Generation of inositol phosphates in bitter taste transduction

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Physiology & Behavior, Vol. 56, No. 6, pp. 1149-1155, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

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Generation of Inositol Phosphates in Bitter Taste Transduction

A N D R E W I. S P I E L M A N , * t T A U F I Q U L HUQUE,~" H A J I M E N A G A I , t ~ t G L A Y D E W H I T N E Y § A N D J O S E P H G. B R A N D ' S ¶ # t

*Division of Basic Sciences, New York University College of Dentistry, New York, NY, USA, ?~Monell Chemical Senses Center, Philadelphia, PA, USA, ~:Institute for Fundamental Research, Suntory Limited, Osaka, Japan,

§Department of Psychology, Florida State University, Tallahassee, FL, USA, ¶Veterans Affairs Medical Center, Philadelphia, PA, USA, and #School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA

SPIELMAN, A. I., T. HUQUE, H. NAGAI, G. WHITNEY AND J.G. BRAND. Generation ofinositol phosphates in bitter taste transduction. PHYSIOL BEHAV 56(6) 1149-1155, 1994.--It is probable that there is a diversity of mechanisms involved in the transduction of bitter taste. One of these mechanisms uses the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Partial membrane preparations from circumvallate and foliate taste regions of mice tongues responded to the addition of known bitter taste stimuli by increasing the amount of inositol phosphates produced after 30 s incubation. Addition of both the bitter stimulus, sucrose octaacetate and the G-protein stimulant, GTPTS, led to an enhanced production of inositol phosphates compared with either alone. Pretreatment of the tissue samples with pertussis toxin eliminated all response to sucrose octaacetate plus GTPTS, whereas pretreatment with cholera toxin was without effect. Western blots of solubilized tissue from circumvallate and foliate regions probed with antibodies to the a-subunit of several types of G-proteins revealed bands reactive to antibodies against Gt~,_2 and Gao, with no apparent activity to antibodies against Goti 3 . Given the results from the immunoblots and those of the toxin experiments, it is proposed that the transduction of the bitter taste of sucrose octaacetate in mice involves a receptor-mediated activation of a G~-type protein which activates a phospholipase C to produce the two second messengers, IP3 and DAG.

Taste Bitter Inositol phosphates G-Proteins Phospholipase C Inositol 1,4,5-trisphosphate

Signal transduction

INTRODUCTION

A VARIETY of signal transduction mechanisms are used in taste. These include the participation of amiloride-sensitive ion channels in the transduction of salty and possibly sour taste, proton inhibitable channels in sour taste, stimulus-gated ion channels in the transduction of glutamate in mouse and of arginine in catfish, and the participation of G-protein coupled, receptor-mediated production of the second messenger, cyclic AMP, in sweet taste (For reviews see 2,3,4,13,14,21). Transduction of bitter tasting stimuli appears more complicated, in that a variety of mechanisms may be involved in mediating the transduction of this single modality (25). This variety of mechanisms may give the system the versatility to detect the very wide range of structural differences that define the class of chemicals which taste bitter.

The existence of such diversity in transduction mechanisms for bitter taste implies that there must be common factors which, when activated, signal bitterness. Transduction processes may differ, as long as they all lead to firing of the appropriate neurons. The taste cell secretes neurotransmitter which changes the firing rate of the innervating neuron. One common feature of neurotransmitter release is the requirement for an increase in intracellular calcium ion activity. Therefore, any mechanism proposed as a transduction scheme for bitter taste must have within it a

mechanism for sufficient change in intracellular calcium ion concentration.

For a number of years, our laboratories have been testing the hypothesis that the lipid-derived messenger, inositol 1,4,5-trisphosphate (IP3), is involved as a second messenger in bitter taste transduction. Direct evidence supporting this hypothesis was first presented by us in 1990 (26). Given what is known about the polyphosphoinositol-derived second messengers, IP3 and diacylglycerol (DAG) (6), it is probable that these lipid-derived second messengers are mediators between the receptor event and the increase in intracelhilar calcium ion required for release of neurotransmitter.

In addition to our own studies suggesting that IP3 is involved in bitter taste transduction, work from other laboratories also sup- ports this hypothesis. The bitter stimulus, denatonium, was shown to increase intracellular calcium ion concentrations in some rat taste cells (1). One way in which intracellular calcium ion concentration can increase is by an IP3-induced release of sequestered calcium from intracellular stores. In addition, components of the inositol phosphate-Ca pathway, such as Ca-ATP- ase and IP3 receptors, were identified in rat taste buds (12). This latter study reported a modest but significant increase in IP3 production due to stimulation with the bitter compound, denatonium.

JRequests for reprints should be addressed to Joseph G. Brand, Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308, USA.

1149

1150 SPIELMAN ET AL.

These studies led credence to the hypothesis that IP3 was involved in bitter taste transduction, and supported our own data of that time demonstrating larger increases in IP3 production in mouse taste tissue stimulated by the bitter tasting compound, sucrose octaacetate (26).

Since these early studies, we have demonstrated increases in IP3 production to bitter stimuli that were specific to taste tissue in mouse, and have shown that these changes take place on the millisecond time scale (24). In this manuscript, we report data on production of IP3 to several bitter-tasting compounds. The data implicate the participation of a G-protein in this transduction pathway, and suggest that the G-protein is of the Gi type. We place these observations in the context of earlier data from our laboratories and from those of others in suggesting that one transduction mechanism for bitter taste involves a receptor-activated, G-protein mediated production of second messengers derived from metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2).

MATERIALS AND METHODS

Animals

Two different strains of mice were used in these studies. Adult male and female mice belonging to the B6.SW congenic strain (35) (laboratory of G. Whitney, Tallahassee, FL) are sensitive to several bitter compounds including sucrose octaacetate, denatonium and strychnine, as determined in two-bottle preference tests (34,35). Adult male mice of the strain C57BL/6J (Jackson Lab- oratories, Bar Harbor, ME) are also sensitive to several bitter tasting compounds including denatonium and strychnine, but are less sensitive to sucrose octaacetate (34).

Chemicals

The taste stimuli, sucrose octaacetate, denatonium benzoate, strychnine HCI, and caffeine, as well as the reagents GTP.yS and cholera toxin, were purchased from Sigma Chemical Co. Pertus- sis toxin was purchased from List Biological, Campbell, CA. Antibodies to the a-subunit of various G-proteins were purchased from Vector Laboratories, Burlingame, CA. All other chemicals were reagent grade. All chemicals were used without additional purification.

Inositol Phosphate Assay

Mouse taste tissue taken from the foliate and circumvallate papillae (27), and lingual epithelial tissue removed from the area of the intermolar eminence (an area devoid of taste buds) were prelabeled with [3H] myoinositol and the tissue homogenate as- sayed for generation of inositol phosphates in the presence of four bitter stimuli. Circumvallate and foliate papillae and control tissues from 18-20 tongues were removed (27), placed in mod- ified Krebs-Henseleit buffer (KHB), pH 7.4 [containing (mM): NaCI 118, KCI 4.7, CaCI2 1.3, KH2PO4 1.2, MgCI2 1.2, NaHCO3 25, glucose 11.7, Na pyruvate 1], and incubated for 2 h at 37°C in the presence of [3H]myoinositol (NEN DuPont, Wilmington, DE). During the last 10 min of the incubation, the tissue was exposed to 10 mM LiCl-containing KHB. The tissue was washed in ice cold divalent cation-free KHB (Ca- and Mg-chloride were replaced with 1.2 mM each of EDTA and EGTA), disrupted in a glass/glass tissue homogenizer and centrifuged at 1,000 × g for 20 min at 4°(2. The supernatant (10-12 #g protein/assay tube) was used to monitor phospholipase C activity by exposing it to various agonists for 30 s. The standard assay mixture contained (mM): MOPS 25, pH 7.1, MgCI2 3, KC180, CaCI2 0.6 and EGTA 2 (giving a calculated free Ca 2+ concentration of about 50 nM,

which is approximately the resting level of free Ca 2+ in taste cells (1)). The reaction was terminated by addition of 10% TCA, fol- lowed by 0.5% BSA to facilitate handling of the precipitate. The tubes were placed on ice for l0 min, then centrifuged at 1,000 × g for 5 min at 4°(2. The supernatant contained water-soluble inositol phosphate isomers. Over 85% of the total inositol phosphates eluted in the IP3 fraction (1,4,5- and 1,3,4-trisphosphate) when separated by anion exchange chromatography (9).

Western Blotting

Membrane preparations from 10 circumvallate and 20 foliate papillae were collected (27) and homogenized in KHB, centrifuged at 1,000 × g for 10 min. The supernatant was centrifuged at 48,000 × g for 30 min, and the pellet was washed and centrifuged a second time under the same conditions. The final pellet was redissolved and separated on a 12.5% SDS polyacrylamide gel (17). Proteins were transferred to nitrocellulose membranes (31), and detected by incubating for 30 min at 22°(2 with antiserum to Gotil.2, Gai3 and Gao, or normal rabbit serum (control) at 1:1,000 dilution, by the avidin-biotin technique according to the manufacturer's recommendations (Vector Laboratory, Bur- lingame, CA).

RESULTS AND DISCUSSION

Total inositol phosphate (IP) production was increased 2.2- fold over basal levels when 10 ~M SOA was present in the assay mixture (Fig. 1). Ten mM caffeine induced a 480% increase, while in the presence of 10/~M denatonium and 100 #M strychnine, increases of 525% and 710%, respectively, were observed (Fig. 1). Tissue devoid of taste buds showed no significant changes in levels of total IP with or without the presence of the taste stimuli. For control tissue devoid of taste buds derived from the B6.SW strain, total IP levels (dpm/mg protein) were: basal-- 870 _ 632 (mean ± SE); SOA--1087 ± 658 (mean ± SE); CAF--1100 (average of 2 experimental values); DEN - 1100 (average of 2 experimental values); STR--1450 (average of 2 experimental values). These data suggest that the inositol phosphates, probably inositol 1,4,5-trisphosphate, may play a direct role in bitter taste transduction.

Since many intracellular signalling mechanisms, including receptor activation of phospholipase C to produce IP3, are associated with GTP-binding regulatory proteins, we examined the in- fluence of GTP-yS on SOA-induced generation of total IP. Addition of 10 /~M GTP3,S, a nonhydrolizable GTP analog, caused a 220% increase in inositol phosphate production over basal levels (Fig. 2). This increase was similar to that elicited by SOA alone. However, addition of both SOA and GTP-yS resulted in a 580% increase. The differences between this value and basal level or the level due to SOA-stimulation were statistically significant (p < 0.008 and p < 0.02, respectively). (Control tissue devoid of taste buds from B6.SW strain gave the following re- suits (dpm/mg protein, mean ___ S.E.): BAS--870 ± 632; S O A - - 1087 ± 658; SOA + GTPTS-1266 ± 1040. These mean values are not significantly different from one another.) The sensitivity of the SOA response to GTP3,S in the taste tissue, and the fact that SOA is probably membrane impermeable, suggest that sucrose octaacetate stimulates the taste cell through a cell surface receptor coupled to a G-protein.

To investigate the nature of this GTP-binding protein, we tested the sensitivity of the IP response to pertussis (PTX) and cholera (CTX) toxins (Fig. 3). The response to simultaneous addition of SOA and GTP3,S was blocked when the tissue was preincubated in the presence of 7.5/zg/ml of PTX but not when preincubated in the presence of 25 #g/ml of CTX. These results

IP3 IN BITI'ER TASTE TRANSDUCTION 1151

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SIR FIG. 1, The effect of various bitter compounds on phosphatidylinositol turnover in mouse taste tissue. Synthesis of total inositol phosphates (IP) was monitored either without added stimuli (basal (BAS) activity) or in the presence of 10 #M sucrose octaacetate (SOA), 10 mm caffeine (CAF), 10 #M denatonium (DEN) or 100 #M strychnine (STR). Values for SOA and BAS are derived from 8-10 experiments from tissue of the B6.SW strain. Values for CAF, DEN and STR are derived from 4 -6 experiments performed in two different strains of mice, B6.SW and C57BL/6J. Stars indicate a significant difference from basal with p < 0.008. Significance levels were tested using the Wilcoxon paired t-test. IP levels are mean +_ S.E.

are consistent with a G-protein mediated bitter taste response of the G~ type.

To identify the presence of Ga-subunits in the mouse taste tissue, Western blotting of solubilized tissue proteins from both

strains of mice (B6.SW and C57BL/6J) was performed using antiserum specific to the a-subunits of Gil-2, Gi3 and Go. Western blotting revealed a strongly reactive band migrating at an apparent molecular mass of 40,000 Da for lanes probed for Ga, .2 and

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BAS SOA G FIG. 2. The effect of sucrose octaacetate on phosphatidylinositoi turnover in mouse (B6.SW) taste tissue homogenates. Total IP was measured in the absence (basal activity, BAS) or presence of 10 #M sucrose octaacetate (SOA), 10 #M GTPTS (G), or a combination of both (S + G) at the same concentrations. Each bar represents the mean +_ SE of 8-10 experiments performed in duplicates. SOA + GTPyS stimulated levels were significantly higher (*) compared with basal (p < 0.008) or SOA-stimulated levels. (p < 0.02).

1152 SPIELMAN ET AL.

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S+G S+G÷P $+G+C FIG. 3. The effect of bacterial toxins on SOA + GTP'yS-stimulated phosphoinositide turnover in mouse taste tissue (B6.SW strains). During the last 30 min of a 2 h incubation, taste papillae were exposed to either 7.5 #g/ mL of PTX (S+G+P) or 25 #g/mL of CTX (S+G+C). Subsequent steps were performed as described in the "Methods" section. Each bar represents the mean _+ SE of 3 duplicate experiments.

Gao in solubilized proteins of taste tissue from both strains of mice (Fig. 4). No reactivity was detected using antibodies against Goti3. While this assay demonstrates the presence of Get,_2 and Gao-type proteins in taste tissue, it does not directly identify the G-protein involved in SOA signal transduction.

Some bitter stimuli, such as caffeine, are cell permeant and could act through intracellular mechanisms similar to those described in skeletal muscle and neurons (16,20,33). Other bitter tasting compounds including denatonium, strychnine and sucrose octaacetate probably do not penetrate the cell membrane due to their hydrophilicity. This suggests that a transmembrane signalling mechanism, perhaps involving a cell surface receptor, is required.

Several G-proteins that activate phospholipase C (7,8,22) could be involved in IPs-mediated bitter taste transduction. Gotq was shown to specifically activate the f l l isozyme of phospholipase C (29). Although Gotq is PTX-insensitive in other systems (30), its involvement with the phosphatidylinositol pathway led us to investigate its presence in taste tissue by immunohistochemistry (23). The results of these immunohistochemistry studies demonstrated that Gotq is expressed in taste bud cells at levels comparable with GOtil. 2 (data not shown). In agreement with the Western blot data presented here, immunohistochemical activity to Goti 3 was not detected.

PTX-sensitive G-proteins have been implicated in activation of phospholipase C in other systems such as hippocampal long- term potentiation (10), neutrophils and HL60 cells (8,32). In the latter two cell types, this G-protein was identified as either Goti2 or Gai3 (8,32).

A novel G-protein, called gustducin, has recently been cloned from rat gustatory tissue (19). Based on the nucleotide sequence of this G-protein, it appears to have both PTX- and CTX-binding sites. In view of the predicted toxin sensitivity, gustducin is un- likely to be involved in SOA-taste mediation in the mouse strains used in these studies. However, this G-protein, being primarily

taste cell-specific, may participate in signal transduction of other bitter compounds or of compounds of other modalities.

BAS

FIG. 4. Western blotting of mouse taste tissue. Forty #g of membrane preparation from the circumvallate and foliate taste papillae of B6.SW and C57BL/6J mice were separated on a 12.5% SDS-polyacrylamide gel and Western blotted. Lane 1 contained molecular weight markers with apparent molecular mass (in kDa from top to bottom) 97; 58; 40; and 29. Lanes 2 and 3 contain tissue from B6.SW and C57BL/6J mice, respectively, probed for Ga~_2 using antibodies active to these G-protein a-subunits. Lanes 4 and 5 contain tissues from B6.SW and C57BId6J mice, respectively, probed for immunoreactivity to antibodies to the a- subunit of Gi3. Lanes 6 and 7 contain tissue from B6.SW and C57BId6J mice, respectively, probed for immunoreactivity to antibodies of the a- subunit of Go. Antibodies were at a dilution of 1:1000. Immunoreactivity is seen at about 40,000 Da in Lanes 2, 3, 6, and 7 (arrows). No reactivity to antibodies against the a-subunit of Gi3 was observed in these tissues.

IP3 IN BITTER TASTE TRANSDUCTION 1153

A M e c h a n i s m f o r B i t t e r T a s t e T r a n s d u c t l o n

IP K ÷ j

IP 4 C a 2÷ J

?

C a 2÷

Na ÷

FIG. 5. A mechanism for bitter taste transduction. Bitter taste stimuli (solid triangles) interact with a receptor (R) located on the apical membrane of the taste receptor cell. This binding interaction stimulates a G-protein (G) whose a- subunit then stimulates the enzyme phospholipase C (PLC). This enzyme catabolizes the membrane-bound lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to form the two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The messenger DAG stimulates the enzyme protein kinase C (PKC) which phosphorylates available sites. These phosphorlyations may be on the receptor, to downregulate it, or on ion channels, to activate or inhibit them. Activation of sodium or calcium channels and inhibition of potassium channels will lead to depolarization. Continued depolarization activates voltage-gated ion channels. The messenger molecule IP~ may release calcium from intracellular stores. This calcium could activate calcium-regulated calcium channels and also further activate PLC, a calcium regulated enzyme, leading to the production of additional second messenger. The IP3 may be metabolized to the inactive form, IP2, or phosphorylated to the active form, IP4. The latter messenger may gate addition ion channels, either on intracellular organelles or the plasma membrane. The result of these activities is to depolarize the cell to induce a sufficient rise in intracellular calcium ion activity for neurotransmitter to be released.

Based on the toxin sensitivity of our IP3 assay, we can elim- inate gustducin and Gaq as being involved in SOA signal transduction. Furthermore, lack of Goti3 in mouse taste tissue (Fig. 4) (23) allows us to propose that the G-protein involved in SOA taste transduction is either Ga~j or Gai2. The results of this study suggest that transduction of bitter compounds, such as sucrose octaacetate, may be mediated through a cell surface receptor coupled to the phosphoinosi t ide pathway through a PTX-sensit ive, CTX-insensi t ive Gi-type protein.

A Model of Bitter Taste Transduction

The structural and physical diversity of bitter compounds (5) raises a challenging question: Can there be a single transduction pathway for all bitter compounds? We recently proposed that several different mechanisms may be necessary to mediate re- sponses to all bit ter stimuli (25). The diversity of bitter sensing mechanisms may be an evolutionary response to the myriad of bitter and potentially toxic compounds in the plant and animal kingdom. One such mechanism to detect these could be the one described in this study. The consequences of IP3 and DA G production in other systems are consistent with their potential role as mediators of bitter taste transduction, in particular, the acti-

vation/inactivation of ion channels and the ability to increase intracellular calcium ion activity.

We had earlier shown (28) that isolated taste cells from the circumvallate region of the mouse tongue possess voltage-activated outward currents. Approximately half of these cells also contained rapidly inactivating voltage-dependent inward currents. Based on kinetics and voltage-dependence, the outward currents were identified as delayed rectifier potassium currents. Application of 10 # M denatonium benzoate to these cells resulted in a strong depression of the vol tage-dependent outward currents. Whether or not this block was due to a direct action of denatonium on the cel l ' s potassium channels or to denatonium-induced changes in intracellular levels of IP3 and DAG, leading ultimately to block of the outward current, was not apparent.

A number of known potassium channel blockers are also bitter tasting, indicating that the ability of the compounds to directly block different types of potassium channels may be an important mechanism of bitter taste transduction (25). The closure of potassium channels seems to be a common feature for taste cells being stimulated by a varietY of taste active compounds (14,15).

Our studies and those of others lead us to propose a model for the involvement of the polyphosphoinosi tol signalling pathway in the transduction of bitter stimuli (Fig. 5). This model

1154 SPIELMAN ET AI..

assumes the presence of receptors of as yet unidentified character. These receptors are linked with a G-protein, possibly a Gail or a Ga,2 type. When stimuli bind to the receptor, the a-subunit of the G-protein activates a phospholipase C which then catabolizes membrane-associated PIP2 into the two second messengers, IP3 and DAG. IP3 could go on to release calcium from intracellular stores or could be further phosphorylated to IP4 (which in some cells is a stimulator of plasma membrane associated calcium channels (6,18)) allowing influx of calcium from extracellular spaces. DAG (as in many other cells) may activate the enzyme, protein kinase C, which can phosphorylate available sites. Some of these sites may be on ion channels which may then be activated or inhibited, others may be on the receptors themselves, causing downregulation of these. The activated ion channels may allow influx of positive charge, depolarizing the cell. Inhibition of outward-going potassium channels will block potassium leak- age, again leading to depolarization. The depolarizing event will activate voltage sensitive calcium channels while the IP3-induced release of intracellular calcium may also activate calcium-sensitive calcium channels and can also activate PLC and induce further release of sequestered calcium. These events will lead to sufficient increases in intracellular calcium ion activity, trigger- ing neurotransmitter release. The entire system relaxes by metabolism of the second messengers (as observed recently in an aquatic model of taste (11)), by dephosphorylation events, and by removal and/or sequestering of the surfeit of intracellular ions.

CONCLUSIONS

The great diversity of structures that all impart a sensation of bitterness (5) has often been used as one reason for hypothesizing a variety of bitter taste transduction mechanisms. Yet an analo- gous argument could be made for olfaction, where thousands of molecules impart thousands of odorous sensations. It is now hy-

pothesized that this diversity in sensation in olfaction is brought about by the action of hundreds of receptors, linked individually to perhaps only two or three different transduction schemes. Thus the diversity in olfaction is solved by a diversity at the receptor recognition step.

Could the same or similar mechanisms be used to detect the large number of bitter tasting stimuli? To a degree, perhaps so. But bitter taste is imparted by chemicals of a broader structural range than those stimulating the olfactory system. In the latter system, only volatile organic compounds are recognized as olfactory stimuli. For bitterness, on the other hand, the range of stimulus structures is truly vast: from simple inorganic salts such, as CsI and MgSO4, to large organic molecules, peptides and even proteins. Can diverse receptors coupled to one or two transduction mechanisms be designed to detect this great diversity? Or is it more likely that bitter stimuli will interact with a very large number of transduction mechanisms (25)? Many bitter stimuli are known to interact with ion channels, G-proteins, second messenger metabolizing enzymes, etc., in cells other than taste cells. They may do so in taste cells as well. It is possible that these interactions may give rise to sufficient metabolic and ionic changes in the taste cells to lead to neurotransmitter release. The studies presented here implicate the lipid-derived second messengers, IP3 and DAG, as being involved in one of the mechanisms for bitter taste transduction.

ACKNOWLEDGEMENTS

We thank Dr. Richard Mattes for statistical analyses, Gloria Turner, Maged Ayad and Steve DeMyer for technical help, Drs. D. Lynn Kali- noski, John Teeter and Diego Restrepo for reviewing this manuscript and Dr. David Manning for helpful discussions. Work in the authors' laboratories was supported by funds from NIH, New York University and the Veterans Affairs Department.

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Generation of inositol phosphates in bitter taste transduction