Why Taste Is Pharmacology.

The chapter presents an argument supporting the view that taste, defined as the receptor-mediated signaling of taste cells and consequent sensory events, is proper subject matter for the field of pharmacology. The argument develops through a consideration of how the field of pharmacology itself is to be defined. Though its application toward the discovery and development of therapeutics is of obvious value, pharmacology nevertheless is a basic science committed to examining biological phenomena controlled by the selective interactions between chemicals - regardless of their sources or uses - and receptors. The basic science of pharmacology is founded on the theory of receptor occupancy, detailed here in the context of taste. The discussion then will turn to consideration of the measurement of human taste and how well the results agree with the predictions of receptor theory.

A Pharmacological Perspective on the Study of Taste.

The study of taste has been guided throughout much of its history by the conceptual framework of psychophysics, where the focus was on quantification of the subjective experience of the taste sensations. By the mid-20th century, data from physiologic studies had accumulated sufficiently to assemble a model for the function of receptors that must mediate the initial stimulus of tastant molecules in contact with the tongue. But the study of taste as a receptor-mediated event did not gain momentum until decades later when the actual receptor proteins and attendant signaling mechanisms were identified and localized to the highly specialized taste-responsive cells of the tongue. With those discoveries a new opportunity to examine taste as a function of receptor activity has come into focus. Pharmacology is the science designed specifically for the experimental interrogation and quantitative characterization of receptor function at all levels of inquiry from molecules to behavior. This review covers the history of some of the major concepts that have shaped thinking and experimental approaches to taste, the seminal discoveries that have led to elucidation of receptors for taste, and how applying principles of receptor pharmacology can enhance understanding of the mechanisms of taste physiology and perception.

Rapid Throughput Concentration-Response Analysis of Human Taste Discrimination.

Human taste threshold measurements often are used to infer tastant receptor functionality. However, taste thresholds can be influenced by receptor-independent variables. Examination of the full range of taste-active concentrations by taste discrimination has been hampered by logistics of testing multiple concentrations in replicate with human subjects. We developed an automated rapid throughput operant methodology for taste discrimination and applied it to concentration-response analysis of human taste. Tastant solutions (200 µl) drawn from a 96-well plate and self-administered to the tongue served as discriminative stimuli for money-reinforced responses on a touch-sensitive display. Robust concentration-response functions for "basic taste" stimuli were established, with particular focus on agonists of the taste 1 receptor member 2-taste 1 receptor member 3 heterodimer receptor (TAS1R2/R3). With a training cue of 100 mM sucrose, EC50 values of 56, 79, and 310 µM and 40 mM were obtained for rebaudioside A, sucralose, acesulfame potassium, and sucrose, respectively. Changing the sucrose training cue to 300 mM had no impact, but changing to 30 mM resulted in slight leftward shifts in potencies. A signal detection method also was used to determine values of d', a probabilistic value for discriminability, which indicated that 5 mM was near the limits of detection for sucrose. With repeated testing, both EC50 values and 5 mM sucrose d' values were established for each individual subject. The results showed little correspondence between threshold sensitivities and EC50 values for sucrose. We conclude that concentration-response analysis of taste discrimination provides a more reliable means of inferring receptor function than measurement of discriminability at the lowest detectable tastant concentrations. SIGNIFICANCE STATEMENT: Many inferences about human tastant receptor functionality have been made from taste threshold measurements, which can be influenced by variables unrelated to receptors. We herein report a new methodology that enables rigorous concentration-response analysis of human taste discrimination and its use toward quantitative characterization of tastant agonist activity. Our data suggest that taste discrimination concentration-response functions are a more reliable reflection of underlying receptor activity than threshold measures obtained at the lowest detectable tastant concentrations.

Pharmacologic antagonism of the oral aversive taste-directed response to capsaicin in a mouse brief access taste aversion assay.

Chemosensory signaling by the tongue is a primary determinant of ingestive behavior and is mediated by specific interactions between tastant molecules and G protein-coupled and ion channel receptors. The functional relationship between tastant and receptor should be amenable to pharmacologic methods and manipulation. We have performed a pharmacologic characterization of the taste-directed licking of mice presented with solutions of capsaicin and other transient receptor potential vanilloid-1 (TRPV1) agonists using a brief access taste aversion assay. Dose-response functions for lick-rate suppression were established for capsaicin (EC(50) = 0.5 microM), piperine (EC(50) = 2 muM), and resiniferatoxin (EC(50) = 0.02 microM). Little or no effect on lick rate was observed in response to the full TRPV1 agonist olvanil. Capsaicin lick rates of wild-type and transient receptor potential melastatin-5 (TRPM5) knockout mice were equivalent, indicating that TRPM5, a critical component of aversive signaling for many bitter tastants, did not contribute to the capsaicin taste response. The selective TRPV1 antagonists N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (10 microM) and (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (AMG9810) (10 microM) effectively blocked capsaicin- and piperine-mediated lick suppression. However, (E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)-N-phenylprop-2-enamide (SB 366791) and capsazepine, also TRPV1 antagonists, were without effect at test concentrations of up to 30 and 100 microM, respectively. Our results demonstrate that TRPV1-mediated oral aversiveness presents a pharmacologic profile differing from what has been reported previously for TRPV1 pain signaling and, furthermore, that aversive tastes can be evaluated and controlled pharmacologically.

Avian and Human Homologues of the P2Y1 Receptor: Pharmacological, Signaling, and Molecular Properties.

[Table: see text] The P2Y receptor on turkey erythrocyte membranes was the first P2 receptor to be shown to activate phospholipase C (PLC) in a strictly guanine nucleotide-dependent manner and remains the only G protein-coupled P2 receptor for which G protein-coupling kinetics have been defined. This membrane receptor has provided a model system for detailed pharmacological analyses of a series of chain-extended 2-thioether derivatives of adenine nucleotides that exhibit remarkable selectivity and potency for P2Y receptors. This model system also has led recently to identification of a novel series of P2 receptor antagonists. The turkey erythrocyte receptor is the species homologue of the chick P2Y1 receptor originally cloned by Webb and coworkers [Webb et al., 1993]. We also have cloned the human homologue of the P2Y1 receptor, which exhibits identical pharmacological and second messenger signaling properties to that of the avian P2Y1 receptor.

TRP channels as targets for therapeutic intervention in obesity: focus on TRPV1 and TRPM5.

The disease of obesity is one of the greatest healthcare challenges of our time. The increasing urgency for effective treatment is driving an intensive search for new targets for anti-obesity drug discovery. The TRP channel super family represents a class of proteins now recognized to serve many functions in physiology related to maintenance of health and the development of diseases. A few of these might offer new potential for therapeutic intervention in obesity. Among the TRP channels, TRPV1 appears most closely associated with body weight homeostasis through its influence on energy expenditure. TRPM5 has been thoroughly characterized as a critical component of taste signaling and recently has been implicated in insulin release. Because of its role in taste signaling, we argue that drugs designed to modulate TRPM5 could be useful in controlling energy consumption by impacting taste sensory signals. As drug targets for obesity, both TRPV1 and TRPM5 offer the advantage of operating in compartments that could limit drug distribution to the site of action. The potential for other TRP channels as anti-obesity drug targets also is discussed.

A generalized model and high throughput data analysis system for functional modulation of receptor-agonist systems suitable for use in drug discovery.

Positive allosteric modulators (PAMs) of receptors represent a class of pharmacologic agents having the desirable property of acting only in the presence of cognate ligands. Discovery and optimization of the structure activity relationships of PAMs is complicated by the requirement of a second ligand to manifest their action, and by the need to quantify both affinity and intrinsic efficacy. Multivariate regression analysis is a statistical method capable of simultaneously obtaining affinity and intrinsic efficacy parameters from curve fits of multiple agonist dose-response functions generated in the presence of varying concentrations of PAMs. Capitalizing on the advantages of multivariate regression analysis for PAM optimization requires a theoretical framework and a system that facilitates efficient flow of information from data generation through data analysis, storage, and retrieval. We describe here the experimental design, mathematical model and informatics workflow enabling a multivariate regression approach for rapidly obtaining affinity and intrinsic efficacy values for PAMs in a drug discovery setting.

A high throughput in vivo assay for taste quality and palatability.

Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli. Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity. Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints. We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm. Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor. As a rat's tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser. The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever. Each disruption of the laser was counted as a lick. Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy. Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness. All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue. The technology is capable of generating multiple concentration-response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.

Triphenylphosphine oxide is a potent and selective inhibitor of the transient receptor potential melastatin-5 ion channel.

Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic ß-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC50 = 12 µM) and murine TRPM5 (IC50 = 30 µM) heterologously expressed in HEK293 cells, but had no effect (up to 100 µM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)⁻ mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.

Quantitative assessment of TRPM5-dependent oral aversiveness of pharmaceuticals using a mouse brief-access taste aversion assay.

Many orally administered pharmaceuticals are regarded by humans as aversive, most often described as 'bitter'. Taste aversiveness often leads to patient noncompliance and reduced treatment effectiveness. 'Bitter' taste is mediated by T2R G-protein coupled receptors through a peripheral signaling pathway critically dependent upon function of the TRPM5 ion channel. The brief-access taste aversion (BATA) assay operationally defines aversive taste as suppression of the rate at which a rodent licks from sipper tubes that deliver tastant solutions or suspensions. We have used a mouse BATA assay for rapid quantification of oral aversiveness from a set of 20 active pharmaceutical ingredients (APIs). Robust lick-rate dose-response functions were obtained from both C57BL/6J wild type (WT) and C57BL/6J/TRPM5-/- (TRPM5 knockout) mouse strains, generating reliable determinations of potency and relative maximal oral aversiveness for each API. A subset of APIs was also evaluated in a human bitterness assessment test; effective concentrations for half-maximum responses (EC50s) from both the human test and WT mouse BATA were equivalent. Relative to WT potencies, EC50s from TRPM5 knockout mice were right-shifted more than 10-fold for most APIs. However, APIs were identified for which EC50s were essentially identical in both mouse strains, indicating a TRPM5-independent alternative aversive pathway. Our results suggest the BATA assay will facilitate formulation strategies and taste assessment of late development-phase APIs.

The pharmacology and signaling of bitter, sweet, and umami taste sensing.

Over the last decade, many of the molecular components that mediate the transduction of taste signaling have been elucidated. The chemosensory receptors for taste have been identified as G protein-coupled receptors (GPCRs) and ion channels that are expressed on the surface of highly specialized taste sensory cells. Tastant molecules act as agonists, binding to and stabilizing active conformations of receptors, resulting in the initiation of signal transduction cascades. Taste signaling, therefore, should be amenable to the methods of pharmacology. This review focuses on the GPCR-mediated signaling of bitter, sweet, and umami tastes and emphasizes the opportunities for pharmacologic evaluation.

Pharmacological analysis of calcium responses mediated by the human A3 adenosine receptor in monocyte-derived dendritic cells and recombinant cells.

Extensive characterization of adenosine receptors expressed by human monocyte-derived dendritic cells (MDDCs) was performed with quantitative polymerase chain reaction, radioligand binding, and calcium signaling. Transcript for the A3 adenosine receptor was elevated more than 100-fold in immature MDDCs compared with monocyte precursors. A3 receptor transcript was substantially diminished, and A2A receptor transcript increased, by lipopolysaccharide maturation of MDDCs. Saturation binding of N(6)-(3-[(125)I]iodo-4-aminobenzyl)-adenosine-5'-N-methyluronamide ([(125)I]AB-MECA) to membranes from immature MDDCs yielded B(max) of 298 fmol/mg of protein and K(D) of 0.7 nM. Competition against [(125)I]AB-MECA binding confirmed the site to be the A3 receptor. Adenosine elicited pertussis toxin-sensitive calcium responses with EC(50) values ranging as low as 2 nM. The order of potency for related agonists was N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) >/= I-AB-MECA > 2Cl-IB-MECA >/= adenosine > 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxyamidoadenosine (CGS21680). The order of efficacy was adenosine >/= CGS21680 > IB-MECA >/= I-AB-MECA > 2Cl-IB-MECA. Calcium responses to 2Cl-IB-MECA and CGS21680, and the lower range of adenosine concentrations, were completely blocked by 10 nM N-(2-methoxyphenyl)-N-[2-(3-pyridyl)quinazolin-4-yl]urea (VUF5574) but not by 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) or 8-cyclopentyl-1,3-dipropylxanthine. Pretreatment with 100 nM 2Cl-IB-MECA eliminated responses to CGS21680 but not to monocyte inhibitory protein-1alpha. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Galphaq-i3 protein, which was required to establish A3-mediated calcium signaling. The pharmacological profile of calcium signaling elicited by adenosine-related agonists in the double-recombinant cells was essentially identical to that obtained from immature MDDCs. Our results provide an extensive analysis of A3-mediated calcium signaling and unequivocally identify immature MDDCs as native expressers of the human A3 receptor.