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2-dependent rise in
IP3 and 
-gustducin-dependent fall in cyclic
nucleotides
1 Department of Basic Science and Craniofacial Biology, Division of Biological Science, Medicine, and Surgery, New York University College of Dentistry, New York, New York 10010; 2 Monell Chemical Senses Center, Philadelphia 19104-3308; and 3 Philadelphia Veterans Affairs Medical Center, and University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Current evidence points to the existence of multiple processes
for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an 
-gustducin
(Ggust)-mediated stimulation of phosphodiesterase in
bitter taste transduction. Additionally, a taste-enriched G protein
-subunit, G13, colocalizes with Ggust
and mediates the denatonium-stimulated production of inositol
1,4,5-trisphosphate (IP3). Using quench-flow techniques, we
show here that the bitter stimuli, denatonium and strychnine, induce
rapid (50-100 ms) and transient reductions in cAMP and cGMP and
increases in IP3 in murine taste tissue. This decrease of
cyclic nucleotides is inhibited by Ggust antibodies,
whereas the increase in IP3 is not affected by antibodies
to Ggust. IP3 production is inhibited by
antibodies specific to phospholipase C-
2
(PLC-2), a PLC isoform known to be activated by
G-subunits. Antibodies to PLC-3 or to
PLC-4 were without effect. These data suggest a
transduction mechanism for bitter taste involving the rapid and
transient metabolism of dual second messenger systems, both mediated
through a taste cell G protein, likely composed of
Ggust//13, with both systems being
simultaneously activated in the same bitter-sensitive taste receptor cell.
taste transduction; denatonium; second messenger; rapid kinetics; taste receptors; inositol 1,4,5-trisphosphate; phospholipase C
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CONTINUING PROGRESS IS BEING made toward understanding the biochemical and molecular biological mechanisms of cellular taste transduction (7, 14, 20, 23, 25, 45). Several properties of the peripheral taste process make studying this chemical sense intriguing. For example, taste sensations can be elicited by a variety of structurally diverse compounds, some of which do not show absolute specificity toward a given modality (i.e., sweet, bitter, salty, sour, umami). It is also becoming clear that the taste system uses a variety of transduction mechanisms to signal the presence of taste-active compounds. Among gustatory stimuli, those that trigger the modality of bitterness are most diverse in structure (6, 48). Of the natural compounds that taste bitter, many are essential in plant defense mechanisms, with some being toxic. Indeed, bitter taste can be considered as a warning and defensive modality, providing a final analytical detector just before ingestion. A variety of bitter-tasting mechanisms may have evolved in response to this structural diversity and potential toxicity (7, 12, 48).
During the past two decades, bitter taste mechanisms have been the focus of an increasing number of studies. These studies used several techniques, including psychophysics (4, 10, 27, 59), behavioral genetics (e.g., Refs. 26 and 56), neurophysiology (13, 21, 35, 36, 44, 49, 55, 58), calcium imaging (2, 37), biochemistry (17, 29, 31, 39, 41, 43, 47, 51), and molecular biology (1, 11, 15, 16, 28-30, 42, 43, 58). These diverse studies support the assumption that several receptor/transduction processes are involved in bitter taste.
Several of these studies point to the possible involvement of
receptor-second messenger pathways in bitter taste transduction. In
particular, there is strong evidence from calcium imaging, biochemical,
and neurophysiological studies that the polyphosphoinositol pathway is
involved in mediating bitterness (2, 16, 17, 33, 34, 36, 41, 42,
47, 50, 51). Still other work convincingly demonstrates that a
gustatory tissue-enriched G protein, Ggust, mediates
bitter taste transduction. Because of Ggust's sequence
homology with the transducins, its activity is assumed to be via an
activation of phosphodiesterases (PDE), thus implicating cyclic
nucleotides as additional second messengers in bitter taste (21,
28, 29, 43, 45, 58).
We reasoned that both the polyphosphoinositol system and the cyclic nucleotide systems may be simultaneously activated in response to bitter compounds.
An important issue in supporting the relevance of several second messenger systems to bitter taste is measurement of the second messenger metabolism in real time. An outside limit to the initiation of a taste transduction response by most compounds can be set at ~250-500 ms after stimulus interaction with a receptor (19). Real-time assessment of biological processes in the millisecond time frame can be followed using stop-flow and quench-flow techniques. We and others have successfully developed and used these rapid kinetics procedures to measure second messenger metabolism in the millisecond time frame (8, 9, 16, 40-42, 51, 54).
Here we report second messenger metabolism in response to bitter stimuli in taste and control tissue from mouse SWR strain. Results indicate that bitter compounds such as denatonium and strychnine affect two second messenger systems simultaneously: they rapidly and reversibly elevate levels of inositol 1,4,5-trisphosphate (IP3) and suppress levels of cAMP and cGMP. The G protein subunits activated by these bitter stimuli and the enzymes that mediate the metabolism of these second messengers are delineated using antibodies specific to each component.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tissue collection.
Vallate and foliate taste papillae, along with control nongustatory
lingual tissue, were collected from 6- to 8-wk-old SWR mice (Hilltop
Lab Animals, Scottsdale, PA) by either carefully removing the papillae
by a punch procedure (41, 46, 51) or peeling off the
dorsal epithelium (5, 16). Peeling of the tissue was
performed after subdermal injection of 4 mg/ml collagenase (type I;
Boehringer Mannheim, Indianapolis, IN) and 1 mg/ml trypsin inhibitor
(Worthington Biochemicals, Lakewood, NJ) in 50 mM MOPS buffer, pH 6.9, containing (in mM) 100 NaCl, 2.5 CaCl2, and 2.5 MgCl2 (MOPS I buffer). The tongue was incubated in this
injection buffer for 20 min at 37°C, after which the entire posterior
epithelium was peeled away under a dissecting microscope. From each
peeled epithelium, one vallate and two foliate papillae and appropriate
nongustatory control tissue were surgically isolated. Control tissue
was removed from the dorsal eminence of the peeled epithelium, a region
devoid of taste papillae. The tissue samples were washed in ice-cold
MOPS II buffer [50 mM MOPS, pH 6.9, containing 100 mM NaCl, 2.5 mM
MgCl2, 1 mM 1,4 dithiothreitol (DTT), 10 mM EGTA, and 81 µM CaCl2 to give a calculated free Ca2+
concentration of 0.010 µM] and a protease inhibitory cocktail [1
mg/ml, specific for serine, cysteine, aspartic and metallo-proteinases (Sigma, St. Louis, MO)]. In this study, tissue homogenates generated from the punch removal technique were used to collect data described in
Figs. 1-4. Tissue homogenates generated from the
epithelial peeling procedure were used for studies described by Figs.
5-7. Data from Figs. 1-4 were gathered early in this study.
Subsequently (Figs. 5-7), we improved our tissue processing by
removing some of the potential epithelial contaminants. This
improvement also allowed us to use fewer mice per experiment. We
checked to determine that the two preparations led to equivalent
results.
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Chemicals.
Denatonium benzoate, strychnine HCl, and caffeine were purchased from
Sigma. Rabbit polyclonal IgG antibodies to Ggust (IgG), to phospholipase C-2 (PLC-2),
PLC-3, and PLC-4 (and their respective
blocking peptides), and normal (control) rabbit IgG (nIgG) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
[3H]IP3 and kits for assaying cyclic
nucleotides (cAMP and cGMP) and IP3 were purchased from NEN
(Boston, MA). All other chemicals were of the highest purity available
and were purchased from either Sigma or Calbiochem (San Diego, CA).
Quench-flow rapid kinetics. A quench-flow module (QFM5, BioLogic; Molecular Kinetics, Pullman, WA) equipped with five syringes was used to measure the subsecond kinetics of second messenger formation. (For description and operation details, see Refs. 41, 51, 54). In our QFM5 system, the first syringe was filled with MOPS III buffer supplemented with freshly prepared 1 mM ATP, 1 µM GTP, and 0.05% sodium cholate (hereafter called basal buffer). Bitter stimulants or pharmacological agents were dissolved into this basal buffer. The following stimulants and concentrations were tested (in mM): 2 and 10 strychnine and 1, 2, and 10 denatonium. For measures of cyclic nucleotides, the PDE inhibitor caffeine (25 mM) was added to the stimulus and control buffers for both taste and control tissues to raise the basal levels of the cyclic nucleotides (41).
The second syringe contained taste tissue or control homogenate in MOPS III buffer at a concentration of 35-50 µg/ml, except for studies shown in Figs. 5-7, where the protein concentration was 85-100 µg/ml. Tissue was maintained at 4°C at all times and loaded into the second syringe in small batches just seconds before injection at 22°C. The third syringe contained 9% perchloric acid to quench the reactions. The reaction was initiated by mixing 60 µl basal buffer or basal buffer containing stimulants with 60 µl tissue. After 0, 25, 50, 100, 200, and 500 ms, the reaction was terminated by injection of 60 µl of 9% perchloric acid. For each time point this was repeated twice, so the collected volume for each time point was 360 µl. The activation of all syringes was controlled by a personal computer, using a software developed by the manufacturer (BioLogic) and drive sequences developed in our laboratory. For the zero time point, tissue was first quenched with ice-cold perchloric acid and then stimulated. When antibodies to Ggust or to the PLC-s (10 µg/ml
antibody per 35-100 µg protein/ml) or nIgG (same concentration)
were used, the homogenate was preincubated with the antibodies for 90 min at 4°C and subsequently exposed to a particular buffer or
stimulant. When blocking peptides were used, each blocking peptide was
incubated with its appropriate antibody at 4°C overnight, and then
the peptide-treated antibodies were incubated with the tissue under the
conditions indicated above for antibodies not pretreated with blocking
peptides. For studies involving blocking peptides alone, the same
procedure was followed, except no antibody was added. After quenching,
330 µl (out of a total 360 µl) of the product was stored at
20°C until assayed.
Extraction and assay of cAMP and cGMP. On the day of the assay, samples were thawed and centrifuged at 1,000 g for 5 min. Cyclic nucleotides and IP3 (shown in Figs. 6 and 7) were extracted from the supernatant by mixing with 82.5 µl of 10 mM EDTA, pH 7.0, and 10 µl of Universal Indicator, with pH adjusted further to pH 7.0 using 1.5M KOH and 60 mM HEPES (38). The mix was centrifuged at 1,200 g for 5 min at 4°C. The supernatant was assayed either for cGMP and cAMP (using a 125I-labeled RIA kit from NEN) or for IP3 (see below).
Extraction and assay of IP3. Each sample was centrifuged at 1,000 g for 5 min, and the supernatant was mixed 5:1 with 10 mM Tris-EDTA, pH 9.0 (vol/vol), and further mixed with an equal volume of a freshly prepared mixture of 1:1 freon: tri-n-octylamine. Samples were vortex-mixed for 15 s and spun at 2,000 g for 1 min at 25°C. The upper phase (400 µl) was assayed for IP3.
IP3 was assayed using either a commercially available kit (NEN) or an IP3-binding protein extracted from bovine adrenal gland. For studies using the adrenal extract, an 3H-labeled IP3 tracer was used. Extraction of this binding protein followed the protocol described by Palmer and Wakelam (38). Because the binding protein is contained within a partial membrane preparation of the adrenal cortex, a differential centrifugation procedure was employed. Briefly, bovine adrenal cortex was dissected from six glands at a time and homogenized in 200 mM NaHCO3 and 1 mM DTT at 4°C. The homogenate was centrifuged at 5,000 g for 15 min. The supernatant was diluted to 40 ml with the NaHCO3 homogenization buffer and spun at 35,000 g for 20 min. The pellet was resuspended in 40 ml NaHCO3 buffer and centrifuged twice more. The final pellet from the final spin was resuspended in 12 ml of buffer at a protein concentration of 20-40 mg/ml. The binding protein was aliquoted at 30 mg/tube and frozen at80°C until used.
Each tube containing binding protein was thawed only once.
All steps for the IP3 assay were performed in siliconized
tubes. The assay followed a published procedure (38) with
minor modifications. The assay volume was half of that described
(38) to conserve binding protein. Centrifugation
of the final pellet was increased to 12,000 g for 4 min to
improve reproducibility between duplicates.
Data analyses.
Data analyses were performed by comparing the areas under the
time/concentration curves between the basal and stimulated levels (0-200 or 0-500 ms) of IP3 anabolism or cyclic
nucleotide catabolism. Six to eighteen measurements per point were
made. Variation among triplicates was usually between 1 and 7% but not
greater than 10%. It was possible to calculate the residual random
error of the basal and stimulated regions. Two-sample (nonpaired)
Student's t-tests were employed with statistical
significance designated at the 95% level ( = 0.05). The actual
P values are reported in the figure legends.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Bitter stimulus-induced metabolism of second messengers. Stimulation of the mouse gustatory tissue with 1, 2, and 10 mM denatonium resulted in a rapid and transient increase in IP3 production. Figure 1A shows that for 10 mM denatonium, levels of IP3 were increased by the first time point measured, 25 ms, and peaked at 75-100 ms. These levels then declined, returning close to basal levels after 200 ms. IP3 levels in gustatory tissue not stimulated by denatonium remained unchanged. When the same experiment using 10 mM denatonium was performed with nongustatory control tissue, IP3 levels were not significantly affected (Fig. 1B).
Likewise, 2 or 10 mM strychnine HCl rapidly induced elevated levels of IP3, which peaked at 100 ms. However, unlike the IP3 levels seen for denatonium, those for strychnine showed a slower return to basal levels (Fig. 2A, using 10 mM strychnine). The peak IP3 levels doubled compared with basal IP3 production when 10 mM strychnine was the stimulus. IP3 levels of gustatory tissue not stimulated by strychnine did not fluctuate. With nongustatory control tissue, 10 mM strychnine induced an IP3 increase that was about half of that seen in the gustatory tissue (Fig. 2B). From 50 to 100 ms, this increase was significantly different from the basal unstimulated controls. On the other hand, the effect of strychnine and/or denatonium on cyclic nucleotide levels demonstrated the opposite effect. When gustatory tissue was stimulated by strychnine and/or denatonium, cyclic nucleotide levels were suppressed from 0 to 100 ms. Figure 3A shows the time course of the relative levels of cGMP when gustatory tissue was stimulated by a mixture of 2 mM denatonium and 2 mM strychnine. This mixture of denatonium and strychnine decreased cGMP levels to 54% of the prestimulated (caffeine alone) levels, with a maximum effect noticeable at about 50 ms (Fig. 3A). The time course for denatonium plus strychnine-induced changes in the level of cAMP (data not shown) was similar to that seen for cGMP. Figure 3B shows bitter-induced changes in cAMP levels at the 50-ms time point only. By 50 ms, denatonium alone at 2 mM suppressed cAMP levels to 55% of their prestimulated (caffeine only) value, whereas strychnine alone at 2 mM reduced this level to 40%. In Fig. 3B, as in Figs. 4 and 5, the zero value refers to the basal level of cyclic nucleotide in the homogenate before either caffeine and/or denatonium or strychnine addition. The 100% level is the normalized amount of cyclic nucleotide available after caffeine (alone) inhibition of the PDEs.Role of Ggust in bitter-stimulated second
messenger metabolism.
To understand the mechanism of cyclic nucleotide catabolism and
IP3 anabolism in taste tissue, we tested the possible role of Ggust in generating these rapid changes in second
messenger levels. Because specific antibodies to Ggust
exist, we preincubated taste and control tissues with antibodies to
Ggust or with a preimmune IgG fraction (see
MATERIALS AND METHODS) in an attempt to rescue the bitter
stimulus-mediated metabolism of cGMP, cAMP, and IP3.
gust, but
not preimmune IgG, blocked the denatonium (1 mM)-induced suppression cyclic nucleotide levels to their prestimulated (i.e., caffeine stimulated) levels (Fig. 4, A and B). Figure
4A demonstrates that, at the 50-ms time point, denatonium
suppresses cAMP levels (as shown also in Fig. 3) but that this decrease
was blocked by preincubation of the tissue with an antibody to
Ggust. The decrease was not blocked, however, by
preincubating the taste tissue with preimmune IgG. The same pattern is
seen when cGMP is measured (Fig. 4B). Nongustatory tissue
did not demonstrate suppression of cAMP or cGMP by denatonium, and
there was no effect of the Ggust antibodies nor
of preimmune IgG on the levels of these messengers after denatonium stimulation (data not shown).
Similar to denatonium, 2 mM strychnine suppressed levels of cAMP (Figs.
3B and 5A) and cGMP (Fig. 5B). In the
case of strychnine, however, the suppression of cAMP was not rescued by
the Ggust antibodies (Fig. 5A), whereas the
strychnine suppression of cGMP was rescued by the
Ggust antibodies (Fig. 5B).
Data shown in Fig. 6 demonstrate that
antibodies to Ggust had no effect on the denatonium-
(Fig. 6A) or strychnine (Fig. 6B)-induced
increases in IP3. Measurements made at the 50-ms time point
show that these bitter-induced increases in IP3 remained unchanged after the tissue was preincubated either with preimmune IgG
or with antibodies specific to Ggust.
Role of PLC-2 in bitter stimulus-induced production
of IP3.
Generation of IP3 and diacylglycerol (DAG) is the result of
the action of the enzyme PLC on the minor membrane lipid
phosphatidylinositol 4,5-bisphosphate. There are several
types of PLCs defined by their sensitivities to modulatory compounds
and by their structural attributes (18, 42, 57).
Antibodies to PLC-2, PLC-3, and to PLC-4 were used to determine if one or more of these
inhibited the denatonium-induced rise in IP3 levels in
taste homogenates. Figure 7
shows data at the 50-ms time point for denatonium-induced changes in
IP3 levels in the absence of any antibodies or blocking peptides and in the presence of denatonium plus antibodies to PLC-2, PLC-3, and PLC-4.
The data show that only in the presence of antibodies to
PLC-2 was the denatonium-induced rise in IP3 levels inhibited. This inhibition could be blocked when antibody to
PLC-2 was preincubated with its blocking peptide.
Blocking peptide-pretreated antibodies to PLC-3 and
PLC-4 and blocking peptide alone did not affect
denatonium-induced IP3 increase. In addition,
antibodies to PLC-2 did not affect cAMP or cGMP levels
(Fig. 7B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Because of the diverse chemical structures of substances that taste bitter, it has long been assumed that there are multiple receptor mechanisms for the modality of bitterness. The literature generally supports this suggestion (for reviews, see Refs. 7 and 48). For example, there is both direct and inferential evidence that bitterness may be transduced by direct interaction of bitter compounds with biophysical properties of the membrane (e.g., Refs. 23 and 32), by interaction with intracellular components, bypassing a receptor step (31, 39, 41, 48), by direct modulation of stimulus-gated plasma membrane ion channels (48, 49, 55), and by more traditional G protein-coupled receptor (GPCR) processes (1, 11). One such receptor/transduction mechanism using the GPCR route is one that leads to the metabolism of second messengers (for reviews, see Refs. 7, 20, 24, 25, 45). Our previous work suggests that both the polyphosphoinositides and/or the cyclic nucleotides act as second messengers in bitter taste GPCR transduction (41, 47, 51).
The involvement of IP3 as a possible second messenger for
bitter taste signal transduction has received support from several studies (2, 16, 17, 33, 34, 36, 41, 42, 47, 51). For
example, we previously demonstrated that taste tissue homogenates from
mice show a rapid and transient bitter stimulus-induced production in
the second messenger IP3 (51). This increase
occurs within the physiologically relevant first 200 ms, making it
likely that IP3 could act as a second messenger in bitter
taste. The finding that the gustatory-enriched G protein
Ggust (28) was involved in bitter taste
transduction (58) and that this G protein showed high
sequence homology with the transducins suggested that taste PDE
(43), acting on cyclic nucleotide levels, may also be a
messenger system for bitter taste (45). Our new findings, described in this current work, support the suggestion that some bitter-tasting compounds may simultaneously alter second messenger levels of both the polyphosphoinositol system and the cyclic nucleotide systems in a G protein-dependent manner.
Second messenger metabolism in the millisecond time frame. The data presented here confirm that denatonium and strychnine can rapidly and transiently increase IP3 levels in taste tissue from mice. Data shown in Figs. 1 and 2 demonstrate that IP3 is increased after tissue stimulation by both denatonium and strychnine. Both of these responses were tissue specific, being significant at the P < 0.005 level. The data show rising IP3 levels from the first point of measurement, 25 ms, to 100 ms, after which the values for IP3 begin to decline rapidly for denatonium, but less rapidly for strychnine. Some increases in IP3 levels are seen with nongustatory controls, although the magnitude of these is much less than that seen with gustatory tissues. Such activity in nongustatory tissue for some bitter compounds has been noted previously (33, 51) and is likely due to the fact that many bitter stimuli are pharmacologically active.
Data from this study plus those from previous ones (50, 51) show that rapid increases in IP3 levels in rodent taste tissue are detected for the bitter-tasting stimuli, denatonium, strychnine, sucrose octaacetate, caffeine, quinine, naringin, and benzyltriethylammonium chloride (50, 51, 52, 53). In addition to the bitter taste-induced changes in IP3 levels, we show here, for the first time, that these same bitter stimuli can significantly reduce levels of both cAMP and cGMP within the millisecond time frame (Figs. 3-5). Data of Fig. 3A show that cGMP levels reach their minimum values by 50 ms, returning thereafter to near baseline levels by 150 ms. The decrease in nucleotide levels is likely due to the stimulation of a PDE by Ggust (see below). The rapid increase in cyclic
nucleotide levels after 50 ms may be due to the activation of several
systems including a gradual decline in PDE activity and/or by a
parallel increase in cyclic nucleotide synthesis. The data presented
here cannot be used to distinguish between these possibilities, and
further work is required to resolve this question.
One of the major hypotheses on bitter taste transduction states that
bitter stimuli induce a receptor-mediated activation of a PDE through
the action of the taste G protein, Ggust. If this were
the case, then one would expect to observe a decrease in cyclic
nucleotides due to stimulation by bitter compounds. Initial attempts to
show such decreases using homogenates were only suggestive (34,
53). Only minimal lowering of cyclic nucleotides was seen,
likely because the initial levels of cyclic nucleotides in the
homogenate were very low, due to the very active PDEs. On the basis of
our previous experimental work in taste, we knew that potent PDEs are
present in these tissues, and that methyl xanthines were able to
inhibit these, but only at high levels.
At the suggestion of a colleague (Dr. Heinz Breer, Stuttgart, Germany)
we adopted the common procedure of adding a xanthine inhibitor of PDE
[caffeine (41)] to raise the levels of cyclic nucleotides in our homogenate preparation. We chose to use 25 mM
caffeine in these studies because at this concentration, consistently high and readily assayed concentrations of cyclic nucleotides could be
sustained, allowing us to at least approach the testing of the
hypothesis outlined above. Although 25 mM is a high level, it is not so
high as to block the bitter stimulus-induced,
Ggust-mediated activation of PDE to affect a rapid, and
even reversible, drop in cyclic nucleotides. Although we cannot rule
out the possibility that caffeine may affect other systems in the
homogenate, the fact that a taste-specific,
Ggust-mediated drop in cyclic nucleotides is seen in
gustatory and not in control (nongustatory) tissue makes the use of
this compound justified for this initial test of the gustducin hypothesis.
Just what the conditions are within the cell that might permit
Ggust signaling to effect a drop in cyclic nucleotides
is unknown. But, unlike the homogenate, which, when being produced, likely quickly depletes cyclic nucleotides because no synthesis is
occurring, in the cell, both synthesis and normal PDE-mediated degradation of cyclic nucleotides are at a steady state. Upon taste
stimulation, the action of Ggust further stimulates PDE to effect a transient drop in cyclic nucleotides. Our homogenate cannot
accurately mimic these dynamic events. In the face of depleted cyclic
nucleotides, we need to not only allow some synthesis to occur (by
adding ATP at time 0) but also partially inhibit PDE to
measure the drop in nucleotides.
Adding caffeine simultaneously with either denatonium or strychnine
resulted in a taste tissue-specific decrease in cyclic nucleotide
levels. In contrast to the ability of denatonium and strychnine to
lower cyclic nucleotide levels in taste tissue, caffeine and other
xanthines raise levels of cyclic nucleotides, suggesting an alternate
mechanism for bitter taste transduction for these bitter tasting and
membrane-permeable PDE inhibitors (e.g., Refs. 22 and
41).
We estimated the stimulus thresholds for both the IP3
increases and the cyclic nucleotide decreases, for both denatonium and strychnine, to be in the range of 0.1 to 0.5 mM. These values are
consistent with, though somewhat higher than, behavioral avoidance thresholds reported by Whitney et al. (56). The
observation of slightly higher threshold values from the in vitro work
presented here is not surprising given the likely weaker coupling of
the major transduction constituents in the homogenate compared with their coupling in vivo.
G protein-mediated second messenger responses.
Data in Figs. 4 and 5 demonstrate that the denatonium- and
strychnine-induced decreases in cyclic nucleotides are dependent upon
Ggust. When antibodies to Ggust
were preincubated with the taste homogenate, neither denatonium nor
strychnine was able to induce a reduction in the levels of either cAMP
or cGMP. Antibodies from nonimmune serum were ineffective. This block
by antibodies to Ggust in taste tissue homogenates was
not seen with homogenates from nontaste tissue controls. The failure of
the Ggust antibodies to rescue the strychnine-induced
decreases in cAMP (Fig. 5) may imply additional control elements for
transducing signals from this and other bitter compounds.
gust were ineffective at
altering the denatonium- and strychnine-induced increases in
IP3 of taste homogenates (Fig. 6). These data, therefore,
suggest that bitter stimuli alter levels of cyclic nucleotides by a
Ggust activation of a taste PDE but that stimulation of
a PLC is Ggust independent.
A recent study demonstrated that the bitter-induced increase in
IP3 levels was dependent upon the /-subunit of a G
protein (16). Antibodies to a taste
G13-subunit prevented the denatonium-stimulated rapid
production of IP3. This observation and the data of Rossler et al. (42) from rat taste tissue suggest that the PLC
type responsible for taste-induced production of IP3 is a
PLC--type phospholipase. When antibodies to the PLC- types 2, 3, and 4 were preincubated with taste tissue homogenates, only the
antibodies to the PLC-2 subtype inhibited the
denatonium-induced increase in IP3. Use of blocking
peptides confirmed that this effect was specific to the antibodies to
PLC-2 (Fig. 7).
Bitter taste transduction mechanisms.
From the new data presented here and from results of previous work, one
can hypothesize that bitter taste signal transduction for denatonium
and strychnine involve both a Ggust-dependent, PDE-mediated reduction in cyclic nucleotide levels and a
G13-dependent, PLC-2-mediated increase in
IP3. Both of these changes in second messenger metabolism
occur within 100 ms, a time frame consistent with taste transduction.
The data of Huang et al. (16) show that a taste
cell-enriched G13-subunit and the
Ggust-subunit colocalize to the same cells, making it
likely that the heterotrimeric G protein involved in bitter taste is
composed of Ggust and G13. In
addition, the recently identified bitter taste receptors were found to
colocalize with Ggust (1).
gust/G13. The
Ggust activates one or more type of PDE, resulting in
the breakdown of cyclic nucleotides within a time frame of 50-75
ms. This transitory nature of the cyclic nucleotide response may
possibly be due to an adaptation mechanism such as phosphorylation of
the cell-surface receptor(s), inactivation of Ggust,
and/or stimulation of cyclic nucleotide synthesis, mechanisms that
cannot be discerned from the current data. Simultaneously, the
G13 activates a PLC-2, resulting in
the production of the two second messengers, DAG and IP3,
within a time frame of 50-100 ms. The exact targets of these
second messengers are not known. The cyclic nucleotides may, for
example, stimulate the activity of a cyclic nucleotide-inhibitable ion
channel (21) or a cyclic nucleotide-stimulated ion channel (30), altering cellular conductance. The messengers
IP3 and DAG may act to release calcium from intracellular
stores and activate protein kinases, respectively. There is also
evidence for a cyclic nucleotide-stimulated activation of a protein
kinase in taste cells (3). The observation that the
metabolism of these different second messenger pathways is stimulated
by components of the same heterotrimeric G protein, i.e.,
Ggust/G13, leads us to hypothesize that the bitter stimulus-induced reductions in cyclic nucleotides and
the bitter stimulus-induced increases in IP3 occur within the same bitter sensitive cell.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent technical assistance of D. Bayley, P. Iskander, D. Chen, and F. LeGeros.
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FOOTNOTES |
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This study was supported by National Institute of Dental Research Grant DE-10754, the United States-Israel Binational Agricultural Research and Development Fund (IS 2518), and Procter & Gamble (to A. I. Spielman), and the Department of Veterans Affairs and National Institute on Deafness and Other Communication Disorders Grants DC-00356 and DC-03969 (to J. G. Brand.)
Present address for G. Sunavala: Dept. of Biochemistry and Molecular Biology, Louisiana State Univ., Shreveport, LA 71130.
Address for reprint requests: A. I. Spielman, Div. of Biological Science, Medicine, and Surgery, New York Univ. College of Dentistry, 345 E. 24th St., New York, NY 10010. E-mail correpondence to: andrew.spielman{at}nyu.edu or joe.brand{at}monell.org.
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 13 April 2000; accepted in final form 20 October 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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) and Dena (
) are the
means ± SE of 3 quadruplicate experiments. The Dena-stimulated
elevation of IP3 in the gustatory tissue was
significantly different from that of the basal level (buffer);
*P < 0.0005.
