Bitter Taste Transduction of Denatonium in the Mudpuppy
Tatsuya Ogura,1Alan Mackay-Sim,1,2and Sue C. Kinnamon1
1Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523, and Rocky
Mountain Taste and Smell Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, and2School
of Biomolecular and Biomedical Science, Griffith University, Nathan, QLD 4111 Australia
Bitter substances are a structurally diverse group of com-
pounds that appear to act via several transduction mecha-
nisms. The bitter-tasting denatonium ion has been proposed to
act via two different G-protein-regulated pathways, one involv-
ing inositol 1,4,5-trisphosphate and raised intracellular calcium
levels, the other involving phosphodiesterase and membrane
depolarization via a cyclic nucleotide-suppressible cation chan-
nel. The aim of the present study was to examine these trans-
duction mechanisms in taste cells of the mudpuppy Necturus
maculosus by calcium-imaging and whole-cell recording. De-
natonium benzoate increased intracellular calcium levels and
induced an outward current independently of extracellular cal-
cium. The denatonium-induced increase in intracellular calcium
was inhibited by U73122, an inhibitor of phospholipase C, and
by thapsigargin, an inhibitor of calcium transport into intracel-
lular stores. The denatonium-induced outward current was
blocked by GDP-?-S, a blocker of G-protein activation. Neither
resting nor denatonium-induced intracellular calcium levels
were affected by inhibition of phosphodiesterase (with IBMX) or
adenylate cyclase (with SQ22536) or by raising intracellular
cyclic nucleotides directly (with cell permeant analogs). Our
results support the hypothesis that denatonium is transduced
via a G-protein cascade involving phospholipase C, inositol
1,4,5-trisphosphate, and raised intracellular calcium levels. Our
results do not support the hypothesis that denatonium is trans-
duced via phosphodiesterase and cAMP.
Key words: bitter taste transduction; mudpuppy; taste recep-
tor cells; fura-2; calcium imaging; whole-cell recording
There are a large number of compounds that taste bitter to
humans (Belitz and Weiser, 1985). Given the structural diversity
of these compounds, it is expected that there may be multiple
transduction mechanisms or multiple receptor proteins in taste
receptor cells. Unexpected perhaps is evidence that the bitter-
tasting denatonium ion may operate via two separate transduction
pathways. One hypothesis proposes that denatonium activates
phospholipase C (PLC), thus increasing intracellular levels of
inositol 1,4,5-trisphosphate (IP3) to release calcium ions from
intracellular stores (Akabas et al., 1988; Hwang et al., 1990;
Spielman et al., 1994b). The increase in intracellular calcium is
proposed to lead to neurotransmitter release, thereby completing
the transduction step (Akabas et al., 1988; Spielman et al., 1994b).
Another hypothesis is that denatonium activates phosphodiester-
ase to decrease intracellular levels of cyclic nucleotides (Ruiz-
Avila et al., 1995; Wong et al., 1996), which would depolarize the
cell by relieving the cyclic nucleotide block of a membrane cation
channel (Kolesnikov and Margolskee, 1995). The influx of cations
(including calcium) is proposed to depolarize the cell leading to
These two hypotheses predict several conflicting properties of
the responses induced by denatonium. First, although both hy-
potheses predict a denatonium-induced rise in intracellular cal-
cium, the former predicts that this is attributable to calcium
release from intracellular stores, whereas the latter predicts that it
is attributable to calcium influx from the extracellular medium.
Second, although the former hypothesis makes no prediction
about the effect of denatonium on the membrane potential, the
latter hypothesis predicts that denatonium should depolarize taste
cells. Finally, each hypothesis predicts testable pathway-specific
effects of pharmacological agents.
In the present report, bitter transduction was studied to test the
predictions arising from these two hypotheses. Denatonium-
induced responses were examined in taste cells of mudpuppy
Necturus maculosus. Mudpuppies can detect denatonium, and it is
aversive to them (Ogura et al., 1996). Mudpuppy taste receptor
cells are large, easily isolated, and amenable to study (Kinnamon
et al., 1988a), and preliminary experiments indicated that the
majority were responsive to denatonium. Denatonium-induced
responses of these cells were examined using whole-cell patch-
clamp recording and calcium imaging using the Ca2?-sensitive dye
MATERIALS AND METHODS
Isolation of taste receptor cells. Mudpuppies (Necturus maculosus) were
obtained from commercial suppliers and kept in large aquaria maintained
at 10°C. They were fed live minnows weekly. Taste receptor cells were
isolated from mudpuppies, as described previously (Kinnamon et al.,
1988a). Briefly, mudpuppies were anesthetized by immersion in ice water
and decapitated. The lingual epithelium was dissected from the underly-
ing connective tissue. To distinguish mature taste cells from other cell
types, they were labeled at the apical membrane by incubating the
epithelium for 15 min in fluorescein isothiocyanate-conjugated wheat
germ agglutinin [FITC-WGA, 0.5 mg/ml in amphibian physiological sa-
line (APS)] (Kinnamon et al., 1988b). The epithelium was then incubated
in APS that contained collagenase (1 mg/ml, Worthington Biochemical,
Freehold, NJ), albumin (1 mg/ml), and glucose (5 mM), until the mucosal
layers of the nongustatory epithelium could be gently peeled free from
Received Jan. 22, 1997; revised March 3, 1997; accepted March 5, 1997.
This work was supported in part by National Institutes of Health Grants DC00244
and DC00766 to S.C.K. We thank Dr. Peter Guthrie for his help in establishing our
calcium-imaging system and for computer programming, as well as Andrew Bower-
man, Vanessa Madsen, and Megan Litster for excellent technical assistance.
Correspondence should be addressed to Dr. Tatsuya Ogura, Department of
Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523.
Copyright © 1997 Society for Neuroscience 0270-6474/97/173580-08$05.00/0
The Journal of Neuroscience, May 15, 1997, 17(10):3580–3587
the underlying lamina propria, which left the taste buds attached to their
papillae. The remaining tissue was treated with Ca2?-free APS to disso-
ciate the taste buds. Isolated cells were plated in recording chambers
made with coverslips (for calcium imaging) or slides (for whole-cell
recording) coated with Cell-Tak (Collaborative Research, Bedford, MA).
Intracellular [Ca2?] measurement. Isolated cells in the chamber were
loaded with the cell-permeable, Ca2?-sensitive, fluorescent dye fura-2
AM (5 ?M, Molecular Probes, Eugene, OR) for 30 min, then washed with
normal APS for 20 min. Images were acquired with an intensified CCD
camera (IC100-ICCD, Paultek Imaging, Grass Valley, CA) through an
oil-immersion objective lens (Fluor 40?, 1.3 NA, Nikon) of an inverted
microscope (Diaphot TMD, Nikon). The video signal from the camera
was captured with a frame grabber board (Quick Capture, Data Trans-
lation) on an Macintosh computer (Quadra 800, Apple Computer). For
dual-wavelength ratiometric Ca2?measurements, pairs of fluorescent
images were recorded at 350 and 380 nm of excitation light using a filter
wheel (EMPIX Imaging). Emitted light was collected by a 510–580 nm
bandpass filter (Chroma Technology). In our system, space resolution is
?3000 pixels/cell. Pseudocolor Ca2?images (e.g., Fig. 1C) were gener-
ated as 20 level color images from ratios of 350 and 380 nm images using
the public domain National Institutes of Health (NIH) Image program
(developed at NIH and available on the Internet at http://rsb.info.nih.gov/
nih-image/). Intracellular Ca2?concentration ([Ca2?]i) in selected areas
was calculated from the ratio of 350 and 380 nm images (Grynkiewicz et
al., 1985). Ca2?calibration curves were obtained with calcium calibration
kit II (C-3009, Molecular Probes). For plotting the time course of [Ca2?]i
(e.g., Fig. 2), we averaged the [Ca2?]iover most of the cell area, avoiding
the edges of the cell. With a recording chamber volume of ?200 ?l, ?10
sec was required to totally exchange solutions by superfusion.
Cells were bathed in normal APS until the resting intracellular calcium
level was stable. The bath was then perfused with APS containing dena-
tonium benzoate (5 mM, Sigma, St. Louis, MO). This was followed by
washing with normal APS until the intracellular calcium again reached
prestimulus levels. Cells were then perfused with other APS solutions
including Ca2?-free APS, ryanodine (10 ?M for 2–5 min or 200 ?M for 10
min, Calbiochem, La Jolla, CA), thapsigargin (1 ?M for 12–16 min,
Sigma), U73122 (5 ?M for 12–17 min, Calbiochem), isobutyl methoxyx-
anthine (IBMX, 100 or 200 ?M for 1–3 min, Sigma), SQ22536 (2.5 mM for
30–40 min, Calbiochem); dibutyryl-cAMP (db-cAMP, 5 mM for 1–3 min,
Sigma); dibutyryl-cGMP (db-cGMP, 5 mM for 1–3 min, Sigma), and a
mixture of db-cGMP (2 mM for 1–3 min, Sigma) and 8-chlorophenylthio-
cAMP (8-cpt-cAMP, 2 mM for 1–3 min, Sigma). Some cells were incu-
bated in pertussis toxin (PTX, 0.5 or 2 ?g/ml in APS, Sigma) for ?20 hr
Cells were considered to respond to denatonium if it caused an in-
crease in [Ca2?]i, which was ?2 SD values above the mean resting level
of each cell. The effects of drug treatments on the denatonium response
were assessed using Student’s t tests. Statistical values are presented as
mean [Ca2?]i? SEM.
Giga-seal whole-cell recording. Membrane currents were measured us-
lular calcium levels. A, Light image of an
isolated taste cell. Scale bar, 20 ?m. B,
Fluorescence image of the same cell show-
ing the apical tip of the taste cell labeled
with FITC-WGA. The labeled region is
shown in white in this pseudocolor image.
C, Calcium images of the same taste cell
loaded with the Ca2?-sensitive dye fura-2.
The pseudocolor scale of [Ca2?]iis shown
on the right. a, [Ca2?]ibefore and b, im-
mediately after application of 5 mM dena-
tonium benzoate. The increase in [Ca2?]i
begins at the apical tip. c, [Ca2?]i30 sec
after application of denatonium benzoate.
[Ca2?]i increases over entire cell. d,
[Ca2?]iafter washing the cells for 2 min
with APS. [Ca2?]ireturns to resting level.
D, Typical time course of denatonium-
induced calcium responses. Denatonium
benzoate (5 mM) was applied during the
denatonium-induced responses are similar
after repeated application of denatonium.
Denatonium increased intracel-
Ogura et al. • Bitter Taste TransductionJ. Neurosci., May 15, 1997, 17(10):3580–3587 3581
ing giga-seal whole-cell recording (Hamill et al., 1981). Electrodes were
fabricated from microhematocrit capillary tubes (American Scientific
Products, McGaw Park, IL) pulled on a two-stage vertical puller (Nar-
ishige). When filled with intracellular saline, resistances ranged from 1.8
to 3.5 M?. Cells were viewed at a magnification of 400?, using a Nikon
Diaphot inverted microscope fitted with Hoffman optics. Seals of 1–10
G? were obtained by gentle suction, and whole-cell recordings were
achieved by delivery of a short depolarizing pulse to the pipette. Whole-
cell currents were measured at room temperature using an Axopatch 1D
patch-clamp amplifier (Axon Instruments, Foster City, CA). Signals were
prefiltered at 5 kHz (low-pass filter) and recorded digitally at 100 ?sec
intervals unless otherwise specified. Data were stored using a laboratory
computer (11/23, Digital Equipment, Maynard, MA) equipped with a
Cheshire data interface and Basic 23 software (Indec Systems). The
computer also generated all voltage commands. In some experiments,
data were also stored on a Pentium computer (Applied Computer Tech-
nology), using Axoscope software (Axon Instruments). Unless otherwise
noted, leak and linear capacitative currents were subtracted from all
records by computer. Series resistance, which was typically ?10 M?, was
not compensated. Gravity-fed stimuli were bath-applied to the 0.5 ml
recording chamber. To prevent loss of the seal and to prevent perfusion
artifacts during whole-cell recording, the perfusion rate was lowered to
2–3 ml/min. To ensure that data were obtained from viable taste receptor
cells, only cells exhibiting voltage-gated Na?currents and input resis-
tances ?0.5 G? were selected for data analysis.
After whole-cell recording was obtained and membrane capacitance
was electronically compensated, membrane currents were measured in
normal APS by stepping the cell membrane from ?60 to 80 mV in 10 mV
increments. The bath was then perfused with 1 mM denatonium benzoate,
and membrane currents were again measured. Finally, the denatonium
was washed out of the bath with normal APS and the currents again
measured. In some cells, the tip of the pipette was filled with the normal
pipette solution but backfilled with a solution containing 1 mM GDP-?-S
(Sigma). In some experiments, 1 ?M tetrodotoxin (TTX, Sigma) was
added to the bath to block Na?currents. There was no effect of TTX on
the response to denatonium.
For comparison, control and denatonium-induced currents were mea-
sured at 35 msec after the membrane potential was stepped to ?80 mV.
Statistical values are presented as mean currents ? SEM.
Solutions. Normal APS contained (in mM): 112 NaCl, 2 KCl, 8 CaCl2,
3 HEPES, buffered to pH 7.2 with NaOH. Ca2?-free APS contained
either 1 mM BAPTA (for cell isolation) or 1 mM EGTA (for calcium
imaging). Patch pipette solution contained (in mM): 114 KCl, 2 NaCl, 0.09
CaCl2, 2 MgCl2, 1 BAPTA, 1 ATP, 0.4 GTP, 10 HEPES, buffered to pH
7.2 with KOH.
Denatonium increased intracellular calcium levels
We measured [Ca2?]iin isolated mudpuppy taste cells using
calcium imaging with the Ca2?-sensitive fluorescent dye fura-2.
Mature taste cells were identified by their elongate shape and
apical fluorescence of the FITC-WGA applied to the epithelium
before dissociation (Fig. 1A,B). Under control conditions, iso-
lated taste cells had intracellular calcium levels of 71 ? 1 nM (n ?
203). Denatonium benzoate increased [Ca2?]iin ?80% of the
taste cells tested. The increase in [Ca2?]ioccurred initially at the
apical tip of the taste cell, then spread basolaterally until [Ca2?]i
was increased over the entire cell (Fig. 1C). The initial response
occurred in the apical end, even though the denatonium was
bath-applied to the entire cell. The time course of the response is
the periods labeled DN. A, The denatonium-induced calcium response was present in Ca2?-free extracellular solution. B, Thapsigargin (1 ?M) abolished
the denatonium-induced calcium response. C, The denatonium-induced calcium response was not affected by ryanodine (10 ?M). D, The denatonium-
induced calcium response was abolished by the PLC inhibitor U73122 (5 ?M).
Time courses of changes in [Ca2?]i. Measurements of [Ca2?]iin individual cells using fura-2. Denatonium benzoate (5 mM) was applied during
3582 J. Neurosci., May 15, 1997, 17(10):3580–3587Ogura et al. • Bitter Taste Transduction
shown in Figure 1D. The peak response occurred near the first
measurement, which was ?5 sec after application of denatonium
to the recording chamber. Intracellular Ca2?levels began to
decline in the presence of denatonium and then decreased further
to baseline levels after the denatonium was washed from the
chamber (Figs. 1D, 2). The peak [Ca2?]ielicited by 5 mM dena-
tonium benzoate was usually 50–150% of the resting [Ca2?]i
(133 ? 4 nM, n ? 203). Repeated applications of denatonium to
the same cell resulted in similar increases in [Ca2?]i, as long as the
cell was washed for at least 3 min after each application of
denatonium (first application, 130 ? 12 nM; second application,
133 ? 14 nM, n ? 16) (Figs. 1D, 3). Sodium benzoate (5 mM)
failed to increase [Ca2?]iin five taste cells that responded to
denatonium benzoate (5 mM), suggesting that benzoate itself has
no effect on the taste cells. We also measured [Ca2?]iin nontaste
lingual epithelial cells; denatonium failed to increase [Ca2?]iin
any of these cells.
Calcium was released from intracellular stores
To determine whether extracellular Ca2?was required, we mea-
sured [Ca2?]iin response to denatonium in Ca2?-free APS. In this
medium, the resting [Ca2?]iwas reduced (initial level, 73 ? 6 nM;
Ca2?-free APS, 65 ? 2 nM; n ? 17), but not significantly (paired,
one-tailed Student’s t test ? 1.52, df ? 16, p ? 0.07). The
denatonium response persisted in all taste cells tested, although
the magnitude of the response was usually smaller than in Ca2?-
containing APS (Figs. 2A, 3). This reduction in the denatonium
response was statistically significant (paired Student’s t test ?
2.37, df ? 16, p ? 0.05). These data illustrate that the response to
denatonium persisted in Ca2?-free APS and suggest that at least
the major part was mediated by calcium release from intracellular
stores. To investigate the role of calcium stores in the response,
we used thapsigargin, a Ca2?-ATPase inhibitor. Thapsigargin
prevents calcium uptake into the intracellular stores (Chu et al.,
1988; Meyer and Stryer, 1990; Thastrup et al., 1990). This results
in a gradual depletion of calcium from the intracellular stores as
the calcium leaks out and is not replenished. We tested the effect
of thapsigargin on 34 cells that responded to denatonium. Thap-
sigargin (1 ?M) increased [Ca2?]ito a variable extent in all of
these cells. This increase in [Ca2?]iwas slow, and the peak [Ca2?]i
was smaller than the denatonium responses in most cells tested
(initial level, 79 ? 4 nM; after thapsigargin, 108 ? 7 nM; n ? 34).
After a 16 min incubation with thapsigargin, which should be
sufficient for store depletion, 5 mM denatonium failed to increase
[Ca2?]iin 27 of 29 cells tested (Figs. 2B, 3). This effect of
thapsigargin on the response to denatonium was statistically sig-
nificant (paired Student’s t test ? 5.40, df ? 26, p ? 0.0001).
These data strongly suggest that denatonium releases calcium
from internal stores and not from extracellular influx, because
thapsigargin inhibited completely the denatonium response. Thus,
the decrease in the response to denatonium in Ca2?-free APS
may be attributable to a requirement of extracellular Ca2?for
reloading of the intracellular stores, as has been shown in neurons
(Thayer et al., 1988).
Calcium was released from IP3-sensitive stores
Two different types of calcium stores have been identified: one
coupled to an inositol IP3receptor and another coupled to a
ryanodine-sensitive calcium receptor (Sharp et al., 1993; Simpson
et al., 1995). To investigate which intracellular calcium receptors
are involved in the denatonium response, we applied denatonium
in the presence of 10 ?M ryanodine. Ryanodine did not change
resting [Ca2?]i(initial level, 77 ? 6 nM; after ryanodine, 79 ? 6
nM; n ? 12) and did not affect the [Ca2?]iincrease in response to
denatonium (Figs. 2C, 3) (125 ? 22 nM; n ? 12). High concen-
trations of ryanodine have been shown to inhibit Ca2?release
from ryanodine receptor-coupled calcium stores (Sutko et al.,
1985). To determine whether higher concentrations of ryanodine
would inhibit the denatonium response, we incubated taste cells in
200 ?M ryanodine for 11 min, but there was no significant effect on
the denatonium response. Thus, ryanodine receptor-coupled cal-
cium stores are not likely to be involved in the response to
denatonium. To determine whether calcium stores in taste cells
are IP3receptor-coupled, we used the PLC inhibitor U73122,
because IP3is produced via the PLC pathway (Thompson et al.,
1991; Salari et al., 1993). U73122 (5 ?M) increased [Ca2?]iin all
cells (initial level, 78 ? 5 nM; U73122, 97 ? 6 nM; n ? 18). The
increase of [Ca2?]iby U73122 itself might be mediated by calcium
release from IP3receptor-coupled calcium stores, as shown in rat
liver microsomes (De Moel et al., 1995). After a 17 min incubation
with U73122, calcium responses induced by denatonium were
inhibited (Figs. 2D, 3). This effect of U73122 on the response to
depended on intracellular stores. This graph shows
maximum denatonium-induced changes in [Ca2?]i
expressed as a percentage of resting [Ca2?]i. Cells
were tested twice, before (hatched bars) and during
or after (open bars) the treatments indicated, as
illustrated in Figure 2 [Ca2?-free bath solution (0 Ca;
n ? 17), thapsigargin (n ? 34), ryanodine (n ? 12),
and U73122 (n ? 18)]. Control cells were tested
twice, as illustrated in Figure 1D (n ? 16), the first
and second stimulations indicated by the hatched and
open bars, respectively.
Denatonium-induced changes in [Ca2?]i
Ogura et al. • Bitter Taste Transduction J. Neurosci., May 15, 1997, 17(10):3580–3587 3583
denatonium was statistically significant (paired Student’s t test ?
7.37, df ? 17, p ? 0.0001). These results strongly suggest that IP3
is involved in the response of mudpuppy taste cells to denatonium.
Denatonium hyperpolarizes mudpuppy taste cells
We also used giga-seal whole-cell recording to study denatonium
transduction in isolated taste cells. Isolated mudpuppy taste cells
have resting potentials of approximately ?65 mV and input re-
sistances of ?1.4 G? (Kinnamon and Roper, 1988). Under
voltage-clamp conditions, we measured voltage-activated current
in response to 1 mM denatonium. Denatonium increased outward
currents in 20 of 25 cells elicited by step depolarizations from a
holding potential of ?80 mV (Fig. 4A). The increase in outward
current was observed at all potentials positive to ?40 mV (Fig.
4B). The outward current induced by denatonium was close to
50% greater than the current recorded in APS (APS before, 2.7 ?
0.3 nA; 1 mM denatonium benzoate, 4.0 ? 0.5 nA; APS after,
3.4 ? 0.7 nA; n ? 11). The time course of the effect is shown in
Figure 5. The time course of the current response was similar to
the time course of the Ca2?response (e.g., Figs. 1, 2), suggesting
that the current response was produced by an increase in [Ca2?]i
in response to denatonium. Indeed, Ca2?-dependent K?(Cum-
mings and Kinnamon, 1992) and Cl?(Taylor and Roper, 1994)
conductances have been identified in mudpuppy taste cells, and
activation of these conductances would elicit an increase in out-
ward current under our recording conditions. The current in-
creased, then began decreasing, in the continued presence of
denatonium. To confirm that the denatonium-induced increase in
outward current would hyperpolarize the cells, we used current-
clamp recording. As expected, the increase in outward current was
accompanied by a 3–10 mV hyperpolarization of the resting
potential (n ? 2 cells).
In some experiments, denatonium was applied focally to taste
cells with a picospritzer during whole-cell recording. Under these
conditions, denatonium activated an outward current that was
similar to the current that was activated by bath application of
denatonium (data not shown).
G-protein involvement in the response to denatonium
To investigate the involvement of G-proteins in the denatonium
response, we used patch pipettes containing a nonhydrolyzable
GDP analog, GDP-?-S (1 mM) (Burch and Axelrod 1987; Gilman,
1987). Six minutes after the establishment of the whole-cell con-
figuration, denatonium still increased the outward current elicited
by a voltage step to ?80 mV (Fig. 6A). By 15 min, however,
denatonium failed to increase outward currents (Fig. 6B). Three
other cells showed similar results. Control cells held for similar
periods did not show an appreciable decline in the response to
denatonium, making it unlikely that the decline with GDP-?-S
was attributable either to rundown or to desensitization. These
results suggest that G-proteins are required for transduction of
To determine which type of G-protein is involved in the path-
way, we incubated lingual epithelia in PTX, which inhibits Gi, Go,
gustducin, and transducin by ADP ribosylation of the G-protein ?
subunit (Birnbaumer, 1990; Simon et al., 1991). Dissected lingual
epithelia were treated with PTX (0.5 and 2 ?g/ml in APS) for ?20
hr at 4°C. Incubation with PTX affected neither the resting levels
of intracellular calcium (control group, 74 ? 2 nM, n ? 13;
PTX-treated group, 73 ? 3 nM, n ? 24) nor the response to
denatonium (Fig. 7).
cAMP involvement in the response to denatonium
The involvement of cAMP in the denatonium response was ex-
amined by increasing intracellular cyclic nucleotides (using
IBMX, a phosphodiesterase inhibitor, and cell permeant cAMP
and cGMP analogs) and by decreasing intracellular cyclic nucle-
denatonium. The bath contained TTX to block inward Na?currents. A,
Whole-cell recording under control conditions and in response to 1 mM
denatonium benzoate (DN). The cell was held at ?80 mV, and the
membrane was stepped to ?20 mV to elicit outward current. Leak and
linear capacitative currents were subtracted from the record by computer.
B, Current–voltage relationship of the denatonium response in the same
cell, as shown in A. Notice that denatonium increased outward currents at
most voltages (solid circles).
Electrophysiological response of a mudpuppy taste cell to
nium. The cell was held at ?80 mV, and the membrane was stepped to
?20 mV for 175 msec every 3 sec. Data were digitized at 125 Hz and
plotted with Axoscope software. TTX was present in the bath solution to
block Na?currents. Note that 1 mM denatonium elicited an increase in
outward current, which is similar in kinetics to the [Ca2?]iresponse shown
in Figures 1 and 2.
Time course of the electrophysiological response to denato-
3584 J. Neurosci., May 15, 1997, 17(10):3580–3587Ogura et al. • Bitter Taste Transduction
otides (using SQ22536, an inhibitor of adenylate cyclase) (Harris
et al., 1979; Goldsmith and Abrams, 1991). Incubation with IBMX
did not affect resting [Ca2?]i(initial level, 68 ? 3 nM; after IBMX,
64 ? 3 nM; n ? 17). Although IBMX reduced the response to
denatonium in some cells, it had no effect on the mean response
(Fig. 7). Incubation with cyclic nucleotides did not affect resting
[Ca2?]i[db-cAMP (5 mM) before, 66 ? 5 nM and after, 64 ? 4 nM;
n ? 7; db-cGMP (5 mM) before, 62 ? 7 nM and after, 60 ? 7 nM,
n ? 7; 8-cpt-cAMP (2 mM) ? db-cGMP (2 mM) before, 67 ? 2 nM
and after, 65 ? 7 nM, n ? 3]. None of the cyclic nucleotides
affected the denatonium-induced calcium response. (Responses
are grouped together and shown in Fig 7.) Incubation of the cells
with SQ22536 (2.5 mM for 30 or 40 min) did not affect resting
[Ca2?]i(initial level, 54 ? 4 nM; after SQ22536, 55 ? 2 nM;
n ? 3) and did not affect the denatonium-induced calcium
response (Fig. 7).
In this study, we used calcium-imaging and whole-cell patch
recording to examine the transduction of denatonium in isolated
mudpuppy taste cells. The results support the hypothesis that
denatonium increases [Ca2?]ivia a G-protein cascade involving
PLC and IP3(Hwang et al., 1990; Spielman et al., 1994b). Our
evidence does not support the hypothesis that denatonium is
transduced via a pathway involving phosphodiesterase and mem-
brane depolarization via a cyclic nucleotide-suppressible cation
channel (Kolesnikov and Margolskee, 1995; Wong et al., 1996).
The IP3-mediated hypothesis is supported by a number of
observations. First, denatonium benzoate increased intracellular
calcium via calcium release from intracellular stores and not via
calcium influx, as indicated by the effects of thapsigargin and
U73122. Second, denatonium induced an outward current across
the cell membrane and not an inward current, as predicted by the
phosphodiesterase hypothesis. The denatonium-induced response
was not dependent on extracellular calcium; when the concentra-
tion of extracellular Ca2?was reduced to zero, the denatonium
still elicited a calcium response. Third, the denatonium-induced
response was mediated via a G-protein; GDP-?-S blocked the
denatonium-induced outward current. Finally, neither increases
nor decreases in intracellular cAMP levels affected either resting
[Ca2?]ior the denatonium-induced response. In combination,
these data support strongly the hypothesis that denatonium is
transduced via an IP3-mediated rather than a cAMP-mediated
pathway in mudpuppy.
There is evidence in rat and mouse that denatonium acts via the
PLC–IP3pathway. In rat, IP3receptors and Ca2?-ATPase are
found in taste buds of the circumvallate papilla, at their highest
densities near the taste pore (Hwang et al., 1990). Denatonium
enhances intracellular levels of IP3in these cells, (Hwang et al.,
1990), and IP3depletes accumulated Ca2?from intracellular
stores in a dose-dependent manner (Hwang et al., 1990). Dena-
tonium increases [Ca2?]iin some rat taste cells (Akabas et al.,
1988; Bernhardt et al., 1996). In mouse circumvallate and foliate
papillae, denatonium activates PLC (Spielman et al., 1994b).
The concentrations of denatonium required to elicit calcium
responses in mudpuppy taste cells were higher than those re-
quired to elicit calcium responses from rat taste cells (Akabas et
TTX was present in the bath solution to block Na?currents. A, After 6 min of whole-cell recording and B, after 15 min of whole-cell recording. Note
that the GDP-?-S (1 mM in pipette solution) abolished the denatonium response, suggesting that the response was G-protein-dependent.
Effect of GDP-?-S on the denatonium-induced outward current. This cell was held at ?80 mV, and the membrane was stepped to ?80 mV.
not depend on intracellular cAMP. This graph shows
maximum denatonium-induced changes in [Ca2?]iex-
pressed as a percentage of resting [Ca2?]i. Control cells
are those illustrated in Figure 3. PTX cells represent two
groups of cells: a control group incubated overnight in
APS (hatched bar, n ? 13) and a treatment group incu-
bated overnight in PTX (open bar, n ? 24). Other cells
were tested twice before (hatched bars) and during or
after (open bars) the treatments indicated: IBMX (n ?
17); cell permeant cyclic nucleotides (cNMP: db-cAMP,
n ? 4; db-cGMP, n ? 6; 8-cpt-cAMP ? db-cGMP, n ?
3), and SQ22536 (n ? 3).
Denatonium-induced changes in [Ca2?]idid
Ogura et al. • Bitter Taste TransductionJ. Neurosci., May 15, 1997, 17(10):3580–3587 3585
al., 1988) or IP3responses from mouse taste tissue (Spielman et
al., 1994b). However, the concentrations were consistent with
those reported to activate a denatonium receptor in bovine taste
membranes (Ruiz-Avila et al., 1995). In addition, in behavioral
studies, mudpuppies reject food pellets containing 1–10 mM de-
natonium after tasting them, suggesting that it is aversive to them
(Ogura et al., 1996).
In mouse taste cells, another transduction pathway has been
suggested for denatonium; activation of phosphodiesterase via the
G-proteins transducin and gustducin (Ruiz-Avila et al., 1995). By
analogy with phototransduction, phosphodiesterase reduces intra-
cellular cAMP. This relieves a cAMP block of a cyclic nucleotide-
suppressible cation channel and causes the cell to depolarize
(Kolesnikov and Margolskee, 1995). This hypothesis is supported
by the observation that transgenic mice, which lack gustducin, are
less sensitive to denatonium (Wong et al., 1996). This model of
bitter transduction was not supported by our data in mudpuppy.
First, denatonium hyperpolarized taste cells; it did not depolarize
them. Second, the denatonium-induced increase in [Ca2?]iarose
from intracellular release and not from influx through a cation
channel. Finally, modulation of intracellular cyclic nucleotide
levels failed to affect intracellular calcium levels, ruling out a
direct effect of cyclic nucleotides on [Ca2?]i. Our evidence that
intracellular cyclic nucleotide
denatonium-induced response also rules out cyclic nucleotide
modulation of the IP3pathway in mudpuppy taste cells (Linde-
The identity of the G-protein involved in denatonium transduc-
tion in the mudpuppy is moot. In mouse, sucrose octa-acetate,
another potent bitter compound, acts via a PTX-sensitive
G-protein such as Gior Go(Spielman et al., 1994b), but in
mudpuppy, PTX failed to abolish the denatonium-induced re-
sponse. We do not believe that the lack of effect of PTX resulted
from an insufficient incubation period, because a similar incuba-
tion period was sufficient to demonstrate inhibition by PTX of a
G-protein modulated calcium current in mudpuppy taste cells
(Delay et al., 1997). These data suggest that a PTX-insensitive
G-protein, such as a member of the Gqfamily (Simon et al., 1991;
Taylor et al., 1991), may activate PLC in mudpuppy taste cells.
?-Subunits of the Gqfamily have been identified in mouse and rat
taste tissue (Spielman et al., 1994a; Tabata et al., 1996). It has
been suggested that the ??-subunits of gustducin may activate
PLC (Wong et al., 1996); however, because mouse gustducin has
PTX-binding sites, it probably plays no role here. Gustducin has
not been examined in mudpuppy. Additional experiments are
necessary to determine which G-proteins could be responsible for
the denatonium response in mudpuppy taste cells.
Although other bitter stimuli such as caffeine, strychnine, and
sucrose octa-acetate activate PLC in mouse taste cells (Spielman
et al., 1994b), not all bitter compounds are transduced via the IP3
pathway. Some bitter compounds, such as quinine and CaCl2,
directly block the voltage-dependent K?channels that are located
on the apical end of mudpuppy taste cells (Kinnamon et al.,
1988b; Bigiani and Roper, 1991; Cummings and Kinnamon,
1992). These K?channels are open at rest, and blocking them
causes depolarization. In mouse taste cells, denatonium also
blocks voltage-dependent K?channels (Spielman et al., 1989),
although the apical location of these channels has not been
established for mouse. In mudpuppy, it is probable that taste cells
may have both transduction pathways for bitter stimuli because
?80% of cells responded to quinine and CaCl2(Bigiani and
Roper, 1991) and a similar percentage responded to denatonium.
levels failed toaffect the
It is not clear whether the mudpuppy can discriminate among
these bitter taste stimuli. Behavioral studies show that most bitter
stimuli are rejected by the mudpuppy (Bowerman and Kinnamon,
1994; Ogura et al., 1996). Because mudpuppies do not respond
well to appetitive taste stimuli such as sugars, it is not surprising
that most taste cells respond to bitter stimuli.
The IP3pathway is not unique to bitter taste transduction, because
it is activated in some mammalian taste cells by synthetic sweeteners
(Bernhardt et al., 1996). Other sweet stimuli are transduced via an
adenylate cyclase–cAMP cascade (Striem et al., 1991; Cummings et
al., 1993, 1996; Kinnamon and Margolskee, 1996).
The final step in the IP3transduction pathway is yet to be
confirmed. In rat and mudpuppy taste cells, denatonium increases
[Ca2?]iby 50–100 nM (Akabas et al., 1988; Bernhardt et al., 1996;
present study). Although this concentration has been suggested to
lead to transmitter exocytosis from synaptic release sites (Akabas
et al., 1988; Spielman et al., 1994b; Bernhardt et al., 1996; Kin-
namon and Margolskee, 1996), this has yet to be demonstrated
directly. Compared with these relatively small increases of [Ca2?]i
in taste cells, neuroendocrine cells require at least 200 nM (reach-
ing a peak at 1 ?M) for exocytosis (Augustine and Neher, 1992),
whereas goldfish retinal presynaptic terminals require at least 50
?M [Ca2?]i(Von Gersdorff and Matthews, 1994). Total [Ca2?]i
rarely rose above 300 nM in any of the cells we tested. For
technical reasons, the measured [Ca2?]imay underestimate the
true maximum at synaptic release sites, because we measured
[Ca2?]iover the center of the whole cell to avoid edge artifacts
and may have missed contributions from synaptic release sites.
Further, the true maximum may have been underestimated be-
cause of out-of-focus blur from above and below the image plane.
Lindemann (1996b) has shown that peak transients of [Ca2?]iin
taste cells can be underestimated using fura-2, by factors of ?3,
unless deblurring algorithms are used to reduce the focal depth.
In addition, the measured maximum [Ca2?]iwas usually the first
measurement made after denatonium stimulation, and we may
have missed the true maximum because of the slow time resolu-
tion of our system. Also, it is possible that the kinetics of calcium
binding to fura-2 may have influenced our ability to identify the
true maximum [Ca2?]i(Nowycky and Pinter, 1993). Finally, the
data do not rule out the possibility that denatonium elicits calcium
influx through the plasma membrane triggered by store depletion,
as has been observed in several cell types (Striggow and Ehrlich,
1996). Such an influx could mediate large increases in [Ca2?]inear
the membrane that might not be detected by the imaging system
because of blurring of the signal. Nevertheless, the maximum
increases in [Ca2?]iobserved here are similar to those observed in
taste cells in rat and mouse in response to various tastants (Aka-
bas et al., 1988; Bernhardt et al., 1996; Hayashi et al., 1996). This
poses the interesting question of whether these levels of intracel-
lular calcium are sufficient to cause transmitter release, because
these levels are less than those necessary for exocytosis in neu-
roendocrine cells and neuronal presynaptic terminals. However,
taste cells are neither neuroendocrine cells nor neurons and may
not use the same mechanisms for exocytosis. For example, taste
receptor cells lack synaptophysin, which is an integral transmem-
brane protein associated with synaptic vesicles of the size found in
taste cells (Nelson and Finger, 1990). Therefore, the mudpuppy
taste cell is a useful model in which to study this problem, because
denatonium increases [Ca2?]iwithout depolarizing the cell. Mem-
brane depolarization could confound the analysis by increasing
[Ca2?]ilocally at transmitter release sites. These studies are
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