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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Current evidence points to the existence of multiple processes
for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an -gustducin
(G
gust)-mediated stimulation of phosphodiesterase in
bitter taste transduction. Additionally, a taste-enriched G protein
-subunit, G
13, colocalizes with G
gust
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 G
gust antibodies,
whereas the increase in IP3 is not affected by antibodies
to G
gust. 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
G
gust/
/
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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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 G
gust'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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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 GExtraction 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 atData 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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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 G
gust in generating these rapid changes in second
messenger levels. Because specific antibodies to G
gust
exist, we preincubated taste and control tissues with antibodies to
G
gust 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.
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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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 GG 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 G
gust
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 G
gust in taste tissue homogenates was
not seen with homogenates from nontaste tissue controls. The failure of
the G
gust 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.
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
G
13-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 G
13-subunit and the
G
gust-subunit colocalize to the same cells, making it
likely that the heterotrimeric G protein involved in bitter taste is
composed of G
gust and G
13. In
addition, the recently identified bitter taste receptors were found to
colocalize with G
gust (1).
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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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