TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL. 125, 2014
BITTER TASTE RECEPTORS IN THE WRONG PLACE: NOVEL AIRWAY SMOOTH MUSCLE TARGETS FOR TREATING ASTHMA STEPHEN B. LIGGETT, MD TAMPA, FLORIDA
ABSTRACT There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than -agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca2⫹-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by -agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.
INTRODUCTION: BITTER TASTE RECEPTORS Bitter taste receptors (TAS2Rs) have been traditionally thought to be expressed exclusively on taste buds, where they respond to a wide variety of bitter substances. There are 25 TAS2R subtypes, which typically respond with low affinity to tens of thousands of compounds (1). They are thought to have evolved as an aversion mechanism for avoidance of toxins in plants. We have found TAS2Rs expressed on the cell surface of human airway smooth muscle (ASM) cells, where they act to markedly relax the airway (2, 3). It is proposed that these Correspondence and reprint requests: Stephen B. Liggett, MD, Departments of Internal Medicine and Molecular Pharmacology and Physiology, University of South Florida, Morsani College of Medicine, 12901 Bruce B. Downs Blvd., MDC02, Tampa, FL 33612, Tel: 813-9747715, Fax: 813-974-3886, E-mail: [email protected]. Potential Conflicts of Interest: Dr Ligget is an Amgen consultant.
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receptors represent novel targets for bronchodilators in the treatment of asthma and chronic obstructive lung disease. WHY DO WE NEED “ANOTHER” BRONCHODILATOR? Currently, agents that have the potential to bronchodilate (or block bronchoconstriction) can be classified as direct or indirect bronchodilators. Because the bronchoconstriction in asthma is due to the release of local mediators such as histamine, acetylcholine, and leukotrienes, antagonists to these spasmogens are somewhat effective in preventing bronchoconstriction, but are dependent on whether a particular patient is “driven” by a given pathway. Additionally, they are specific to only one pathway. Direct bronchodilators, on the other hand, act to relax ASM and dilate regardless of the spasmogen. Currently, there is only one class of direct bronchodilators for treating obstructive lung disease, which are the -agonists. Although these agents have various properties, such as duration of action or degree of activity, the mechanism of action is the same: an increase in intracellular cyclic adenosine monophosphate which acts via protein kinase A to directly relax the muscle. It is well recognized that asthma treatment is currently in need of additional therapeutic approaches (4), with approximately 50% of asthmatics having less than optimal control when using the current available agents including inhaled corticosteroids and long acting -agonists (5– 8). -agonists are also associated with extensive interindividual variability of the clinical response, tachyphylaxis, bronchial hyperactivity, and increased adverse events (7, 9 –14). TAS2R SIGNALING As shown in Figure 1, TAS2Rs are G-protein coupled receptors that are integrated into the cell membrane, where extracellular agonists activate the receptor which evokes intracellular events. The G-protein for TAS2Rs is gustducin, and upon receptor activation the heterotrimer dissociates into ␣, and the ␥, subunits. In taste buds, the ␥ subunit carries out signaling via coupling to and activating phospholipase C (PLC), which results in an increase in intracellular inositol-1, 4, 5 triphosphate (IP3). IP3 then binds to the IP3 receptor on the endoplasmic reticulum which promotes movement of Ca2⫹ from this storage compartment to the intracellular space. As indicated in Figure 1, we also find the same signaling pathway up to this point in human and mouse ASM. However, the fate of the intracellular Ca2⫹ ([Ca2⫹]i) is different in ASM compared to taste buds. In
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taste buds, [Ca2⫹]i activates a transient receptor potential (TRP) channel, which depolarizes the membrane, causing secretion of neurotransmitter which activates the type III cell of the taste bud that communicates via nerves to the brain (left of the green line in Figure 1). In ASM, we find that [Ca2⫹]i activates one or more channels leading to hyperpolarization of the membrane (3), which relaxes the muscle (right of the dashed blue line). In ASM there is no involvement of a second cell type or a central nervous system connection, nor is there activation of the depolarizing TRP channel. This latter point is important, as some groups have used the presence/absence of a TRP channel as evidence for or against expression of bitter taste receptors in a given cell (15). We have clearly shown that there is a divergence of signaling in these two cell types, so TRP channel identification is not adequate for identification of TAS2Rs, particularly in non-gustatory tissues or cells.
nerves
Type III cell in taste bud
Bitter substances
Ggust
βγ βγ
neurotransmitter release Ca2+ TRP channel
BKCa or Other
FIG. 1. Overall signaling cascade of TAS2R in taste bud cells and human airway smooth muscle. To the left of the dashed green line is the pathway in the type 2 cell of the taste bud. To the right of the blue line is the pathway in human airway smooth muscle cells. The portion between these two lines is common between the two cell-types.
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RESULTS TAS2RS on Human ASM Quantitative reverse transcription polymerase chain reaction (RTPCR) revealed six TAS2Rs with high (subtypes 10, 14, 31) and moderate (subtypes 5, 4, 19) expression in normal primary cultured human ASM cells (3). In initial studies with various known bitter compounds, human ASM cells displayed an increase in [Ca2⫹]i in a dose-dependent manner (Figures 2A, 2B, and 2C). The effect was blocked by the PLC inhibitor U73122, the ␥ inhibitor gallein, and partially blocked by the IP3 receptor antagonist 2APB (Figure 2D). In intact isolated mouse (Figure 3A) and human (Figure 3B) airways studied in the ex vivo myograph system, TAS2R agonists relaxed acetylcholine- and serotonin-mediated contracted airways in a dose-dependent fashion (3). These relaxation effects were also studied using magnetic twisting cytometry, a method that ascertains single cell mechanics in isolated cells (16, 17). In this way, communication from the bronchial epithelium or other cells of the airway is not possible, and quantitative measurements of ASM mechanics can be made. As shown in Figure 4, the bitter tastants chloroquine and saccharin decreased intrinsic cell
FIG. 2. TAS2R agonists promoted increases in [Ca2⫹]i. (A) Dose response to the bitter tastant saccharin. (B) Dose response to the bitter tastant chloroquine. (C) Maximal responses to the indicated agents (n ⫽ 3– 8 experiments). (D) Effects of various inhibitors (see text) on saccharin-promoted [Ca2⫹]i (n ⫽ 3 experiments).
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FIG. 3. TAS2R agonists relax mouse and human airways ex vivo. (A) Response to the bitter tastants denatonium, chloroquine, and quinine in mouse airways (n ⫽ 6 – 8 experiments). (B) Relaxation to the -agonist isoproterenol and the bitter tastants chloroquine and quinine in human airways (n ⫽ 4 experiments).
FIG. 4. Isolated airway smooth muscle mechanics ascertained by magnetic twisting cytometry. (A) Response to the known relaxant isoproterenol and the known contractor histamine, and the bitter tastant saccharin. (B) The relaxation effect of saccharin is blocked by the PLC inhibitor U73112, but not the PKA inhibitor H89. Ca2⫹-dependent K⫹ channel blockers IbTx and ChTx partially block relaxation. Results are from a representative experiment.
stiffness (3). This relaxation was blocked by the aforementioned PLC inhibitor, which ties together the biochemical results with the physiological results. That an increase in [Ca2⫹]i was associated with relaxation was unexpected because virtually all agents that increase [Ca2⫹]i in ASM cause contraction (18). However, depleting the sarco(endo) plasmic reticulum of Ca2⫹ with thapsigargin eliminated the relaxation effect of TAS2R agonists, further strengthening the cause-and-effect between [Ca2⫹]i elevation and relaxation (3). Consistent with the relaxation effect of TAS2R agonists, they were found to cause hyperpolarization of the cell membrane (Figure 5). One mechanism as to how
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FIG. 5. TAS2R agonists hyperpolarize the membrane of human airway smooth muscle. Results are from a representative experiment using 1 mM saccharin and chloroquine, and 100 M histamine.
an increase in [Ca2⫹]i relaxes ASM that we identified is via the large conductance Ca-dependent K⫹ channel (BKCa). A specific BKCa inhibitor, iberiotoxin, caused a partial (but not complete) loss of the hyperpolarizing and relaxation effects of chloroquine. Others have suggested (19) the lack of involvement of BKCa, but in our hands we consistently observe partial effects with iberiotoxin (19); therefore, we currently believe that BKCa, in conjunction with other hyperpolarizing membrane channels, are responsible for the relaxation effect from the increased [Ca2⫹]i due to TAS2R agonism. IMAGING Ca2ⴙ SIGNALS FROM TAS2Rs We have recognized that measuring [Ca2⫹]i in whole cells might obscure subcellular differences between [Ca2⫹]i increases from receptors that cause contraction (such as the H2-histamine) and the TAS2Rs, which also increase [Ca2⫹]i but relax ASM. This was explored using FLUO-3 loaded human ASM and scanning two-dimensional confocal images every 0.22 m at 0.5-second intervals (3). Qualitative differences between saccharin and histamine [Ca2⫹]i images are readily apparent. For saccharin, Ca2⫹ events were observed as early as 2.5 seconds after exposure to the drug, whereas at least 10 seconds were required for histamine. The saccharin signal begins, and is the most intense, on the slender ends and sarcolemmal regions of the cell, whereas histamine caused a less localized, more global, response.
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These preliminary studies suggest that the bronchodilating [Ca2⫹]i signals from TAS2R are in a specialized or confined compartment, which may provide for coupling to a hyperpolarizing channel. TAS2R FUNCTION IN ASTHMA We sensitized BALB/c mice with subcutaneous ovalbumin and challenged them with inhaled albumin which resulted in eosinophilic inflammation and hyperreactivity to inhaled methacholine in the sedated intubated mouse model (3). Figure 6 shows the results of airway resistance measurements in sham and ovalbumin mice. Methacholine caused the expected increase in airway resistance, which was partially reversed by the -agonist albuterol and the TAS2R agonist quinine, in the sham (control) mice. In the ovalbumin sensitized and challenged mice, quinine remained effective in decreasing methacholine-promoted increase in resistance. However, albuterol was much less effective in this inflammatory model of asthma in reversing the contraction by methacholine. Taken together, we conclude that TAS2R function is not perturbed in this model, whereas -agonist is less effective. This suggests that TAS2R agonists may be effective, perhaps even more effective than -agonists, in treating airway obstruction from bronchospasm. To further assess this issue, human ASM cells from normal individuals and from asthmatics were studied for expression of TAS2R mRNA and functional responses (20). TAS2R 10, 14, and 31 transcript levels
FIG. 6. TAS2R function is not altered in a mouse model of asthma. Mice were sedated, intubated, and ventilated and received the indicated drugs by inhalation. (A) Responses in sham (control) mice. (B) Response in ovalbumin sensitized and challenged mice.
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were not different in nonasthmatic versus asthmatic human ASM. Functional studies with subtype-specific agonists showed no loss of TAS2R function in the latter cells. Figure 7A shows a representative experiment, and Figure 7B shows mean results from studies with chloroquine (TAS2R10), diphenhydramine and flufenamic acid (TAS2R14), saccharin (TAS2R31) and quinine (TAS2R10, 14 and 31). Additional studies were performed using live precision cut lung slices (PCLS) of nonasthmatic lungs (20). Here, the area of the bronchi within a PCLS is measured in response to contracting or relaxing drugs. To mimic an asthmatic phenotype, some PCLS were treated with interleukin-13 (IL-13) for 12 hours. Slices were exposed to carbachol to contract, and then either quinine or the -agonist formoterol. Figure 8A shows the previously reported loss of -agonist responsiveness with the IL-13 exposure. In contrast, TAS2R function remains intact, with identical dose-response curves for quinine (Figure 8B). We have also examined TAS2R function under two scenarios often encountered in the treatment of obstructive lung diseases. First, we have shown that TAS2R function remains intact under conditions of extreme 2AR desensitization due to chronic -agonist exposure (21). This suggests little, if any, “cross-talk” between the two pathways. Secondly, we have found minimal TAS2R desensitization due to bitter tastant exposure (20). This suggests that unlike 2-agonists, TAS2R agonists may show little tachyphylaxis during repetitive dosing.
FIG. 7. TAS2R function is not altered in human ASM cells derived from nonasthmatic and asthmatic subjects. The agonists are shown with the TAS2R subtype to which they couple. (A) results from a representative experiment, (B) results from six experiments.
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FIG. 8. TAS2R function is not altered in the IL-13 model of asthma. Precision cut lung slices were derived from a nonasthmatic lung and airway diameter measured. Some slices were pretreated with IL-13 for 12 hours before the study. The response to the -agonist formoterol (A) was depressed with IL-13 treatment, whereas the response to quinine (B) was unaffected (N ⫽ 6, *, P ⬍ .02).
CONCLUSIONS TAS2Rs expressed on ASM have a promising pharmacologic profile as novel bronchodilators for treating obstructive lung disease. TAS2R agonists relax ASM by mechanisms that are distinctly different than
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those of the -agonists, thus they are not “look alike” drugs. This would expand the potential armamentarium of drugs for treatment of asthma and chronic obstructive lung disease. This increase in options should lead to improved outcomes. There are tens of thousands of known bitter compounds that are TAS2R agonists. These include natural compounds, derivatives of natural compounds, and synthetic agents used in treating other diseases. This presents an ideal setting for high-throughput screening and medicinal chemistry to provide new compounds for human studies. ACKNOWLEDGMENTS Funded by the National Institutes of Health grants HL114471, HL045967, and HL071609. The author thanks Charmaine Disimile for manuscript preparation.
REFERENCES 1. Meyerhof W, Batram C, Kuhn C, et al. The molecular receptive ranges of human TAS2R bitter taste receptors. ChemSenses 2010;35:157–70. 2. Deshpande DA, Robinett KS, Wang WC, Sham JS, An SS, Liggett SB. Bronchodilator activity of bitter tastants in human tissue. Nat Med 2011;17:776 – 8. 3. Deshpande DA, Wang WC, McIlmoyle EL, et al. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction. Nat Med 2010;16:1299 –304. 4. Gerthoffer WT, Solway J, Camoretti-Mercado B. Emerging targets for novel therapy of asthma. Curr Opin Pharmacol 2013;13:324 –30. 5. World Health Report 2002—reducing risks, promoting healthy life. Available at: http://wwwwhoint/whr/2002/en/; 2002. 6. National Health Interview Survey Public Use Data File. Available at: http:// wwwcdcgov/NCHS/nhis/nhis_2007_data_releasehtm; 2007. 7. Drazen JM, Silverman EK, Lee TH. Heterogeneity of therapeutic responses in asthma. Br Med Bull 2000;56:1054 –70. 8. Malmstrom K, Rodriguez-Gomez G, Guerra J, et al. Oral montelukast, inhaled beclomethasone, and placebo for chronic asthma. A randomized, controlled trial. Montelukast/Beclomethasone Study Group. Ann Intern Med 1999;130:487–95. 9. Beasley R, Pearce N, Crane J, Burgess C. Beta-agonists: what is the evidence that their use increases the risk of asthma morbidity and mortality? J Allergy Clin Immunol 1999;103:S18 –S30. 10. Cheung D, Timmers MC, Zwinderman AH, Bel EH, Dijkman JH, Sterk PJ. Longterm effects of a long acting b2-adrenoceptor agonist, salmeterol, on airway hyperresponsiveness in patients with mild asthma. N Engl J Med 1992;327:1198 – 203. 11. Kraan J, Koeter GH, van der Mark TW, Sluiter HJ, De Vries K. Changes in bronchial hyperreactivity induced by 4 weeks of treatment with antiasthmatic drugs in patients with allergic asthma: a comparison between budesonide and terbutaline. J Allergy Clin Immunol 1985;76:628 –36. 12. Lipworth BJ. Airway subsensitivity with long-acting beta 2-agonists. Is there cause for concern? Drug Saf 1997;16:295–308.
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13. Salpeter SR, Wall AJ, Buckley NS. Long-acting beta-agonists with and without inhaled corticosteroids and catastrophic asthma events. Am J Med 2010;123:322– 8. 14. Sears MR, Taylor DR. The b2-agonist controversy: observations, explanations and relationship to asthma epidemiology. Drug Saf 1994;11:259 – 83. 15. Tizzano M, Cristofoletti M, Sbarbati A, Finger TE. Expression of taste receptors in solitary chemosensory cells of rodent airways. BMC Pulm Med 2011;11. 16. An SS, Fabry B, Trepat X, Wang N, Fredberg JJ. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness? Am J Respir Cell Mol Biol 2006;35:55– 64. 17. An SS, Laudadio RE, Lai J, Rogers RA, Fredberg JJ. Stiffness changes in cultured airway smooth muscle cells. Am J Physiol Cell Physiol 2002;283:C792–C801. 18. Green SA, Liggett SB. G-protein-coupled receptor signaling in the lung. In: Liggett SB, Meyers DA, eds. The Genetics of Asthma. New York: Marcel Dekker, Inc.; 1996:67–90. 19. Zhang CH, Chen C, Lifshitz LM, Fogarty KE, Zhu MS, ZhuGe R. Activation of BK channels is not required for bitter tastant-induced bronchodilation. Nat Med 2012;18:648 –50. 20. Robinett KS, Deshpande DA, Malone MM, Liggett SB. Agonist-promoted homologous desensitization of human airway smooth muscle bitter taste receptors. Am J Respir Cell Mol Biol 2011;45:1069 –74. 21. An SS, Wang WC, Koziol-White CJ, et al. TAS2R activation promotes airway smooth muscle relaxation despite beta(2)-adrenergic receptor tachyphylaxis. Am J Physiol Lung Cell Mol Physiol 2012;303:L304 –11.
DISCUSSION Ausiello, Boston: As you know, the senses of taste and smell are pretty intimately associated, even with the very large overlap in terms of receptors. So that raises two questions. First of all, is it possible that you could narrow that 10,000 compounds by looking at those compounds that have taste but also are aerosolized, I suppose, in terms of having effect in terms of smooth muscle? And then secondly, have you looked at obvious noxious agents that stimulate these taste buds in terms of their capacity for smooth muscle modification of the airway? Liggett, Tampa: I didn’t quite understand the second part of your question; have I looked at obvious noxious stimuli? Ausiello, Boston: Are there any taste bud sensitizers that also have the capacity to be aerosolized and, in that sense, you have both taste and smell being activated? And could that narrow your capacity for looking at the numerous compounds that may be able to activate these. . . Liggett, Tampa: I have to answer that question in a couple ways. First of all, we do see a few smell receptors on airway smooth muscle. So, I mean, you know, there is a whole other pathway that we might be able to consider. And secondly, to clarify, we do think that if this becomes a drug, it’s going to have to be aerosolized. And most of these drugs can be put in an inhaler form, so we are not too concerned about that. Some other issues, though, would come about in terms of palatability. You are inhaling something that you’re going to taste as bitter. The advantage, we think, is that we only have to hit one of the three highest receptors to get bronchodilation, and we actually would want a very highly specific compound to try to do that. I’m not sure how to integrate the smell component to it, but we will have to make a palatable compound in the end. Rothman, Baltimore: I really enjoyed your talk. Can you hypothesize a little bit
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about the normal ligand for these receptors, and the role of these receptors in normal lung physiology? Liggett, Tampa: So that question comes up every time, and it’s a great question. So we have found that gram-negative bacteria secrete a bitter taste receptor ligand that activates bitter taste receptors. So what we are thinking — this is just a hypothesis — that the receptors evolved so that you don’t get a socked-in pneumonia. So as you begin to have a severe bronchitis, if there is bronchodilation to help clear the cellular debris and the bacteria. That’s the closest we have been able to come so far. Hochberg, Baltimore: Two-part question, I guess. The first goes back to the mouse model. Did you try to administer these agents parenterally as opposed to directly into the lung? Liggett, Tampa: No. Hochberg, Baltimore: So I guess the second part is, would you expect that if these were administered parenterally, you would see a similar effect as you saw with administration directly? Liggett, Tampa: Well, it looks like with the agents we had available — quinine, chloroquine, et cetera — you have to reach, you know, high micromolar concentrations in order to activate these receptors. And you just couldn’t do that in the blood if you took the pills. I mean, if you took that much chloroquine, you’d have all the toxic effects from chloroquine. So our feeling is that we are going to have to come up with a compound through this screening technique that has a higher affinity for these agents, and then maybe we could take them parenterally, orally. I mean, wouldn’t it be great to have a highly effective oral bronchodilator? It would be great. Hochberg, Baltimore: Well, there are a lot of sort of off-target effects of hydroxychloroquine, which was used in the treatment of rheumatoid arthritis, which have been recently reviewed by Nancy Olson, who is now, I guess, at Penn State Hershey, and I’m not aware that she included in her paper an effect on chronic obstructive pulmonary disease or asthma. Liggett, Tampa: Well, there was one trial of chloroquine in asthma, and it showed no benefit. But it was really thought of as an anti-inflammatory, and the dose was pretty low. So pharmacologically, it just won’t work out right now with the compounds we have available to go oral. Gotto, New York: Back in the late 1970s, saccharine got taken off the market. I testified on behalf of the American Heart Association in front of Congressman Rogers’ committee. And then in 1982, when aspartame was introduced, Coca-Cola tried to kill Tab, which I liked to drink, which still has saccharine in it. We were told at the time that 18% to 20% of the population has a mutation that makes saccharine taste bitter, even at low concentrations. Would that be reflected in the bronchial airways by a difference in the receptors? Liggett, Tampa: That’s a really great question. So actually, part of my earlier career was looking at polymorphisms of other G-protein coupled receptors — such as the beta 2-adrenergic — to see if that was a pharmacogenetic locus or intra-individual variability in response to beta agonist. So we’ve looked at the three top taste receptors, and each one of them has at least one single nucleotide polymorphism in it that changes the amino acid. One of them is associated with differential tasting of various compounds, so that needs to be explored to fully bring this to clinical utility.
BITTER TASTE RECEPTORS IN THE WRONG PLACE: NOVEL AIRWAY SMOOTH MUSCLE TARGETS FOR TREATING ASTHMA STEPHEN B. LIGGETT, MD TAMPA, FLORIDA
ABSTRACT There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than -agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca2⫹-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by -agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.
INTRODUCTION: BITTER TASTE RECEPTORS Bitter taste receptors (TAS2Rs) have been traditionally thought to be expressed exclusively on taste buds, where they respond to a wide variety of bitter substances. There are 25 TAS2R subtypes, which typically respond with low affinity to tens of thousands of compounds (1). They are thought to have evolved as an aversion mechanism for avoidance of toxins in plants. We have found TAS2Rs expressed on the cell surface of human airway smooth muscle (ASM) cells, where they act to markedly relax the airway (2, 3). It is proposed that these Correspondence and reprint requests: Stephen B. Liggett, MD, Departments of Internal Medicine and Molecular Pharmacology and Physiology, University of South Florida, Morsani College of Medicine, 12901 Bruce B. Downs Blvd., MDC02, Tampa, FL 33612, Tel: 813-9747715, Fax: 813-974-3886, E-mail: [email protected]. Potential Conflicts of Interest: Dr Ligget is an Amgen consultant.
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receptors represent novel targets for bronchodilators in the treatment of asthma and chronic obstructive lung disease. WHY DO WE NEED “ANOTHER” BRONCHODILATOR? Currently, agents that have the potential to bronchodilate (or block bronchoconstriction) can be classified as direct or indirect bronchodilators. Because the bronchoconstriction in asthma is due to the release of local mediators such as histamine, acetylcholine, and leukotrienes, antagonists to these spasmogens are somewhat effective in preventing bronchoconstriction, but are dependent on whether a particular patient is “driven” by a given pathway. Additionally, they are specific to only one pathway. Direct bronchodilators, on the other hand, act to relax ASM and dilate regardless of the spasmogen. Currently, there is only one class of direct bronchodilators for treating obstructive lung disease, which are the -agonists. Although these agents have various properties, such as duration of action or degree of activity, the mechanism of action is the same: an increase in intracellular cyclic adenosine monophosphate which acts via protein kinase A to directly relax the muscle. It is well recognized that asthma treatment is currently in need of additional therapeutic approaches (4), with approximately 50% of asthmatics having less than optimal control when using the current available agents including inhaled corticosteroids and long acting -agonists (5– 8). -agonists are also associated with extensive interindividual variability of the clinical response, tachyphylaxis, bronchial hyperactivity, and increased adverse events (7, 9 –14). TAS2R SIGNALING As shown in Figure 1, TAS2Rs are G-protein coupled receptors that are integrated into the cell membrane, where extracellular agonists activate the receptor which evokes intracellular events. The G-protein for TAS2Rs is gustducin, and upon receptor activation the heterotrimer dissociates into ␣, and the ␥, subunits. In taste buds, the ␥ subunit carries out signaling via coupling to and activating phospholipase C (PLC), which results in an increase in intracellular inositol-1, 4, 5 triphosphate (IP3). IP3 then binds to the IP3 receptor on the endoplasmic reticulum which promotes movement of Ca2⫹ from this storage compartment to the intracellular space. As indicated in Figure 1, we also find the same signaling pathway up to this point in human and mouse ASM. However, the fate of the intracellular Ca2⫹ ([Ca2⫹]i) is different in ASM compared to taste buds. In
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taste buds, [Ca2⫹]i activates a transient receptor potential (TRP) channel, which depolarizes the membrane, causing secretion of neurotransmitter which activates the type III cell of the taste bud that communicates via nerves to the brain (left of the green line in Figure 1). In ASM, we find that [Ca2⫹]i activates one or more channels leading to hyperpolarization of the membrane (3), which relaxes the muscle (right of the dashed blue line). In ASM there is no involvement of a second cell type or a central nervous system connection, nor is there activation of the depolarizing TRP channel. This latter point is important, as some groups have used the presence/absence of a TRP channel as evidence for or against expression of bitter taste receptors in a given cell (15). We have clearly shown that there is a divergence of signaling in these two cell types, so TRP channel identification is not adequate for identification of TAS2Rs, particularly in non-gustatory tissues or cells.
nerves
Type III cell in taste bud
Bitter substances
Ggust
βγ βγ
neurotransmitter release Ca2+ TRP channel
BKCa or Other
FIG. 1. Overall signaling cascade of TAS2R in taste bud cells and human airway smooth muscle. To the left of the dashed green line is the pathway in the type 2 cell of the taste bud. To the right of the blue line is the pathway in human airway smooth muscle cells. The portion between these two lines is common between the two cell-types.
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RESULTS TAS2RS on Human ASM Quantitative reverse transcription polymerase chain reaction (RTPCR) revealed six TAS2Rs with high (subtypes 10, 14, 31) and moderate (subtypes 5, 4, 19) expression in normal primary cultured human ASM cells (3). In initial studies with various known bitter compounds, human ASM cells displayed an increase in [Ca2⫹]i in a dose-dependent manner (Figures 2A, 2B, and 2C). The effect was blocked by the PLC inhibitor U73122, the ␥ inhibitor gallein, and partially blocked by the IP3 receptor antagonist 2APB (Figure 2D). In intact isolated mouse (Figure 3A) and human (Figure 3B) airways studied in the ex vivo myograph system, TAS2R agonists relaxed acetylcholine- and serotonin-mediated contracted airways in a dose-dependent fashion (3). These relaxation effects were also studied using magnetic twisting cytometry, a method that ascertains single cell mechanics in isolated cells (16, 17). In this way, communication from the bronchial epithelium or other cells of the airway is not possible, and quantitative measurements of ASM mechanics can be made. As shown in Figure 4, the bitter tastants chloroquine and saccharin decreased intrinsic cell
FIG. 2. TAS2R agonists promoted increases in [Ca2⫹]i. (A) Dose response to the bitter tastant saccharin. (B) Dose response to the bitter tastant chloroquine. (C) Maximal responses to the indicated agents (n ⫽ 3– 8 experiments). (D) Effects of various inhibitors (see text) on saccharin-promoted [Ca2⫹]i (n ⫽ 3 experiments).
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FIG. 3. TAS2R agonists relax mouse and human airways ex vivo. (A) Response to the bitter tastants denatonium, chloroquine, and quinine in mouse airways (n ⫽ 6 – 8 experiments). (B) Relaxation to the -agonist isoproterenol and the bitter tastants chloroquine and quinine in human airways (n ⫽ 4 experiments).
FIG. 4. Isolated airway smooth muscle mechanics ascertained by magnetic twisting cytometry. (A) Response to the known relaxant isoproterenol and the known contractor histamine, and the bitter tastant saccharin. (B) The relaxation effect of saccharin is blocked by the PLC inhibitor U73112, but not the PKA inhibitor H89. Ca2⫹-dependent K⫹ channel blockers IbTx and ChTx partially block relaxation. Results are from a representative experiment.
stiffness (3). This relaxation was blocked by the aforementioned PLC inhibitor, which ties together the biochemical results with the physiological results. That an increase in [Ca2⫹]i was associated with relaxation was unexpected because virtually all agents that increase [Ca2⫹]i in ASM cause contraction (18). However, depleting the sarco(endo) plasmic reticulum of Ca2⫹ with thapsigargin eliminated the relaxation effect of TAS2R agonists, further strengthening the cause-and-effect between [Ca2⫹]i elevation and relaxation (3). Consistent with the relaxation effect of TAS2R agonists, they were found to cause hyperpolarization of the cell membrane (Figure 5). One mechanism as to how
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FIG. 5. TAS2R agonists hyperpolarize the membrane of human airway smooth muscle. Results are from a representative experiment using 1 mM saccharin and chloroquine, and 100 M histamine.
an increase in [Ca2⫹]i relaxes ASM that we identified is via the large conductance Ca-dependent K⫹ channel (BKCa). A specific BKCa inhibitor, iberiotoxin, caused a partial (but not complete) loss of the hyperpolarizing and relaxation effects of chloroquine. Others have suggested (19) the lack of involvement of BKCa, but in our hands we consistently observe partial effects with iberiotoxin (19); therefore, we currently believe that BKCa, in conjunction with other hyperpolarizing membrane channels, are responsible for the relaxation effect from the increased [Ca2⫹]i due to TAS2R agonism. IMAGING Ca2ⴙ SIGNALS FROM TAS2Rs We have recognized that measuring [Ca2⫹]i in whole cells might obscure subcellular differences between [Ca2⫹]i increases from receptors that cause contraction (such as the H2-histamine) and the TAS2Rs, which also increase [Ca2⫹]i but relax ASM. This was explored using FLUO-3 loaded human ASM and scanning two-dimensional confocal images every 0.22 m at 0.5-second intervals (3). Qualitative differences between saccharin and histamine [Ca2⫹]i images are readily apparent. For saccharin, Ca2⫹ events were observed as early as 2.5 seconds after exposure to the drug, whereas at least 10 seconds were required for histamine. The saccharin signal begins, and is the most intense, on the slender ends and sarcolemmal regions of the cell, whereas histamine caused a less localized, more global, response.
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These preliminary studies suggest that the bronchodilating [Ca2⫹]i signals from TAS2R are in a specialized or confined compartment, which may provide for coupling to a hyperpolarizing channel. TAS2R FUNCTION IN ASTHMA We sensitized BALB/c mice with subcutaneous ovalbumin and challenged them with inhaled albumin which resulted in eosinophilic inflammation and hyperreactivity to inhaled methacholine in the sedated intubated mouse model (3). Figure 6 shows the results of airway resistance measurements in sham and ovalbumin mice. Methacholine caused the expected increase in airway resistance, which was partially reversed by the -agonist albuterol and the TAS2R agonist quinine, in the sham (control) mice. In the ovalbumin sensitized and challenged mice, quinine remained effective in decreasing methacholine-promoted increase in resistance. However, albuterol was much less effective in this inflammatory model of asthma in reversing the contraction by methacholine. Taken together, we conclude that TAS2R function is not perturbed in this model, whereas -agonist is less effective. This suggests that TAS2R agonists may be effective, perhaps even more effective than -agonists, in treating airway obstruction from bronchospasm. To further assess this issue, human ASM cells from normal individuals and from asthmatics were studied for expression of TAS2R mRNA and functional responses (20). TAS2R 10, 14, and 31 transcript levels
FIG. 6. TAS2R function is not altered in a mouse model of asthma. Mice were sedated, intubated, and ventilated and received the indicated drugs by inhalation. (A) Responses in sham (control) mice. (B) Response in ovalbumin sensitized and challenged mice.
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were not different in nonasthmatic versus asthmatic human ASM. Functional studies with subtype-specific agonists showed no loss of TAS2R function in the latter cells. Figure 7A shows a representative experiment, and Figure 7B shows mean results from studies with chloroquine (TAS2R10), diphenhydramine and flufenamic acid (TAS2R14), saccharin (TAS2R31) and quinine (TAS2R10, 14 and 31). Additional studies were performed using live precision cut lung slices (PCLS) of nonasthmatic lungs (20). Here, the area of the bronchi within a PCLS is measured in response to contracting or relaxing drugs. To mimic an asthmatic phenotype, some PCLS were treated with interleukin-13 (IL-13) for 12 hours. Slices were exposed to carbachol to contract, and then either quinine or the -agonist formoterol. Figure 8A shows the previously reported loss of -agonist responsiveness with the IL-13 exposure. In contrast, TAS2R function remains intact, with identical dose-response curves for quinine (Figure 8B). We have also examined TAS2R function under two scenarios often encountered in the treatment of obstructive lung diseases. First, we have shown that TAS2R function remains intact under conditions of extreme 2AR desensitization due to chronic -agonist exposure (21). This suggests little, if any, “cross-talk” between the two pathways. Secondly, we have found minimal TAS2R desensitization due to bitter tastant exposure (20). This suggests that unlike 2-agonists, TAS2R agonists may show little tachyphylaxis during repetitive dosing.
FIG. 7. TAS2R function is not altered in human ASM cells derived from nonasthmatic and asthmatic subjects. The agonists are shown with the TAS2R subtype to which they couple. (A) results from a representative experiment, (B) results from six experiments.
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FIG. 8. TAS2R function is not altered in the IL-13 model of asthma. Precision cut lung slices were derived from a nonasthmatic lung and airway diameter measured. Some slices were pretreated with IL-13 for 12 hours before the study. The response to the -agonist formoterol (A) was depressed with IL-13 treatment, whereas the response to quinine (B) was unaffected (N ⫽ 6, *, P ⬍ .02).
CONCLUSIONS TAS2Rs expressed on ASM have a promising pharmacologic profile as novel bronchodilators for treating obstructive lung disease. TAS2R agonists relax ASM by mechanisms that are distinctly different than
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those of the -agonists, thus they are not “look alike” drugs. This would expand the potential armamentarium of drugs for treatment of asthma and chronic obstructive lung disease. This increase in options should lead to improved outcomes. There are tens of thousands of known bitter compounds that are TAS2R agonists. These include natural compounds, derivatives of natural compounds, and synthetic agents used in treating other diseases. This presents an ideal setting for high-throughput screening and medicinal chemistry to provide new compounds for human studies. ACKNOWLEDGMENTS Funded by the National Institutes of Health grants HL114471, HL045967, and HL071609. The author thanks Charmaine Disimile for manuscript preparation.
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13. Salpeter SR, Wall AJ, Buckley NS. Long-acting beta-agonists with and without inhaled corticosteroids and catastrophic asthma events. Am J Med 2010;123:322– 8. 14. Sears MR, Taylor DR. The b2-agonist controversy: observations, explanations and relationship to asthma epidemiology. Drug Saf 1994;11:259 – 83. 15. Tizzano M, Cristofoletti M, Sbarbati A, Finger TE. Expression of taste receptors in solitary chemosensory cells of rodent airways. BMC Pulm Med 2011;11. 16. An SS, Fabry B, Trepat X, Wang N, Fredberg JJ. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness? Am J Respir Cell Mol Biol 2006;35:55– 64. 17. An SS, Laudadio RE, Lai J, Rogers RA, Fredberg JJ. Stiffness changes in cultured airway smooth muscle cells. Am J Physiol Cell Physiol 2002;283:C792–C801. 18. Green SA, Liggett SB. G-protein-coupled receptor signaling in the lung. In: Liggett SB, Meyers DA, eds. The Genetics of Asthma. New York: Marcel Dekker, Inc.; 1996:67–90. 19. Zhang CH, Chen C, Lifshitz LM, Fogarty KE, Zhu MS, ZhuGe R. Activation of BK channels is not required for bitter tastant-induced bronchodilation. Nat Med 2012;18:648 –50. 20. Robinett KS, Deshpande DA, Malone MM, Liggett SB. Agonist-promoted homologous desensitization of human airway smooth muscle bitter taste receptors. Am J Respir Cell Mol Biol 2011;45:1069 –74. 21. An SS, Wang WC, Koziol-White CJ, et al. TAS2R activation promotes airway smooth muscle relaxation despite beta(2)-adrenergic receptor tachyphylaxis. Am J Physiol Lung Cell Mol Physiol 2012;303:L304 –11.
DISCUSSION Ausiello, Boston: As you know, the senses of taste and smell are pretty intimately associated, even with the very large overlap in terms of receptors. So that raises two questions. First of all, is it possible that you could narrow that 10,000 compounds by looking at those compounds that have taste but also are aerosolized, I suppose, in terms of having effect in terms of smooth muscle? And then secondly, have you looked at obvious noxious agents that stimulate these taste buds in terms of their capacity for smooth muscle modification of the airway? Liggett, Tampa: I didn’t quite understand the second part of your question; have I looked at obvious noxious stimuli? Ausiello, Boston: Are there any taste bud sensitizers that also have the capacity to be aerosolized and, in that sense, you have both taste and smell being activated? And could that narrow your capacity for looking at the numerous compounds that may be able to activate these. . . Liggett, Tampa: I have to answer that question in a couple ways. First of all, we do see a few smell receptors on airway smooth muscle. So, I mean, you know, there is a whole other pathway that we might be able to consider. And secondly, to clarify, we do think that if this becomes a drug, it’s going to have to be aerosolized. And most of these drugs can be put in an inhaler form, so we are not too concerned about that. Some other issues, though, would come about in terms of palatability. You are inhaling something that you’re going to taste as bitter. The advantage, we think, is that we only have to hit one of the three highest receptors to get bronchodilation, and we actually would want a very highly specific compound to try to do that. I’m not sure how to integrate the smell component to it, but we will have to make a palatable compound in the end. Rothman, Baltimore: I really enjoyed your talk. Can you hypothesize a little bit
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about the normal ligand for these receptors, and the role of these receptors in normal lung physiology? Liggett, Tampa: So that question comes up every time, and it’s a great question. So we have found that gram-negative bacteria secrete a bitter taste receptor ligand that activates bitter taste receptors. So what we are thinking — this is just a hypothesis — that the receptors evolved so that you don’t get a socked-in pneumonia. So as you begin to have a severe bronchitis, if there is bronchodilation to help clear the cellular debris and the bacteria. That’s the closest we have been able to come so far. Hochberg, Baltimore: Two-part question, I guess. The first goes back to the mouse model. Did you try to administer these agents parenterally as opposed to directly into the lung? Liggett, Tampa: No. Hochberg, Baltimore: So I guess the second part is, would you expect that if these were administered parenterally, you would see a similar effect as you saw with administration directly? Liggett, Tampa: Well, it looks like with the agents we had available — quinine, chloroquine, et cetera — you have to reach, you know, high micromolar concentrations in order to activate these receptors. And you just couldn’t do that in the blood if you took the pills. I mean, if you took that much chloroquine, you’d have all the toxic effects from chloroquine. So our feeling is that we are going to have to come up with a compound through this screening technique that has a higher affinity for these agents, and then maybe we could take them parenterally, orally. I mean, wouldn’t it be great to have a highly effective oral bronchodilator? It would be great. Hochberg, Baltimore: Well, there are a lot of sort of off-target effects of hydroxychloroquine, which was used in the treatment of rheumatoid arthritis, which have been recently reviewed by Nancy Olson, who is now, I guess, at Penn State Hershey, and I’m not aware that she included in her paper an effect on chronic obstructive pulmonary disease or asthma. Liggett, Tampa: Well, there was one trial of chloroquine in asthma, and it showed no benefit. But it was really thought of as an anti-inflammatory, and the dose was pretty low. So pharmacologically, it just won’t work out right now with the compounds we have available to go oral. Gotto, New York: Back in the late 1970s, saccharine got taken off the market. I testified on behalf of the American Heart Association in front of Congressman Rogers’ committee. And then in 1982, when aspartame was introduced, Coca-Cola tried to kill Tab, which I liked to drink, which still has saccharine in it. We were told at the time that 18% to 20% of the population has a mutation that makes saccharine taste bitter, even at low concentrations. Would that be reflected in the bronchial airways by a difference in the receptors? Liggett, Tampa: That’s a really great question. So actually, part of my earlier career was looking at polymorphisms of other G-protein coupled receptors — such as the beta 2-adrenergic — to see if that was a pharmacogenetic locus or intra-individual variability in response to beta agonist. So we’ve looked at the three top taste receptors, and each one of them has at least one single nucleotide polymorphism in it that changes the amino acid. One of them is associated with differential tasting of various compounds, so that needs to be explored to fully bring this to clinical utility.
