Wayne C. H. Wang

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation

Receptor-mediated airway smooth muscle (ASM) contraction via G(alphaq), and relaxation via G(alphas), underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G(alphai) expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M(3)-muscarinic receptors and diminished relaxation mediated by beta(2)-adrenergic receptors (beta(2)ARs). A causal effect between G(i) expression and phenotype has not been established, nor have mechanisms whereby G(i) modulates G(q)/G(s) signaling. To delineate isolated effects of altered G(i), transgenic mice were generated overexpressing G(alphai2) or a G(alphai2) peptide inhibitor in ASM. Unexpectedly, G(alphai2) overexpression decreased contractility to methacholine, while G(alphai2) inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G(q) signaling network: decreased phospholipase C and increased PKCalpha, respectively. G(alphai2) overexpression decreased beta(2)AR-mediated airway relaxation, while G(alphai2) inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G(s) and G(i). IL-13 transgenic mice (a model of asthma), which developed increased ASM G(alphai), displayed marked increases in airway hyperresponsiveness when G(alphai) function was inhibited. Increased G(alphai) in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes beta-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G(s)/G(q) activities could provide new asthma therapies.

TAS2R activation promotes airway smooth muscle relaxation despite β 2-adrenergic receptor tachyphylaxis

Recently, bitter taste receptors (TAS2Rs) were found in the lung and act to relax airway smooth muscle (ASM) via intracellular Ca(2+) concentration signaling generated from restricted phospholipase C activation. As potential therapy, TAS2R agonists could be add-on treatment when patients fail to achieve adequate bronchodilation with chronic β-agonists. The β(2)-adrenergic receptor (β(2)AR) of ASM undergoes extensive functional desensitization. It remains unknown whether this desensitization affects TAS2R function, by cross talk at the receptors or distal common components in the relaxation machinery. We studied intracellular signaling and cell mechanics using isolated human ASM, mouse tracheal responses, and human bronchial responses to characterize TAS2R relaxation in the context of β(2)AR desensitization. In isolated human ASM, magnetic twisting cytometry revealed >90% loss of isoproterenol-promoted decrease in cell stiffness after 18-h exposure to albuterol. Under these same conditions of β(2)AR desensitization, the TAS2R agonist chloroquine relaxation response was unaffected. TAS2R-mediated stimulation of intracellular Ca(2+) concentration in human ASM was unaltered by albuterol pretreatment, in contrast to cAMP signaling, which was desensitized by >90%. In mouse trachea, β(2)AR desensitization by β-agonist amounted to 92 ± 6.0% (P < 0.001), while, under these same conditions, TAS2R desensitization was not significant (11 ± 3.5%). In human lung slices, chronic β-agonist exposure culminated in 64 ± 5.7% (P < 0.001) desensitization of β(2)AR-mediated dilation of carbachol-constricted airways that was reversed by chloroquine. We conclude that there is no evidence for physiologically relevant cross-desensitization of TAS2R-mediated ASM relaxation from chronic β-agonist treatment. These findings portend a favorable therapeutic profile for TAS2R agonists for the treatment of bronchospasm in asthma or chronic obstructive lung disease.

A polymorphism of G-protein coupled receptor kinase5 alters agonist-promoted desensitization of β2-adrenergic receptors

Beta-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of beta2-adrenergic (beta2AR) signaling genes. beta2AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for beta-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared with GRK5-Gln41. Consistent with this functional effect, agonist-promoted beta2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated beta2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of beta2AR during persistent agonist exposure, and thus may contribute to beta-agonist variability in asthma treatment of African-Americans.

MicroRNA let-7 establishes expression of β2-adrenergic receptors and dynamically down-regulates agonist-promoted down-regulation

Although β2-adrenergic receptors (β2AR) are expressed on most cell types, mechanisms that establish expression levels and regulate expression by chronic agonist remain unclear. The 3′ UTR of ADRB2 has a conserved 8-nucleotide seed region that we hypothesized is targeted by the let-7 family of miRNAs leading to translational repression. In luciferase assays with transfected cells, luc-β2WT3′UTR had decreased expression when cotransfected with let-7f, but a mutated luc-β23′UTR lacking the seed was unaffected by let-7f; a mutated let-7f also had no effect on luc-β2WT3′UTR expression. ADRB2 mRNA was in greater abundance in immunoprecipitates of Ago2, a core component of the miRNA-induced silencing complex, when cells were transfected with let-7f, but not with a mutated let-7f, indicating a direct interaction with the silencing mechanism. H292 cells transfected with let-7f caused ∼60% decrease in native β2AR expression, but transfection with let-7f–specific locked nucleic acid anti-miRNA increased β2AR expression by ∼twofold. We considered that an increase in let-7f leading to greater repression of translation contributes to agonist-promoted down-regulation. Paradoxically, in cells and in lungs from mice treated in vivo, an ∼50% decrease in let-7f occurs during long-term agonist exposure, indicating a counterregulatory event. Consistent with this notion, let-7f locked nucleic acid transfection caused depressed agonist-promoted down-regulation. Thus, let-7f miRNA regulates baseline β2AR expression and decreases in let-7f evoked by agonist attenuate down-regulation. This positive feedback loop has not previously been described for a G protein-coupled receptor and its miRNA. Methods to decrease let-7f expression in targeted cells may increase therapeutic responses to β-agonist by increasing β2AR expression or minimizing tachyphylaxis.

Bronchodilator activity of bitter tastants in human tissue

Belvisi et al. show in human bronchi that the bitter taste receptor (TAS2R) agonist chloroquine evokes marked relaxation which is in agreement with our findings in mouse airways1. They note, however, an equivalent efficacy (degree of maximal relaxation) with the β-agonist isoproterenol in human bronchi. In our paper, we performed the vast majority of intact airway physiology in mice, where we found that bitter tastants had a greater efficacy compared to isoproterenol. We never stated that TAS2R agonists were more potent than β-agonists, and we note the differences in the potency (the EC50) between isoproterenol and chloroquine are comparable between our study and Belvisi et al. The three-fold greater efficacy of TAS2R agonists compared to the β-agonist isoproterenol in our paper was specifically noted in the text in regard to mouse airways. With the limited number of human airways that we studied, we were not in position to provide quantitative efficacy data in human airways. We have now examined additional human rings after contraction with methacholine (Fig. 1a-c) and can adequately compare the efficacy of bitter tastants and isoproterenol. We find a 77 ± 4.3 % relaxation by isoproterenol (n=15 experiments), while chloroquine and quinine achieved essentially 100% relaxation (106 ± 4.7 and 99 ± 2.9 %, respectively (Fig. 1d). So, we still note a somewhat greater efficacy of bitter tastants compared to isoproterenol, but recognize that it is certainly less than the three-fold difference we observed in mice1, and attribute this difference to a species-effect. Thus we agree that in the ex vivo human airway ring model, the efficacy of these TAS2R agonists is similar to that of isoproterenol. We would like to point out, however, that the full β-agonist isoproterenol is not used clinically. The most commonly utilized inhaled β-agonist is albuterol, a partial agonist with, at best, a ~60% efficacy for relaxation in human airways compared to isoproterenol2,3. So from a clinical standpoint, it remains to be seen whether inhaled bitter tastants are more efficacious than this benchmark inhaled β-agonist. Figure 1 TAS2R agonist-mediated relaxation of human and mouse airways. a-c) Dose-dependent relaxation of human airway rings by isoproterenol (ISO), chloroquine (Chloro) and quinine. Shown are representative experiments in rings contracted with 0.1 mM methacholine ... We note differences in the methods of Belvisi et al compared to ours that might explain the modest decreased efficacy of isoproterenol that we find in human airways compared to quinine or chloroquine: inclusion of indomethacin in the myograph bath, contraction of the bronchi with different agents, the setting of a greater resting tension, and in the two experiments performed, they apparently combined data from both open strips of main bronchi and closed rings from lower order bronchi. Ironically, the authors normalized the data in their Fig. 1a,b, to the papaverine response, which further complicates interpretation because papaverine is recognized4 as an agonist for the two highest expressing TAS2Rs in airway smooth muscle (TAS2R10 and 14, ref.1). Finally, we caution about interpretations of data derived from two experiments (with a representative experiment shown), as there can be variability in contractile and relaxation responses in human airway rings from intrinsic inter-individual factors, chronic drug effects, and disease states. In contrast to our paper1, additional human airway data shown in Fig. 1a-d, and the correspondence from Belvisi et al., Morice et al. found little relaxation of human bronchi to chloroquine or quinine within the expected time-frame for agonist activation of G-protein coupled receptors such as a TAS2R, and, a lack of reversibility of the effect. They also find a minimal response to saccharin. In regards to saccharin, it should be noted that there is not an orthologous TAS2R for saccharin in mice, so we do not utilize this bitter tastant for physiologic studies in mice. We have limited experience with saccharin in intact human airways and would not be surprised to find that this agent acts as a partial agonist at these receptors for this response. The issue is not particularly relevant, given the wide variety of TAS2R agonists that are available, and the fact that saccharin does not activate a bitter taste receptor in mice or guinea pigs, which are commonly used for models of human airways disease. The kinetic data with chloroquine and quinine in human airways reported by Morice et al. is in contrast to what we have found1. In fact, these investigators suggest these agents promote “irreversible” relaxation or cell injury. However, we have shown that blockade of [Ca2+]i re-uptake, or antagonism of the BKCa channel, inhibits mouse airway relaxation to chloroquine, and in isolated human airway smooth muscle cells the relaxation to bitter tastants is blocked by a PLC inhibitor1. The results of these interventions, which are pathway specific, would be highly unlikely if TAS2R agonists cause nonspecific effects or cell injury. The assertions of Morice et al. are based on their finding of a slow onset of action and an inability to reverse the relaxation effect by washing and then re-contracting with methacholine in human airways obtained from surgical specimens. Since the primary set of observations from our paper were with inbred mice, we first turned to our ex vivo method in this species (where there is no confounding by genetic differences, chronic drug treatment, or disease) to specifically address the rate and reversibility issues. In new studies using a single concentration of chloroquine (3 mM) or quinine (1 mM), the time to maximal relaxation was found to be 4.9 ± 0.24 min and 4.4 ± 0.38 min for the two drugs, respectively (n=4, Fig. 1e). After contraction with acetylcholine and relaxation by chloroquine or quinine, we utilized four exchanges of Krebs buffer in the bath, and then re-challenged with the same dose of acetylcholine. The mean time from the last wash to 90% of the maximal contraction by the subsequent acetylcholine dose was 7.2 ± 0.85 min for chloroquine and 7.8 ± 0.48 min for quinine (n=4). The maximal extent of that subsequent contraction to acetylcholine compared to the first challenge was 95 ± 1.5%, and 96 ± 2.3%, respectively (P > 0.05, Fig. 1e). Having derived these kinetic values in mice, which are compatible with what we had previously observed1, we then applied this same approach to human airway rings, using 0.1 mM methacholine as the contractile agent (Fig. 1f). The times to maximal response to chloroquine and quinine were 8.6 ± 0.05 and 5.3 ±0.14 min, respectively (n=4), which is comparable to what was observed in mouse airways. These values clearly differ from Morice et al., who give mean times of 34 and 23 minutes for the two bitter tastants. Furthermore, like in the mouse airways, in human airways we observed ~90% reversal of the relaxant effect after washout and re-stimulation with methacholine (Fig. 1f). Again, these results differ markedly from those reported by Morice et al. who failed to observe any return of the contractile effect to methacholine once rings were exposed to chloroquine or quinine. Thus we demonstrate rapid on-rates, with virtually complete reversibility, in these human airways. Taken together, both mouse and human models indicate a rapid response to TAS2R agonists in a manner consistent with a typical agonist-GPCR interaction in airways, marked efficacy, and complete reversibility. Indeed, once re-contracted, airways can be relaxed again in a dose-dependent manner by another TAS2R agonist (Fig. 1e,f) or isoproterenol1. The suggestion by Morice et al. that these bitter tastants evoke cell injury is not compatible with these functional studies. There are differences between our technique and Morice et al. that might explain the slow onset of action and apparent irreversibility that they report for chloroquine and quinine. For our original studies1 and those reported here, we set the resting (passive) tension at ~5-10 mN (equivalent to ~0.50 to 1.0 g), while they utilized tensions of 2-3 g. This lower baseline stretch placed on rings in our studies might favor TAS2R mediated relaxation and functional recovery, while the greater tensions used by Morice et al. may not be conducive to relaxation by this specific mechanism or may promote cell injury in the context of the higher concentrations of TAS2R agonists that are required. We have thus now shown the relative efficacies of isoproterenol and two TAS2R agonists in relaxing human airway smooth muscle. The efficacy of the bitter tastants, in human airways studied under our conditions, is modestly greater than the full β-agonist, but the relevance of this difference at the physiologic or clinical level has not been defined. We show rapid on- and off-kinetics of the relaxation to these agonists, and functional recovery of contraction and a repeat relaxation, in mouse and human airways. Together with our original studies1 using inhibitors of the TAS2R pathway, we demonstrate airway relaxation via a receptor-mediated mechanism. These results continue to show that this previously unknown pathway may provide a novel mechanism to relax smooth muscle in the treatment of obstructive airway disease. Ultimately, human trials will be required to ascertain the clinical effectiveness of TAS2R agonists in the context of diseases such as asthma and chronic obstructive pulmonary disease.

Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway β2-adrenergic receptor physiologic signaling

Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize β2-adrenergic receptor (β2AR) signaling, and these are thought to be mechanisms involved with cell and organ homeostasis and tolerance to agonists. However, there is little direct evidence that these events are relevant to β2AR physiological function, such as airway smooth muscle (ASM) relaxation leading to bronchodilation. To maintain cell- and receptor-specificity without altering the natural complement of kinases/arrestins, transgenic mice were generated expressing the human WT and mutated β2ARs lacking PKA and/or GRK phosphorylation sites on ASM at ≈4-fold over background. Functional gains in response to β-agonist from the selective loss of these mechanisms were determined in mouse airways. Relaxation kinetics were altered in all mutant airways compared with β2WT. At low receptor occupancy, β2PKA(-) had enhanced agonist-promoted relaxation, while β2GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with β2GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the β2PKA(-)/GRK(-) airways had the greatest relaxation efficiency, indicating a graded effect of GRKs as agonist concentration increased. ASM cAMP levels paralleled relaxation phenotypes. No interaction between PKA phosphorylation of β2AR and GRK-promoted events was identified by β-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant β2AR physiologic function, acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve β-agonist treatment of obstructive lung disease.

Reply to: Activation of BK channels may not be required for bitter tastant-induced bronchodilation.

Reply to: Activation of BK channels may not be required for bitter tastant–induced bronchodilation

Targeted transgenesis identifies Gαs as the bottleneck in β2-adrenergic receptor cell signaling and physiological function in airway smooth muscle

G protein-coupled receptors are the most pervasive signaling superfamily in the body and act as receptors to endogenous agonists and drugs. For β-agonist-mediated bronchodilation, the receptor-G protein-effector network consists of the β2-adrenergic receptor (β2AR), Gs, and adenylyl cyclase, expressed on airway smooth muscle (ASM). Using ASM-targeted transgenesis, we previously explored which of these three early signaling elements represents a limiting factor, or bottleneck, in transmission of the signal from agonist binding to ASM relaxation. Here we overexpressed Gαs in transgenic mice and found that agonist-promoted relaxation of airways was enhanced in direct proportion to the level of Gαs expression. Contraction of ASM from acetylcholine was not affected in Gαs transgenic mice, nor was relaxation by bitter taste receptors. Furthermore, agonist-promoted (but not basal) cAMP production in ASM cells from Gαs-transgenic mice was enhanced compared with ASM from nontransgenic littermates. Agonist-promoted inhibition of platelet-derived growth factor-stimulated ASM proliferation was also enhanced in Gαs mouse ASM. The enhanced maximal β-agonist response was of similar magnitude for relaxation, cAMP production, and growth inhibition. Taken together, it appears that a limiting factor in β-agonist responsiveness in ASM is the expression level of Gαs. Gene therapy or pharmacological means of increasing Gαs (or its coupling efficiency to β2AR) thus represent an interface for development of novel therapeutic agents for improvement of β-agonist therapy.