAMP suppresses the effect of
L-AP4 stimulation, we do not believe that cAMP modulates
the Cl
conductance directly because changes in
membrane conductance were usually not observed in these cells in
response to 8-bromo-cAMP application.
In contrast to the above-described effects of L-AP4, we
observed that some cells responded to L-AP4 with an
increase in holding current and membrane depolarization. This response
was not reported in taste cells of rat vallate papillae (Bigiani
et al. 1997), although in taste cells of mice vallate papillae,
L-AP4 could either decrease or increase the intracellular
Ca2+ level (Hayashi et al. 1996
).
Whether mGluR4 also mediates this response is not yet determined.
Interestingly, taste cells express two different forms of mGluR4; a
long form that is similar to mGluR4 in the brain, and a short form in
which a significant portion of the extracellular N-terminus has been
truncated. This short form may be specific to taste cells
(Fedorov and Chaudhari 1998
). Both forms of mGluR4 have
been expressed in heterologous cells, and activation of both receptors
causes decreases in intracellular cAMP. Activation of taste-mGluR4
requires higher concentrations of glutamate for activation
(Landin and Chaudhari 1999
), similar to those required
to elicit umami taste. Thus this receptor may be more important for
glutamate taste transduction. The inward currents induced by
L-AP4 in our study required higher concentrations of
L-AP4 than the outward current. It is tempting to speculate that the inward current generated by L-AP4 results from
activation of taste-mGluR4.
The present study suggests that both NMDA receptors and metabotropic
glutamate receptors are present in taste cells and may be co-activated
in response to glutamate taste stimulation. An important caveat in this
interpretation is that glutamate was applied to the entire taste cell
membrane, rather than selectively to the apical membrane as occurs in
vivo. It is possible that some responses to glutamate observed in the
present study were mediated by receptors located on the basolateral
membrane rather than the apical membrane. The neurotransmitter in taste
cells has not been identified, but in most other sensory receptors the transmitter is glutamate. Group III mGluRs are expressed on the presynaptic membranes of glutamatergic neurons in many regions of the
brain, where they function to inhibit glutamate release (Pin and
Duvoisin 1995). The inhibitory responses to L-AP4
could be mediated by such an inhibitory autoreceptor located on the basolateral membrane, possibly the brain form of mGluR4. Further studies will be required to determine the location of NMDA receptors and both forms of mGluR4 in taste cell membranes. However, a recently published paper showed that both L-AP4 and NMDA induced
measurable responses in chorda tympani nerve recordings when these
compounds were applied to the surface of a rat tongue (Sako and
Yamamoto 1999
). These data imply that receptors for both NMDA
and L-AP4 are located at the apical membrane of taste
cells, but further studies will be required to determine which type of
L-AP4 response (hyperpolarizing or depolarizing) is
mediated by receptors on the apical membrane.
One of the important features of umami taste is potentiation by
5'-ribonucleotides, such as guanosine 5'-monophosphate (5'-GMP) and
inosine 5'-monophosphate (5'-IMP). This potentiation has been observed
in afferent nerve recordings when MSG is applied together with 5'-GMP
or 5'-IMP (Hellekant and Ninomiya 1991) and also when L-AP4 is applied together with the nucleotides (Sako
and Yamamoto 1999
). In preliminary studies, we have observed
synergy between MSG and 5'-GMP in a subset of glutamate-responsive
taste cells. Both subset I and II responses could be potentiated
(Lin and Kinnamon 1996
). Further studies will be needed
to examine the mechanisms involved in the synergy.
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ACKNOWLEDGMENTS |
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We thank Drs. Nirupa Chaudhari and Stephen Roper for sharing unpublished data and for helpful discussions throughout the course of this study.
This study was supported by National Institute on Deafness and Other Communication Disorders Grant DC-03013.
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FOOTNOTES |
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Address for reprint requests: W. Lin, Dept. of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 May 1999; accepted in final form 29 June 1999.
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REFERENCES |
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