Therapeutic Potential and Challenges of Bitter Taste Receptors on Lung Cells

Abstract

Airway smooth muscle (ASM) hyperresponsiveness and airway remodeling are pathological drivers of disease progression and mortality in asthma. Importantly, approximately 50% of affected individuals are unable to reliably manage disease symptoms using the current standard of care. Recently, T2Rs have been identified as a novel class of G protein-coupled receptors expressed in the airway that on activation can induce ASM relaxation and reduction in airway tone. Further, agonists of T2Rs may also remedy airway remodeling, which has been difficult to manage with currently available medications. In this review, we will discuss the recent developments in T2R biology and their role in cellular physiology (particularly ASM) and expand on the therapeutic potential of T2R agonists in treatment of asthma.

Introduction

Airway smooth muscle (ASM) is the principal cell type influencing airway tone and the physiology of respiration. Studies have unequivocally established a pivotal role for ASM in the pathophysiology of obstructive airway diseases such as asthma and COPD, and this includes regulation of bronchoconstriction and thickening of the airway wall, which is one of the major components of airway remodeling. G protein-coupled receptors (GPCRs) play a central role in regulating the functions of ASM. In asthma exacerbations, endogenous agonists (e.g. acetylcholine) and inflammatory mediators (e.g. histamine, leukotrienes) exert pro-contractile actions on ASM by activating signaling through specific GPCRs (coupled to heterotrimeric G protein G_q). Therefore, for many decades agonists of GPCRs that promote pro-relaxant outcomes (typically G_s-coupled) and antagonists of pro-contractile GPCRs have been overwhelmingly favored for therapeutic targeting to reverse airway contraction [1]. The approaches to date have provided relief from exacerbations, however, this protection is not conferred on ~50% of asthmatics presumably because of the lack of effects of current therapies on multiple features of asthma. For example, beta agonists (β agonists) are potent bronchodilators, but lack effect on airway remodeling. Over time, individuals suffering from asthma exacerbations develop features of airway remodeling such as the accumulation of smooth muscle mass, deposition of extracellular matrix proteins including collagen and fibronectin, epithelial and subepithelial changes, and goblet cell metaplasia, which combine to further reduce airway diameter. Consequently, there is a need to develop novel targets that can address multiple pathological features of asthma. Receptors of bitter taste sensation (T2Rs) represent one such promising alternative.

Investigations into ectopic expression of T2Rs in lungs and their functional role in airway cells has provided an opportunity to exploit T2Rs in developing new asthma therapeutics [2]. It is understood that bitter taste sensation in the oral cavity evolved in higher animals to stimulate rejection of possibly spoilt or toxic food. Consequently, it was speculated that the ectopic expression of T2Rs in lungs may play a similar role in dispelling harmful agents entering the airways [3]. Indeed, research studies have shown that solitary chemosensory cells (SCCs) and immune cells (resident and tissue infiltrating) express T2Rs and respond to acyl-homoserine lactones (quorum sensing bitter tasting molecule) synthesized by bacteria [3–5] thereby possibly regulating innate immune functions in the airways. Research studies from our laboratory have demonstrated that ASM cells express multiple T2R subtypes and their activation results in bronchodilation and inhibition of ASM proliferation [2,6–8]. Further, in pre-clinical models, agonists of T2Rs have been shown to mitigate allergen-induced airway inflammation, remodeling and hyperresponsiveness, which are the three classical pathological features associated with allergic asthma [8]. Collectively, T2Rs provide unique opportunities for developing anti-asthma therapeutics owing to the distinct biology and effectiveness of T2Rs in mitigating multiple features of asthma pathobiology.

Therapeutic opportunities facilitated by unique signaling and functional effects

i). Redundancy in T2Rs provides a therapeutic advantage.

A decade ago, we first reported the expression of T2Rs in ASM cells [2]. Further, we discovered that ASM cells express distinct receptor subtypes, with multiple receptors (at least 3–4) expressed at relatively higher levels compared to the major target of bronchodilator action, the beta 2 adrenergic receptor (β₂AR). A total of 25 well-characterized subtypes of T2Rs exist; however, what unique signaling and functional effects, if any, are mediated by a given subtype of receptor is not clearly established. Nonetheless, different bitter tastants activate one or a set of T2R subtypes. This diversity amongst available bitter tastants provides a unique opportunity to select a subtype or a group of subtypes of T2Rs for therapeutic targeting. In the case of β agonists, the drugs target only one receptor (i.e. β₂AR), which undergoes homologous desensitization due to chronic use and possesses genetic polymorphisms limiting the therapeutic efficacy [9]. Bitter tastants on the other hand activate multiple receptor subtypes expressed on a target cell and redundancy in T2Rs in ASM potentially improves the efficacy of bronchodilation.

ii). T2R-mediated bronchodilation not dependent on cAMP-PKA mechanism.

The unique signaling features of T2Rs indicate divergence from our understanding of traditional GPCR signaling and related outcomes in ASM (Figure 1). Therapeutic facilitation of ASM relaxation has generally been achieved through the activation of G_s-coupled GPCRs, which activates adenylyl cyclase resulting in cAMP accumulation and subsequent activation of the protein kinase A (PKA)-dependent pathway. Over the past four to five decades, anti-asthma drug development has primarily focused on targets that are dependent on cAMP-PKA pathway [10]. This approach has resulted in development of drugs with improved duration/magnitude of efficacy albeit belonging to the same therapeutic target. Therefore, limitations for use of β agonists such as β2AR desensitization still remain a concern. T2Rs offer a unique paradigm shift in this context. ASM relaxation and bronchodilation elicited via T2Rs does not involve generation of cAMP or activation of PKA [2]. Interestingly, murine rings contracted with acetylcholine experienced a larger reduction in contractile force when treated with the T2R agonist chloroquine and β-agonist isoproterenol in tandem than with either agonist individually [2]. Therefore, T2R agonists could be potentially used as an adjunct therapy to β agonists [11]. In fact, T2R agonists are able to induce bronchodilation under conditions when β₂ARs are desensitized due to chronic exposure to β agonists or when β₂AR signaling is reduced in the presence of inflammatory cytokines such as IL-13 [12,13].

Figure 1. Regulation of contractile G_q-coupled GPCR signaling by G_s-coupled GPCRs and T2Rs.

Binding of contractile agonists to the G_q-coupled receptor (e.g. histamine to H1 receptor) activates PLCβ via the Gα_q subunit, which leads to production of IP₃, ultimately resulting in a diffused global rise in cytosolic calcium which is linked to cross-bridge cycling and force generation responsible for contraction. Additionally, G_q-PLCβ signaling activates the contractile-related proteins, PKC and RhoA. Traditional G_s-coupled β2AR agonists stimulate cAMP accumulation within cells which activates the main effector, protein kinase A (PKA), which inhibits multiple foci within the contractile signaling pathway. Activation of T2Rs with agonists such as saccharine results in Gβγ-PLCβ-IP₃-dependent cytosolic calcium accumulation, which appears to be localized. This spatial management of T2R-induced calcium facilitates ASM relaxation. T2R activation also inhibits G_αq-mediated contractile signaling by inhibiting calcium entry via voltage-gated calcium channels (VDCC), by inhibiting IP₃-mediated calcium oscillations or by promoting calcium sequestration into mitochondria when stimulated concomitantly with a G_q ligand. Red and blue arrows indicate contractile- and relaxation-related activation respectively. Question marks emphasize the mechanisms that remain to be elucidated.

iii). Multimodal regulation of ASM tone by T2Rs.

Activation of G_q-coupled GPCRs facilitates contractile outcomes through increased enzymatic activity of phospholipase C beta (PLCβ and elevation in intracellular calcium concentration ([Ca²⁺]_i). Interestingly, activation of T2Rs results in an increase in [Ca²⁺]_i, however, this increase paradoxically concludes with relaxation of ASM, an outcome that is conserved across species. At rest, [Ca²⁺]_i is maintained at low concentrations in ASM cells. The binding of agonists to prototypical G_q-coupled GPCRs leads to G alpha subunit (Gα)-mediated PLCβ activation, PLCβ-mediated cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol triphosphate (IP₃). Subsequently, IP₃ diffuses through the cytosol and binds to its specific receptor on the sarcoplasmic reticulum (SR), leading to release of Ca²⁺ from IP₃-sensitive stores. Following this rapid increase in [Ca²⁺]_i, calcium binds to calmodulin. This complex in turn activates the calmodulin kinase (CaMK) and myosin light chain kinase (MLCK) pathway, resulting in phosphorylation of regulatory myosin light chain (MLC), a critical component involved in actin-myosin cross-bridge formation and ASM contraction. Further, extracellular calcium influx through voltage-dependent calcium channels (VDCC) and store-operated calcium channels is required in order to sustain the response initially caused by the IP₃-mediated calcium release [14]. These observations emphasize the diverse range of mechanisms available to facilitate depolarization of ASM cells and sustain calcium-mediated ASM contraction.

Calcium handling in ASM following activation of T2Rs is relatively poorly understood. Studies in taste cells have unequivocally established that T2R-induced calcium elevation in type II taste cells is indispensable for taste sensation [15,16]. The unique nature of T2Rs is also related to the G protein that is involved in mediating T2R signaling. Studies have suggested that T2Rs likely couple with heterotrimeric G protein gustducin (G_gust; and not G_q or G_s) although evidence to the contrary also exists [17]. Further, although gustducin expression is confirmed in ASM cells, the functional role of gustducin in mediating T2R signaling is not directly established. G_i-coupled signaling is primarily known for its antagonistic effects on G_s-coupled signaling through its direct inhibition of adenylyl cyclase. A recent study by the Liggett lab demonstrated that other members of the G_i family of G proteins are more prevalent compared to G_gust and that T2Rs couple to G_αi1, G_αi2, and G_αi3, and not G_gust in ASM cells [17]. This evidence comes from cell-based studies and in vivo relevance of G_i proteins in T2R signaling needs to be established in relevant knockout mouse models. Downstream of T2R-G protein coupling, signaling advances in a G beta gamma subunit (Gβγ)-, PLCβ- and IP₃-dependent manner [2], in contrast to Gα-mediated PLCβ activation downstream of G_q. This signaling cascade results in [Ca²⁺]_i influx (comparable in magnitude to G_q-coupled receptors in cultured ASM cells); however, the qualitative dynamics of spatial localization of calcium diverge from what is classically observed with G_q-coupled receptor signaling. Using a high-resolution real-time confocal imaging technique, we showed that treatment of ASM cells with saccharin (a T2R agonist) results in rapid (2.5 s) calcium increase in the discrete sarcolemmal regions. In contrast, treatment of ASM cells with contractile agonist histamine demonstrated a relatively delayed increase in [Ca²⁺]_i which diffused throughout the cell devoid of any localized accumulations. Further, in saccharin-treated ASM cells, spatially and temporally discernible [Ca²⁺]_i movements occurred prior to sustained local rise in [Ca²⁺]_i [2]. These differences could potentially account for the distinct functional outcomes following activation of these pathways. However, in murine ASM cells and in lung slices, T2R agonists fail to elicit a calcium response at low agonist concentrations [18]. Interestingly, T2R activation in murine ASM cells followed the canonical T2R signal transduction pathway involving T2R107-Gβγ-PLCβ-IP₃ [18], but not in murine lung slices [19]. Additional studies are needed to establish the species-specific differences in T2R subtypes, agonist selectivity and optimum concentration of agonists required to elicit a signaling/functional effect.

Interestingly, concomitant stimulation of ASM cells with a T2R agonist and a contractile agonist (e.g. histamine, acetylcholine) results in inhibition of calcium elevation induced by the contractile agonist [20]. Based on these studies, multiple mechanisms by which T2R activation induces relaxation in ASM have been proposed [2,19,21–23] (Figure 1). Studies in our laboratory suggest that activation of T2Rs results in a rapid initial increase in [Ca²⁺]_i from IP₃ stores and subsequent activation of large conductance calcium-activated potassium (BK_Ca) channels leading to hyperpolarization of ASM cells [2]. Others have suggested that the opening of BK_Ca channels may not be necessary for T2R-mediated relaxation [22]. The proposed alternative mechanism suggests that in addition to affecting contractile-related proteins via PLCβ activation, T2R-induced Gβγ can directly inhibit the influx of extracellular calcium through VDCCs at the plasma membrane [22]. Another mechanistic insight is that bitter tastant-induced Gβγ causes inhibition of G_q agonist-mediated calcium release from intracellular stores (IP_3-mediated) in murine lung slices [19]. Therefore, T2R signaling would attenuate G_q-coupled signaling by inhibiting mechanisms involved in elevation of [Ca²⁺]_i necessary for sustained ASM contraction. It has been discerned that T2Rs attenuate bronchoconstrictive agonist-mediated ASM contraction despite inducing [Ca²⁺]_i transients similar to those observed with bronchoconstrictive agonists; however, the differences in the kinetic and spatial dynamics of [Ca²⁺]_i mobilization that may account for these observations have yet to be fully understood. In fact, a recent study using human ASM cells demonstrated that absinthin, a T2R46 specific agonist, not only reduced cytosolic calcium elevation induced by histamine, but also increased calcium influx into mitochondria [23]. Furthermore, T2R46-mediated mitochondrial calcium uptake is mediated via the mitochondrial uniporter. These findings underscore a complex interplay of calcium regulatory mechanisms in different compartments of ASM cells upon activation of T2Rs in the presence or absence of contractile agonists.

iv). T2Rs are amenable to genetic and pharmacological manipulation.

T2R specificity of [Ca²⁺]_i mobilization, membrane hyperpolarization, and relaxation by certain bitter tastants in ASM cells have been established using molecular- (siRNA) and antibody- (anti-T2R subtype) based approaches [2]. For example, silencing expression of T2R10 resulted in a significant reduction in [Ca²⁺]_i stimulated by its agonist strychnine. Further, this outcome is consistent when blocking receptor activation using anti-T2R10 (but not anti-T2R7 or isotype control) polyclonal sera. In a recent study, the authors used 3β-hydroxydihydrocostunolide as an antagonist of T2R46 in human ASM cells [23]. Both siRNA-mediated knockdown and pharmacological inhibition of T2R46 resulted in attenuation of absinthin-induced inhibition of cytosolic calcium elevation by histamine. Collectively, these studies validate the ability of certain bitter tastants to facilitate canonical T2R-mediated signaling and relaxation in ASM cells via T2Rs. Evidences such as these are critical in developing drugs targeting T2Rs for use in obstructive lung diseases.

v). T2R agonists are effective against multiple contractile agents.

Efficacy of ASM relaxation and bronchodilation by T2R agonists has been investigated in multiple model systems using both human and rodent cells and tissues [21]. Although belonging to structurally diverse groups of chemicals, T2R agonists efficaciously antagonize the contractile response induced by acetylcholine (endogenous agonist), histamine, serotonin and endothelin [2,20]. Of note, a number of asthmatic mediators are G_q-coupled GPCR agonists namely histamine, leukotrienes, serotonin and bradykinin. Functional studies of T2R agonism collectively suggest that the efficacy of T2R-mediated bronchodilation is maintained independent of the contractile agonist. This feature of T2Rs should favor the development of drugs targeting T2Rs for asthma.

vi). T2Rs possess anti-proliferative effects in ASM.

Many currently available asthma therapeutics fail to address airway remodeling, a seemingly irreversible pathological feature of asthma that affects both fixed as well as dynamic airway resistance [24]. One of the critical features of T2Rs that makes them a prolific target for developing asthma therapeutics is their ability to inhibit ASM growth and inhibit/reverse airway remodeling [6,25]. These studies have demonstrated that T2Rs can inhibit specific check points within the signaling pathways associated with cellular proliferation. More specifically, we have shown that activation of T2Rs inhibits phosphorylation of Akt and p70-S6 kinase, which shuts down transcriptional programs that contribute to cellular proliferation [6]. Further, T2R agonists arrest cell cycle progression by regulating expression of cyclin D1. Importantly, the antiproliferative actions of T2R agonists are preserved in ASM cells obtained from asthmatic donors [25]. As mentioned earlier, T2R46 activation promotes calcium uptake in mitochondria [23] and in support of this observation our studies demonstrated that the T2R agonists-induced anti-mitogenic effect also involves a mitochondrial mechanism, more specifically observed as excessive autophagy in ASM cells [7]. Anti-proliferative effects of T2Rs have also been established in a pre-clinical model of allergen-induced asthma in mice [8]. Repeated allergen inhalation in mice results in airway inflammation and progressive remodeling changes. We have shown that treatment of mice with T2R agonists both during exposure to allergen (pre-treatment model) as well as following establishment of allergic inflammation (therapeutic model), can result in significant mitigation of multiple asthma-like features in murine airways/lungs [8]. Of particular note, various markers (α-actin and myosin heavy chain, collagen, fibronectin and PAS staining) suggestive of airway remodeling resulting from repeated inhalation of allergen, were inhibited by T2R agonists.

vii). T2R activation promotes ciliary beating in airway epithelium.

Another salient feature of human asthma is excessive mucus accumulation. Shah and co-workers demonstrated that T2R subtypes are expressed on the cilia of the human respiratory epithelium and their stimulation resulted in increased ciliary beat frequency [3]. The ciliated epithelium in human airways express T2Rs, and canonical T2R signal transduction components including gustducin, PLCβ and TRPM5. Stimulation of ciliated epithelial cells with bitter tastants such as denatonium, quinine, salicin and nicotine results in elevation of intracellular calcium levels. Interestingly, the bitter tastants-induced increase in intracellular calcium enhanced ciliary beat frequency [3]. However, these findings have yet to be confirmed in human subjects. Although additional in vivo studies are needed to establish the causal relationship between T2R signaling and ciliary functions, these data support the notion that T2R activation promotes mucus clearance.

viii). T2R signaling and functions are not compromised in human asthma.

The discovery of T2Rs in ASM, SCCs and immune cells (resident and inflammatory) has fueled significant interest into exploring the role of T2Rs in airway diseases, particularly in asthma. All these cell types play important roles in the pathogenesis and progression of asthma pathobiology, and from a therapeutic perspective, activation of T2Rs on these cells promotes favorable outcomes. Moreover, T2Rs appear to be accessible targets during asthmatic condition and their expression has been shown to be significantly upregulated in circulating leukocytes isolated from severe asthmatics [26]. Further, in asthmatic ASM cells and tissues (precision cut lung slices), T2R expression and functionality is unperturbed, even under conditions that typically compromise β₂AR function [12,13]. Collectively, these studies underscore the pharmacological accessibility of the T2Rs for therapeutic intervention in asthma. The next critical step in T2R research is to determine which specific T2R subtypes are most important with respect to asthma pathogenesis and potential anti-asthma therapy.

Collectively, in addition to causing an efficacious bronchodilation, T2R agonism has been shown to mitigate features of airway remodeling including ASM proliferation and mucus accumulation suggesting simultaneous mitigation of multiple pathologies in human asthma. This further underscores the potential clinical usefulness of T2R agonists in the treatment of obstructive pulmonary diseases.

Challenges of T2R pharmacology

One of the principal challenges in exploiting T2Rs for therapeutic ends has been the lack of receptor subtype-specific agonists. Currently available agonists exhibit promiscuity in activating distinct T2Rs, making it challenging to examine the relative contribution of individual receptor subtypes in driving bronchodilatory and anti-proliferative actions in ASM. Further, the structural organization of T2Rs allows for compounds of diverse classes to interact with the ligand binding pocket, which further complicates discovery of a specific class of compounds that can be structurally refined to provide receptor specificity. T2Rs are also broadly-tuned receptors and demonstrate low affinity to currently available bitter tastants [27]. While evolutionary pressures may have dictated broad tuning for maintaining responsiveness to diverse pathological insults, this feature severely impedes potency of available compounds for therapeutic purposes. This broad-tuning of the receptor is owing to a large agonist binding pocket, which allows for interactions with agonists of distinct classes. Further, some ligands (e.g. flufenamic acid) are known to activate one subtype of T2R expressed on ASM whereas other ligands activate more than one (e.g. chloroquine, quinine). Receptor subtypes and agonist specificity are scenarios that are being considered when studying the efficacy of these agonists for asthma. Although a crystal structure of the T2R prototype is lacking, efforts are ongoing to offer structural improvements to certain classes of T2R agonists that could potentially unlock a novel class of receptor subtype-specific agonists [27–29]. These approaches have purposed both chemical improvements and molecular modeling to identify more suitable analogs and mimetics of currently available agonists. These approaches aimed at ‘fine-tuning’ have proven useful and analogs of the bitter tastant flufenamic acid have been recently synthesized and have demonstrated potential for the targeted activation of T2R14 [30]. Further, in silico modeling also suggests differential amino acid requirements of the binding pocket on T2R14 for different ligands. For example, diphenhydramine binds to a binding pocket formed by Trp66, Phe71, Trp89, and Gln266 amino acid residues, which is less accessible to the other T2R14 agonists. Flufenamic acid was unable to fit into this binding pocket as deeply as diphenhydramine due to poor interactions with Trp66 and Phe71 [31]. The advent of bitter tastant derivatives with higher binding affinities for their respective T2Rs may provide an avenue to address the concerns of cytotoxicity and off-target effects that persist with the currently available bitter tastants. The agonist-T2R interaction studies mentioned above will provide a basis for developing T2R agonists with improved specificity and efficacy.

Future directions

T2R agonists show potential as an effective therapy for the treatment of asthma. However, additional investigation is warranted in a few key areas of ASM T2R biology and pharmacology. Despite the therapeutic advantages attributed to T2R diversity, the variability among T2R subtypes with regard to activation of upstream signaling components in the T2R signaling pathway (Gβγ, PLCβ, IP₃) needs to be further investigated. This is particularly pertinent to IP₃-mediated intracellular calcium localization and varying phenotypical effects in different cells/tissue types that may or may not be relevant to different functional effects due to activation of a specific T2R subtype(s). Our studies suggest that there are differences in the localization of [Ca²⁺]_i in ASM treated with saccharin compared to histamine [2]. However, the mechanistic differences in [Ca²⁺]_i dynamics (such as specific subcellular compartmentalization) between T2Rs and G_q-coupled receptors in ASM have yet to be fully elucidated. With the observed inhibition of ASM contraction by T2R activation in mind, there are multiple theories that suggest that T2R signaling inhibits G_q-coupled [Ca²⁺]_i flux by blocking IP₃-mediated stores, blocking extracellular calcium channels and/or by redirecting cytosolic calcium into the mitochondria. Specifically, investigation into the differential effects of T2Rs and G_q-coupled receptors on mitochondrial dynamics such as oxidative phosphorylation, reactive oxygen/nitrogen species production and calcium flux may provide additional insight into where these pathways diverge. Additionally, a knowledge gap exists in terms of cell-type specific expression and functional effects of T2R subtypes that needs to be addressed in future studies. Finally, while most studies focus on comparing efficacy of T2R-mediated bronchodilation to β2AR agonists since they are the current standard of care, other commonly used bronchodilators include antagonists of the muscarinic receptor, cysteinyl leukotriene type I receptor, and xanthine derivatives (such as aminophylline). However, there is currently no literature that compares the efficacy of bronchodilation induced by T2R agonists to anticholinergic and xanthine derivative bronchodilators. This would be a good direction for future studies to consider once more information is known about T2R agonists alone and/or in tandem with β2AR agonists.

Concluding remarks

T2Rs possess unique physiological properties and studies within the last decade have sought to systematically and comprehensively unravel the enigma surrounding these receptors. Collectively, these studies have presented T2Rs as a promising target for mitigating pathological features of obstructive airway diseases. While these receptors can be targeted on ASM to promote bronchodilation and anti-proliferative outcomes, the therapeutic potential of T2Rs is amplified. This therapeutic potential is directly correlated to T2R expression and regulatory actions on other cell types relevant to the disease, including the ones on immune cells and ciliary epithelial cells. The testing of compounds structurally similar to T2R agonists and the development of novel T2R subtype-specific mimetics shows promise in addressing safety concerns and engaging a step closer towards developing a comprehensive therapeutic option for asthma control.