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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Varkevisser, Brian and Sue C. Kinnamon. Sweet Taste Transduction in Hamster: Role of Protein Kinases. J. Neurophysiol. 83: 2526-2532, 2000. Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweeteners stimulate production of inositol 1,4,5-tris-phosphate (IP3) and diacylglycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K+ conductance, but the intermediate steps in the transduction pathways have not been examined. In this study, the loose-patch recording technique was used to examine the role of protein kinases and other downstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from ~35% of fungiform taste buds in response to sucrose (200 mM) or NC-00274-01 (NC-01, 200 µM), a synthetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PDBu (10 µM). In all sweet-responsive taste buds tested (n = 11), PDBu elicited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmaleimide I (0.15 µM) resulted in inhibition of the NC-01 response by ~75%, whereas the response to sucrose either increased or remained unchanged. These data suggest that activation of PKC is required for the transduction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined in the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 µM). Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transduction of sugars, but may play a modulatory role in both pathways, such as adaptation of the response. We also examined whether Ca2+-calmodulin dependent cAMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, by examining responses to sucrose and synthetic sweeteners in the presence of the CaM-PDE inhibitor W-7 (100 µM). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchanged. These data suggest that the pathways for sugars and sweeteners are negatively coupled; the Ca2+ that is released from intracellular stores during stimulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.
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INTRODUCTION |
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Recent biochemical and electrophysiological
studies have demonstrated that there are two intracellular pathways for
sweet taste transduction: sugars stimulate an increase in the
intracellular concentration of cyclic AMP (cAMP) (Bernhardt et
al. 1996; Naim et al. 1991
), whereas synthetic
sweeteners and some amino acids stimulate the production of
1,4,5-inositol trisphosphate (IP3) and
diacylglycerol (DAG) (Bernhardt et al. 1996
;
Uchida and Sato 1997
). Several lines of evidence suggest
that both second-messenger pathways are expressed in the same
sweet-sensitive taste cells. Calcium-imaging studies have shown that
the same taste cells respond to both sucrose and the synthetic
sweetener SC-45647 (Bernhardt et al. 1996
), and
electrophysiological studies have shown that the same taste cells
respond to both cAMP and synthetic sweeteners (Cummings et al.
1996
; Tonosaki and Funakoshi 1988
). Presumably, these second messengers are activated when sweet stimuli bind to G
protein-coupled receptors on the taste cell membrane. Recently, a
putative sweet taste receptor was cloned (Hoon et al.
1999
), but the receptor has not been characterized functionally.
Synthetic sweeteners and cAMP depolarize taste cells by closing a
resting K+ conductance, and these responses show
cross adaptation (Cummings et al. 1996). Thus the cAMP
and IP3/DAG second-messenger pathways appear to
target the same K+ channels, but the mechanisms
involved are not known. Taste cells have been shown to express both
cyclic nucleotide-activated (Misaka et al. 1997
) and
cyclic nucleotide-suppressed (Kolesnikov and Margolskee
1995
) ion channels, which suggests that nucleotides can
interact directly with ion channels in taste cells. Yet, in studies of
frog taste cells, K+ channels are closed by
cAMP-dependant protein kinase (Avenet et al. 1988
),
suggesting that protein kinase A (PKA)-mediated phosphorylation of
K+ channels can occur. For synthetic sweeteners,
one consequence of elevating IP3 is release of
Ca2+ from intracellular stores, which has been
measured in rat taste buds (Bernhardt et al. 1996
). An
increase in intracellular Ca2+ coupled with the
production of DAG has been shown in other systems to activate protein
kinases, specifically protein kinase C (PKC). However, whether PKC
activation is required for K+ channel closure in
response to synthetic sweeteners is not known.
The loose-patch technique for recording from taste buds in situ has
provided important information about transduction mechanisms in mammals
for NaCl (Avenet and Lindemann 1991), acids
(Gilbertson et al. 1992
), and sweeteners
(Cummings et al. 1993
). The technique consists of
recording action currents, reflecting taste cell action potentials,
from single fungiform taste buds in situ. In hamsters, action currents
are generated to NaCl, citric acid, sucrose, and several synthetic
sweeteners, but not to most bitter compounds (P. Avenet and S. Kinnamon, unpublished data). Previous studies have shown that, whereas
most hamster taste buds are NaCl sensitive, only ~35% are sweet
sensitive. The responses to sweeteners in any single taste bud are
reliable and repeatable for up to periods of 2 h. In addition,
responses show dose dependency, with increased frequency of action
currents at higher concentrations. Sweet-sensitive taste buds also
respond to membrane-permeant analogues of cAMP and cGMP, but
sweet-insensitive taste buds are unresponsive to these second
messengers. The responses to both sweeteners and second messengers show
adaptation at higher concentrations (Cummings et al.
1993
).
In this study we used the loose-patch technique to investigate the role
of protein kinases in sweet taste transduction. Specifically, we asked
the following questions. 1) What is the role of PKC in the
transduction of synthetic sweeteners? 2) What is the role of
PKA in the transduction of sugars? 3) Do the sugar and
sweetener transduction pathways interact with each other? Preliminary
accounts of this work were published in abstract form
(Varkevisser and Kinnamon 1998; Varkevisser et
al. 1997
).
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METHODS |
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Preparation and recording method
Golden Syrian hamsters ranging from 4 to 10 wk in age were
killed by CO2 asphyxiation and cervical
dislocation. Their tongues were excised ~4 mm posterior to the field
of the fungiform papilla, rinsed with distilled water, and mounted in a
bipartitioned silicone elastomer (Sylgard) recording chamber. The
dorsal surface of the exposed anterior half of the tongue was
illuminated and viewed with a dissecting microscope at ×5-50
magnification. The loose-patch recording technique (Avenet and
Lindemann 1991; Cummings et al. 1993
;
Gilbertson et al. 1992
) was used to record from the
fungiform papillae. The recording electrode, pulled on a Sachs-Flaming
PC84 micropipette puller and fire polished to a tip diameter of 70-120 µm, was positioned over a fungiform papilla. Strong suction was applied to the pipette via a suction pump connected to the loose-patch device just above the pipette. Contained within this pipette were the
perfusion pipette and a AgCl wire connected to the headstage ground.
Perfusion pipettes were fabricated from fused silica tubing (Polymicron Technologies, Phoenix, AZ; 224 µm OD, 100 µm ID) pulled by hand over a gas flame. The open end was fitted to polyethylene tubing that was connected to the pipette assembly. The tip was cut to a diameter of 40-50 µm. The tip of this inner pipette was positioned to within 500 µm of the tip of the recording pipette to allow the solution to reach the taste bud before removal by suction. Solutions were held in eight 50-ml syringes, each with a fitted stopcock, and were connected by polyethylene tubing to the pipette assembly. The solution reservoirs were pressurized such that a turn of one stopcock pushed the solution of choice through the perfusion pipette into the tip of the recording electrode and thus over the taste pore itself. Action currents, reflecting taste cell action potentials, were seen within 15-25 s after switching the stopcocks to deliver stimuli. Subthreshold responses usually could not be resolved. Because of variability in the stimulus delivery, response latencies could not be ascertained with this technique. The recording pipette, perfusion pipette, and ground electrode were all fitted into one end of the pipette assembly.
An agar bridge impregnated with Tyrode's solution and connected to a
AgCl wire was placed against the cut proximal end of the tongue. The
lead was connected to the headstage of a patch-clamp amplifier
(Axopatch 1D, Axon Instruments) with a 100-M feedback resistor. A
AgCl wire in the recording pipette served as the ground electrode. This
reversal of leads minimized the power line interference (cf.,
Avenet and Lindemann 1991
). Currents were recorded with the amplifier in voltage-clamp mode at a pipette potential of 0 mV. The
amplifier was kept in tracking mode to minimize DC shifts resulting
from junction potentials generated during solution changes. The signal
was filtered with a low-pass Bessel eight-pole filter (Frequency
Devices, 902 LPF) at a corner frequency of 270 Hz. The analogue signal
from the amplifier was digitized at 125 Hz by an A/D board (Digitata
1200 interface, Axon Instruments), viewed on-line, and stored on a PC
(Applied Computer Technology) using Axoscope software.
Solutions
The control solution, designed to mimic the low ionic strength
of hamster saliva, consisted of 30 mM
N-methyl-D-glucamine chloride (NMDG-Cl) and 5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES) titrated to a pH of 7.4 with HCl. Taste stimuli dissolved in this control solution were 100 mM NaCl, 200 mM sucrose, and 200 µM NC-00274-01 (NC-01), a synthetic sweetener. These
concentrations were shown previously to be strongly preferred by
hamsters and to elicit robust responses in loose-patch recordings of
taste buds in situ (Cummings et al. 1993). The
membrane-permeant drug, phorbol-12,13-dibutyrate (PDBu), diluted in the
control solution to a concentration of 10 µM, was used as an
activator of PKC. The inhibitors used were: bisindolylmaleimide I (Bis
I; 0.15 µM), a specific inhibitor of PKC; H-89 dihydrochloride (10 and 19 µM), a specific inhibitor of PKA; and W-7 hydrochloride (W-7,
100 µM), a specific inhibitor of
Ca2+-calmodulin dependent cAMP
phosphodiesterase (CaM-PDE). Inhibitor concentrations were
chosen based on the vendor's specifications. For most experiments we
chose the highest concentration that would ensure specificity.
Inhibitors were perfused before and during the application of the test
stimulus, after control stimuli were tested. Thus a typical experiment
began with verifying that the taste bud was sweet sensitive, followed
by the application of the inhibitor, then the simultaneous application
of inhibitor with sweet stimuli. All inhibitors were obtained from
Calbiochem (La Jolla, CA). NC-00274-01 was a generous gift of the
NutraSweet Corporation. All other chemicals were obtained from Sigma
Chemical Corporation (St. Louis, MO).
Data analysis
To determine the effects of various protein inhibitors on the
response to sucrose and the synthetic sweetener NC-01, action currents
were monitored in response to taste stimuli before and during
application of the inhibitor. If spontaneous activity was not present,
the response duration was defined as the interval between the first and
last spikes of the response. In taste buds that exhibited spontaneous
activity, a response was defined as a twofold increase in activity over
basal (unstimulated) activity. Thus the beginning of a response was
defined as the moment spike frequency increased to twice that of
background activity; the end of the response occurred when the rate
decreased to <50% of the stimulated response. This response magnitude
was chosen to ensure that responses could be delineated clearly from
spontaneous activity. Because response rates tend to saturate at high
concentrations of taste stimuli (Cummings et al. 1993),
experiments with a high rate of spontaneous activity were omitted from
analysis. Sweet stimulus response rates were analyzed by counting the
number of spikes within the response and dividing by the total duration of the response. During experiments that involved using a protein inhibitor, a response was analyzed only if the effect of the inhibitor was reversible. For display purposes, response rates during application of the inhibitor were normalized by dividing the response rate during
application of the inhibitor by the response rate in the absence of the
inhibitor. Therefore a value of >1 indicates an increase in the rate
of response, whereas a value <1 indicates a decrease in the interspike
rate of the response to the taste stimulus. For statistical analysis,
spike rates (spikes/s) were analyzed for each taste bud in the presence
and absence of the inhibitor. Statistical analysis of all experiments
was performed using a one-tailed Wilcoxon signed rank test.
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RESULTS |
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Experiments were performed using the loose-patch technique for
recording from taste buds in situ (Avenet and Lindemann
1991). By placing a recording electrode over the apical pore of
fungiform papillae, we recorded population action currents from single
taste buds in response to sweet taste stimuli in the presence and
absence of protein inhibitors. The advantage of this approach is that the sweet stimulus could be restricted to the taste pore, as occurs in
situ. This allowed us to use sucrose as a taste stimulus, which is
sweet only at concentrations that would cause osmotic damage if applied
to isolated taste cells. The disadvantage of the technique is that we
are unable to distinguish which taste cells within the taste bud
participate in a response, or if the same taste cells participate in
each response during repetitive stimulation. In addition, responses to
the same taste stimuli vary in different taste buds. Some taste buds
exhibit robust responses to sweet stimuli, whereas others show only
weak responses or no response at all. Thus all responses to sweet
stimuli in the presence of protein inhibitors were normalized to
responses before application of the inhibitors.
Each taste bud was first tested by stimulation with 100 mM NaCl,
because previous studies showed that most taste buds respond vigorously
to NaCl (Avenet and Lindemann 1991; Cummings et
al. 1993
). This was done to ensure that cells within the taste
bud were capable of generating action potentials. A total of 283 taste buds was tested in this manner. Taste buds that were responsive to NaCl
were then tested for responses to NC-01 and sucrose. Of the total
number of taste buds responding to NaCl (198) only a small fraction
(<35%) responded to the sweet stimuli. Taste buds that were sweet
sensitive usually responded to both sucrose and NC-01, as shown
previously (Cummings et al. 1993
).
Role of protein kinase C in sweet taste transduction
In rat membrane preparations, application of synthetic sweeteners
leads to the production of DAG and IP3
(Bernhardt et al. 1996), a second messenger that leads
to the opening of IP3-gated Ca2+ channels in intracellular stores. One effect
of elevated Ca2+ in the presence of DAG is
activation of protein kinases, specifically PKC. To determine whether
PKC activation is required for generation of taste cell action
potentials in response to synthetic sweeteners, we first examined
whether activation of PKC mimicked the effect of sweet taste stimuli.
Because not all taste buds are sweet responsive, we correlated
responses to NC-01 and sucrose with responses to the membrane-permeant
PKC activator, PDBu (10 µM).
Figure 1 depicts recordings from two fungiform taste buds. Both were first stimulated with NaCl to demonstrate viability of the taste bud. Following a wash of >3 min, each taste bud was stimulated sequentially with NC-01, sucrose, and PDBu, with intervening washes. PDBu elicited action currents in the sweet-sensitive taste bud (Fig. 1A) but had no effect on the sweet-insensitive taste bud (Fig. 1B). Of the 11 sweet-sensitive taste buds tested (Fig. 1C), all responded to PDBu with action currents. In contrast, only 4 of 19 sweet-insensitive taste buds tested responded to PDBu with action currents. Responses in sweet-insensitive taste buds may indicate a role for PKC in the transduction of taste qualities other than sweet.
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The results described above suggest that PKC may be involved in the sweet transduction pathway, but whether it plays a role in the transduction of synthetic sweeteners or sugars cannot be determined from such correlative studies. To test more specifically PKC's involvement in sweet taste transduction, Bis I (0.15 µM), a membrane-permeant inhibitor of PKC, was used to determine whether PKC activation is required for sweet taste transduction. Figure 2 illustrates the effect of Bis I on responses to sucrose and NC-01. In the taste bud illustrated in Fig. 2, A and B, responses to NC-01 before application of the inhibitor were more robust than responses to sucrose (Fig. 2A). However, in the presence of Bis I, responses to NC-01 decreased, whereas responses to sucrose showed a large increase (Fig. 2B). This large enhancement of the sucrose response occurred in only 2 of the 11 experiments, although there was a small increase in response to sucrose in several experiments. The effects of the inhibitor were reversible (Fig. 2C). Overall, the response to sucrose remained unchanged (P = 0.1016) during inhibition of PKC, whereas the response to NC-01 decreased to ~25% of the control response (P = 0.0020; n = 11, Fig. 2D). These data suggest that activation of PKC is directly involved in the transduction of synthetic sweeteners. In addition, the enhancement of the sucrose response in the presence of Bis I in some taste buds suggests that the transduction pathway for sugars may be negatively coupled to the transduction of synthetic sweeteners in these buds, possibly via activation of PKC.
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Role of protein kinase A in sweet taste transduction
There is abundant evidence that sucrose stimulates cAMP
(Bernhardt et al. 1996; Naim et al. 1991
;
Striem et al. 1989
) and that cAMP elicits action
potentials in taste cells (Cummings et al. 1993
) by
closure of a resting K+ conductance
(Cummings et al. 1996
). What is not clear is whether phosphorylation is required for this response. cAMP-dependent protein
kinase (PKA) has been shown to phosphorylate K+
channels in frog taste cells (Avenet et al. 1988
), but
this response was not connected to sweet stimulation. To determine
whether PKA is required for transduction of sugars in hamster taste
buds, we perfused H-89 (10 and 19 µM), a membrane-permeant inhibitor of PKA, onto sweet-sensitive taste buds. Quite to our surprise, H-89
did not inhibit the response to either sucrose or NC-01 in any of the
six taste buds tested. Instead, H-89 increased the response
to sucrose and NC-01 in most taste buds. In the experiment shown in
Fig. 3, the response to NC-01 increased
~1.5-fold in the presence of H-89, whereas the response to sucrose
increased over 10-fold. The degree of increase was quite variable in
magnitude, especially for sucrose. The increase in response rate to
sucrose varied from 20% to over 1,400%, whereas the increase in
response to NC-01 was somewhat less variable, ranging from little or no increase to approximately a 400% increase. The effects of H-89 were
reversible. Overall, the increase in response in the presence of H-89
was statistically significant (NC-01: P = 0.0313;
sucrose: P = 0.0156). What is very clear from these
data are that PKA is not required for the transduction of sugars,
because there was no decease in the response to sucrose in the presence
of H-89. These data suggest that cAMP may close
K+ channels directly in response to sucrose
stimulation.
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Role of CaM-dependent phosphodiesterase in sweet transduction
Although not statistically significant, in a few of the taste buds
tested with Bis I, there was an enhancement of the sucrose response
coupled with the inhibition of the NC-01 response. This raises the
possibility that the pathway for transduction for synthetic sweeteners
inhibits the pathway for transduction of sugars. We have already
demonstrated that PKC activation can inhibit the transduction of
sucrose in some taste buds, because some taste buds showed increases in
response to sucrose after inhibition of PKC (i.e., Fig. 2). In
addition, further inhibition may result from the
IP3 that is produced in response to stimulation
with synthetic sweeteners. IP3 causes an increase
in intracellular Ca2+ due to release of
Ca2+ from intracellular stores. This increase in
intracellular Ca2+ may activate CaM-PDE,
resulting in a decrease in intracellular cAMP levels, which would
inhibit the sucrose response. Thus we examined whether inhibition of
CaM-PDE influences the response to sucrose. W-7 (100 µM), a specific
membrane-permeant inhibitor of CaM-PDE (Itoh and Hidaka
1984), was perfused onto sweet-sensitive taste buds before and
during sweet taste stimulation. Figure 4 illustrates the effect of W-7 on responses to sucrose and NC-01 in a
sweet-responsive taste bud. In the presence of W-7 (Fig. 4B), there was no effect on the response to NC-01, whereas
the response to sucrose increased fivefold. This effect of W-7 was reversible. Analysis of 10 taste buds showed an average 2-fold increase
in the rate of response to sucrose (P = 0.0010),
whereas that to NC-01 remained unchanged (P = 0.2852).
Taken together, these data suggest that activation of CaM-PDE during
stimulation with NC-01 may inhibit responses to sucrose by decreasing
intracellular cAMP levels.
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DISCUSSION |
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Present models indicate that taste cells utilize two separate
second-messenger pathways for the transduction of sweet stimuli: sugars
operate via cAMP, whereas synthetic sweeteners utilize the
IP3 and DAG pathway. Both pathways converge,
blocking the same resting K+ conductance leading
to deplarization of the taste cell. In this study, we used the
loose-patch technique for recording from taste buds in situ
(Avenet and Lindemann 1991; Cummings et al.
1993
; Gilbertson et al. 1992
) to examine the
role of downstream regulatory proteins in sweet taste transduction.
Our study provides additional support for distinct, but interdependent pathways in the transduction of sugars and synthetic sweeteners. Moreover, we provide strong evidence for a role of PKC in the transduction pathway for synthetic sweeteners. PDBu, a potent activator of PKC, elicited action currents in sweet response taste cells. More direct evidence was obtained from inhibitor studies; responses to sweeteners during inhibition of PKC decreased 75% compared with control responses, whereas responses to sugars remained unaffected. It is possible that PKC phosphorylates the sweet-sensitive K+ channels, leading to closure and membrane depolarization, but this awaits confirmation by single-cell recording.
In contrast to sweeteners, our results do not support the need for phosphorylation of channels in the transduction pathway for sugars. Inhibition of PKA resulted instead in an enhancement of responses to both sugars and synthetic sweeteners. One caveat in this interpretation is whether H-89 is getting into the cells at a concentration sufficient to block PKA. We believe that the increase in the response to both sweeteners and sugars is evidence that the H-89 is getting into the cells. Responses were enhanced whether the inhibitor was in place for a few minutes or >1 h, which should be sufficient time for the inhibitor to permeate the taste cells. Further, we have used two different concentrations (10 and 19 µM) of the inhibitor and found no differences between the two concentrations. However, we cannot rule out the possibility that H-89 is not blocking all isoforms of PKA, although it is the most broad-spectrum membrane-permeant PKA inhibitor that is currently available.
The tendency for H-89 to increase the response to NC-01 was not totally
unexpected, because several studies with other systems have shown that
PKA-mediated phosphorylation can inhibit the production of
IP3 when both second-messenger systems are
expressed in the same cells (Campbell et al. 1990;
Liu and Simon 1996
). In taste cells, a recent study
shows that some bitter compounds both increase IP3 and inhibit cAMP, suggesting that a decrease
in cAMP may be required for full activation of the
IP3 response (Yan et al. 1999
). In
contrast, the inhibitory effect of PKA on the response to sugars was
unexpected, because a previous study had shown that PKA can phosphorylate and close K+ channels in taste
cells (Avenet et al. 1988
). It is likely that PKA plays
a modulatory, rather than a mediatory role in the response to sugars.
One possibility is that PKA phosphorylates components in the sugar
transduction pathway, causing adaptation of the response. In support of
this hypothesis, whole cell recordings show strong adaptation in
response to membrane-permeant cAMP analogues (Cummings et al.
1996
).
So, what is the role of cAMP in the transduction of sugars? Numerous
studies have shown that an increase in the intracellular concentration
of cAMP leads to depolarization of the cell (Avenet and
Lindemann 1987; Cummings et al. 1996
;
Tonosaki and Funakoshi 1988
), an increase in membrane
resistance (Tonosaki and Funakoshi 1988
), and that cAMP
mimics the effects of sweet stimuli when using the loose-patch
technique (Cummings et al. 1993
). Therefore we believe
that cAMP, in leading to cell depolarization, bypasses PKA and binds
directly to the sweet-sensitive K+ channels. A
direct cyclic nucleotide-blocked cation channel has been described in
frog taste cells (Kolesnikov and Margolskee 1995
), but
its role in sweet transduction has not been investigated.
In some taste cells we observed an enhancement of the sucrose response
while inhibiting PKC (e.g., Fig. 2B). This raises the possibility that the IP3/DAG pathway for
synthetic sweeteners inhibits the cAMP pathway for sugars. Such cross
talk between these signaling pathways has been observed in other
systems (Selbie and Hill 1998). One way this could occur
is if the Ca2+ that is released from
intracellular stores during stimulation by synthetic sweeteners
activates Ca2+-dependent enzymes that inhibit the
cAMP pathway. Calcium-dependent enzymes in taste cells include both PKC
and CaM-dependent PDE. Activation of CaM-dependent PDE would lead to
decreases in intracellular cAMP, thus decreasing the response to
sucrose. Our data suggest that CaM-dependent PDE may modulate the
transduction of sugars, because the CaM-PDE inhibitor W-7 significantly
enhanced the response to sucrose in all taste buds tested. Although
inhibition of PKC also produced an increase in the response to sucrose
in some taste buds, the targets of PKC modulation in taste buds have
not been elucidated.
Finally, our results suggest a possible role for gustducin in sweet
transduction. Gustducin, a chemosensory-specific G protein in taste
cells (McLaughlin et al. 1992), has significant sequence homology to transducin, which is also expressed in taste cells (McLaughlin et al. 1993
). Both gustducin and transducin
activate PDE, decreasing intracellular cAMP. Targeted disruption of the gustducin gene in mice results in a profound decrease in the
sensitivity to both sugars and sweeteners (Wong et al.
1996
). We propose, as proposed earlier by Lindemann
(1996)
, that the reason for the decreased sensitiviy in the
gustducin knockout mice is that the knockouts have an increased
concentration of intracellular cAMP, which leads to a constitutive
activation of PKA. Thus the sweet-sensitive taste cells may be in a
constant state of adaptation and unable to respond effectively to
either sugars or sweeteners.
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ACKNOWLEDGMENTS |
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We thank Drs. Tatsuya Ogura and Thomas Cummings for helpful suggestions throughout the study and Dr. Kathryn Partin for helpful comments on the manuscript. We also thank the NutraSweet Corporation for the generous gift of the synthetic sweetener NC-00274-01.
This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-00244 to S. C. Kinnamon and by a minority supplement to B. Varkevisser.
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FOOTNOTES |
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Address for reprint requests: S. C. Kinnamon, 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 15 June 1999; accepted in final form 11 January 2000.
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REFERENCES |
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