The Cellular and Molecular Basis of Bitter Tastant-Induced Bronchodilation

Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca²⁺ signals, as revealed in cultured ASM cells, to activate large-conductance Ca²⁺-activated K⁺ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca²⁺ events nor alter spontaneous local Ca²⁺ transients. Interestingly, they increase global intracellular [Ca²⁺]_i, although to a much lower level than bronchoconstrictors. We show that these Ca²⁺ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca²⁺]_i induced by bronchoconstrictors, and this lowering of the [Ca²⁺]_i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca²⁺ channels (VDCCs), resulting in reversal in [Ca²⁺]_i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca²⁺ signaling pathways via Gβγ to increase [Ca²⁺]_i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca²⁺]_i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.     

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 (IP?) 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(+) (BK(Ca)) 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.     

Biochemical interactions among intercellular adhesion molecules expressed by airway epithelial cells

Intercellular adhesion between adjacent airway epithelial cells plays a critical role in maintaining the barrier function of the respiratory mucosa. In the current study, we examined the expression and interaction of cell surface adhesion molecules (E-cadherin, ICAM-1, and MUC1) and their intracellular binding partners (alpha-catenin, beta-catenin, gamma-catenin, and ezrin) in 16HBE14o-, HBE1, 1HAEo-, BEAS-2B, A549, and NCI-H292 human airway epithelial cells. Expression of E-cadherin and MUC1, both in whole cell lysates and biotinylated surface proteins, was observed in 16HBE14o-, HBE1, A549, and NCI-H292 cells, while ICAM-1 was detected only in NCI-H292. In contrast, alpha-, beta-, and gamma-catenin and ezrin were expressed in all of the cells. E-cadherin formed coimmunoprecipitation complexes with beta- and gamma-catenin, whereas MUC1 only associated with beta-catenin. ICAM-1, but not MUC1, coimmunoprecipitated with ezrin in NCI-H292 cells. We conclude that airway epithelial cell-cell adhesion involves a complex network of protein-protein interactions mediated by a diverse array of membrane-bound and cytosolic protein partners.     

Taste and smell GPCRs in the lung: Evidence for a previously unrecognized widespread chemosensory system

Taste and smell receptor expression has been traditionally limited to the tongue and nose. We have identified bitter taste receptors (TAS2Rs) and olfactory receptors (ORs) on human airway smooth muscle (HASM) cells. TAS2Rs signal to PLCβ evoking an increase in [Ca^2 +]_i causing membrane hyperpolarization and marked HASM relaxation ascertained by single cell, ex vivo, and in vivo methods. The presence of TAS2Rs in the lung was unexpected, as was the bronchodilatory function which has been shown to be due to signaling within specific microdomains of the cell. Unlike β₂-adrenergic receptor-mediated bronchodilation, TAS2R function is not impaired in asthma and shows little tachyphylaxis. HASM ORs do not bronchodilate, but rather modulate cytoskeletal remodeling and hyperplasia, two cardinal features of asthma. We have shown that short chain fatty acids, byproducts of fermentation of polysaccharides by the gut microbiome, activate HASM ORs. This establishes a non-immune gut-lung mechanism that ties observations on gut microbial communities to asthma phenotypes. Subsequent studies by multiple investigators have revealed expression and specialized functions of TAS2Rs and ORs in multiple cell-types and organs throughout the body. Collectively, the data point towards a previously unrecognized chemosensory system which recognizes endogenous and exogenous agonists. These receptors and their ligands play roles in normal homeostatic functions, predisposition or adaptation to disease, and represent drug targets for novel therapeutics.     

Effect of the BMPR-II-SMAD3/MRTF pathway on proliferation and migration of ASMCs and the mechanism in asthma

Background

A variety of smooth muscle-specific genes and proteins, including SMAD3, BMPR-II, and MRTF, are involved in airway remodeling in asthma. As a receptor of bone morphogenetic protein (BMP) signaling, BMPR-II has important roles in airway remodeling in asthma. However, the underlying mechanism of BMPR-II in airway smooth muscle cells (ASMCs) in asthma remains incomplete.

Methods

Wistar rats were intraperitoneally injected with ovalbumin antigen suspension and aluminium hydroxide and, stimulated with ovalbumin nebulized inhalation to constructed asthma model. Primary ASMCs were isolated with collagenase I and identified by testing the α-SMA expression. Quantitative polymerase chain reaction (qPCR) and western blot assay were employed to detect the gene expression. CCK8, Transwell and Fluo-4 A assays were introduced to measure the cell viability, migration and intracellular Ca²⁺. Co-Immunoprecipitation (Co-IP) assay was applied to test the interaction among proteins.

Results

First, we observed significant increases in BMPR-II in asthmatic rat model and ASMCs at both the mRNA and protein levels. Second, we observed that silencing of siBMPR-II inhibited proliferation, migratory capacity and intracellular Ca²⁺ concentration in ASMCs. Furthermore, our study demonstrated that siBMPR-II inhibited the Smad3 expression and overexpression promoted the bioactivity of ASMCs. In addition, this study showed that p-Smad3 could interacted with MRTF and siMRTF inhibits the bioactivity of ASMCs. Finally, our results revealed BMPR-II-SMAD3/MRTF pathway affected the bioactivity of ASMCs.

Conclusions

This study indicates that the BMPR-II-SMAD3/MRTF signaling pathway is involved in the process of ASMCs remodeling, providing novel avenues for the identification of new therapeutic modalities.

     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells

To test the hypothesis that the excess inflammatory response in cystic fibrosis airway epithelial cells is the result of differential activation of genes in response to a laboratory strain of Pseudomonas aeruginosa (PAO1), a 48-h time course of genes expressed following PAO1 stimulation (10(9) CFU for 1 h) was studied in two pairs of airway epithelial cells: 9/HTEo- and 16HBE14o-, each with a matched normal and CF phenotype pair. cRNA was hybridized to Affymetrix HG-U95Av2 chips and pairwise comparisons against zero time (no PAO1) were calculated for each time point. PAO1 elicited profound changes in both cell pairs: for 9/HTEo-, 144 genes changed significantly in the normal pair, and 116 for the CF pair; for the 16HBE14o- pair, 57 genes changed significantly for the normal pair and 53 for the CF pair. Changes were much greater in the 9/HTEo- than in the 16HBE14o- pair, but basal levels of expression of inflammatory genes are higher in the 16HBE14o- pair, so 16HBE14o- was used mainly to corroborate the results of 9/HTEo-. Clustering analysis indicated that the pattern of gene expression is similar in the CF cells and their normal counterparts. However, there were substantial quantitative differences in gene expression. Thus, the difference between CF and normal resides in the magnitude, not the pattern, of the changes.     

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival

Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

     

A novel family of mammalian taste receptors

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.     

The human taste receptor hTAS2R14 responds to a variety of different bitter compounds

The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, (-)-alpha-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of (-)-alpha-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances.     

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

Background

Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs).

Methods

Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1.

Results

HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA.

Conclusion

These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.     

Phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression

Rhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-kappaB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis.