Bitter Taste Receptor Agonists Research Articles

GLUT1 inhibitor BAY-876 induces apoptosis and enhances anti-cancer effects of bitter receptor agonists in head and neck squamous carcinoma cells

Head and neck squamous cell carcinomas (HNSCCs) are cancers that arise in the mucosa of the upper aerodigestive tract. The five-year patient survival rate is ~50%. Treatment includes surgery, radiation, and/or chemotherapy and is associated with lasting effects even when successful in irradicating the disease. New molecular targets and therapies must be identified to improve outcomes for HNSCC patients. We recently identified bitter taste receptors (taste family 2 receptors, or T2Rs) as a novel candidate family of receptors that activate apoptosis in HNSCC cells through mitochondrial Ca2+ overload and depolarization. We hypothesized that targeting another component of tumor cell metabolism, namely glycolysis, may increase the efficacy of T2R-directed therapies. GLUT1 (SLC2A1) is a facilitated-diffusion glucose transporter expressed by many cancer cells to fuel their increased rates of glycolysis. GLUT1 is already being investigated as a possible cancer target, but studies in HNSCCs are limited. Examination of immortalized HNSCC cells, patient samples, and The Cancer Genome Atlas revealed high expression of GLUT1 and upregulation in some patient tumor samples. HNSCC cells and tumor tissue express GLUT1 on the plasma membrane and within the cytoplasm (perinuclear, likely co-localized with the Golgi apparatus). We investigated the effects of a recently developed small molecule inhibitor of GLUT1, BAY-876. This compound decreased HNSCC glucose uptake, viability, and metabolism and induced apoptosis. Moreover, BAY-876 had enhanced effects on apoptosis when combined at low concentrations with T2R bitter taste receptor agonists. Notably, BAY-876 also decreased TNFα-induced IL-8 production, indicating an additional mechanism of possible tumor-suppressive effects. Our study demonstrates that targeting GLUT1 via BAY-876 to kill HNSCC cells, particularly in combination with T2R agonists, is a potential novel treatment strategy worth exploring further in future translational studies.

Relevance of Phytochemical Taste for Anti-Cancer Activity: A Statistical Inquiry.

Targeting inflammation and the pathways linking inflammation with cancer is an innovative therapeutic strategy. Tastants are potential candidates for this approach, since taste receptors display various biological functions, including anti-inflammatory activity (AIA). The present study aims to explore the power different tastes have to predict a phytochemical's anti-cancer properties. It also investigates whether anti-inflammatory phytocompounds also have anti-cancer effects, and whether there are tastes that can better predict a phytochemical's bivalent biological activity. Data from the PlantMolecularTasteDB, containing a total of 1527 phytochemicals, were used. Out of these, only 624 phytocompounds met the inclusion criterion of having 40 hits in a PubMed search, using the name of the phytochemical as the keyword. Among them, 461 phytochemicals were found to possess anti-cancer activity (ACA). The AIA and ACA of phytochemicals were strongly correlated, irrespective of taste/orosensation or chemical class. Bitter taste was positively correlated with ACA, while sweet taste was negatively correlated. Among chemical classes, only flavonoids (which are most frequently bitter) had a positive association with both AIA and ACA, a finding confirming that taste has predictive primacy over chemical class. Therefore, bitter taste receptor agonists and sweet taste receptor antagonists may have a beneficial effect in slowing down the progression of inflammation to cancer.

Bitter Taste Receptor Agonist Denatonium Inhibits Stemness Characteristics in Hematopoietic Stem/Progenitor Cells.

Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.

Therapeutic potential of bitter taste receptor agonists on amyloid β-induced rat model of Alzheimer’s disease

ABSTRACT This study was conducted to investigate the effect of bitter taste receptor (T2R) agonists (caffeine and extracts of myrrh and Boswellia serrata) on amyloid β (Aβ)-induced Alzheimer’s disease (AD) in rats. Rats intracerebroventricularly (ICV) injected with a single dose of Aβ (3 μg/μL/rat) had significantly: 1) lower brain weight, 2) higher short-term memory impairment, 3) lower serum dopamine level, 4) higher AChE activity, 5) higher brain lipid peroxide malonaldehyde, 6) lower brain antioxidant activities of catalase, superoxide dismutase and glutathione peroxidase, 7) higher brain phosphor tau protein level, 8) higher brain levels of apoptotic markers (Bax and caspase 3), 9) lower brain level of antiapoptotic marker Bcl2, 10) downregulated brain expression of the bitter receptor (T2R4) gene, 11) upregulated brain expression of tumor necrosis factor α (TNFα) and nuclear factor κB (NF-κB) genes and 12) marked neuronal degeneration associated with severe vacuolation of neutrophils. All deteriorated effects of Aβ were restored following treatment with caffeine (20 mg/kg), myrrh aqueous extract (10 mg/kg) and Boswellia serrata aqueous extract (400 mg/kg) with best effect for myrrh aqueous extract. These data conclude that bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) had therapeutic potential on amyloid β-induced rat model of Alzheimer’s disease.

Nebulized caffeine alleviates airway hyperresponsiveness in a murine asthma model.

The clinical definition of "difficult asthma" has expanded recently to include an ever-growing subset of patients with symptoms that cannot be controlled by conventional means, forcing the medical community to develop innovative therapeutics. Beneficial effects of coffee for subjects with asthma, primarily the effect of methylxanthine components, have long been described. Methylxanthines, including theophylline and caffeine, inhibit phosphodiesterases and downstream cAMP signaling to prevent mast cell degranulation while promoting immunomodulation (Peleman RA, Kips JC, Pauwels RA. Clin Exp Allergy 28: 53-56, 1998; Deshpande DA, Wang WCH, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JSK, Liggett SB. Nat Med 16: 1299-1304, 2010). Caffeine is also a bitter taste receptor agonist, binding to taste-sensing type 2 receptors (TAS2R) before releasing calcium to hyperpolarize airway smooth muscle membranes, inducing bronchodilation (Workman AD, Palmer JN, Adappa ND, Cohen NA. Curr Allergy Asthma Rep 15: 72, 2015; Devillier P, Naline E, Grassin-Delyle S. Pharmacol Ther 155: 11-21, 2015). Theophylline is conventionally used to treat asthma, whereas, according to the literature, the dosage required for orally administered caffeine has yielded modest improvement (Alfaro TM, Monteiro RA, Cunha RA, Cordeiro CR. Clin Respir J 12: 1283-1294, 2018). We sought to determine whether aerosolization of ultrafine caffeine particles (2.5-4 μm) directly to the lungs of susceptible A/J mice challenged with methacholine would improve pulmonary function via forced oscillation technique. In addition, we assessed whether nebulization of caffeine leads to changes in lung pathophysiology and bronchoalveolar lavage cell profiles. We found that mice that received aerosolized caffeine had statistically significant decreases in maximum airway resistance [6.3 vs. 3.9 cmH2O·s/mL at 62.5 mg/mL caffeine; confidence interval (CI) = -4.3, -0.4; P = 0.02] and significant delays in the time required to reach maximum resistance compared with that of controls (64.7 vs. 172.1 sec at 62.5 mg/mL caffeine, CI = 96.0, 118.9; P < 0.0001). Nebulized caffeine yielded a consistent effect on airway hyperresponsiveness at a range of doses without evidence of significant pathology relative to vehicle control.NEW & NOTEWORTHY For decades, coffee has been shown to improve symptoms in patients with asthma. One component, theophylline, is conventionally used to treat asthma, whereas the dosage required for orally administered caffeine has yielded modest improvement. We sought to determine whether aerosolization of caffeine directly to the lungs of susceptible A/J mice challenged with methacholine would alter pulmonary function via forced oscillation technique. We found nebulized caffeine yielded a consistent improvement on murine AHR.

The bitter taste receptor (TAS2R) agonist denatonium promotes a strong relaxation of rat corpus cavernosum

Bitter taste receptors (TAS2R) are found in numerous extra-oral tissues, including smooth muscle (SM) cells in both vascular and visceral tissues. Upon activation, TAS2R stimulate the relaxation of the SM. Nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is involved in penile erection, and type 5 phosphodiesterase (PDE5) inhibitors, a cGMP-specific hydrolase are used as first-line treatments for erectile dysfunction (ED). Nevertheless, PDE5 inhibitors are ineffective in a considerable number of patients, prompting research into alternative pharmacological targets for ED. Since TAS2R agonists regulate SM contractility, this study investigates the role of TAS2Rs in rat corpus cavernosum (CC). We performed immunohistochemistry to detect TAS2R10, isometric force recordings for TAS2R agonists denatonium and chloroquine, the slow-release H2S donor GYY 4137, the NO donor SNAP, the β-adrenoceptor agonist isoproterenol and electrical field stimulation (EFS), as well as measurement of endogenous hydrogen sulfide (H2S) production. The immunofluorescence staining indicated that TAS2R10 was broadly expressed in the CC SM and to some extent in the nerve fibers. Denatonium, chloroquine, SNAP, and isoproterenol cause potent dose-dependent SM relaxations. H2S production was decreased by NO and H2S synthase inhibitors, while it was enhanced by denatonium. In addition, denatonium increased the relaxations induced by GYY 4137 and SNAP but failed to modify EFS- and isoproterenol-induced responses. These results suggest neuronal and SM TAS2R10 expression in the rat CC, where denatonium induces a strong SM relaxation per se and promotes the H2S- and NO-mediated inhibitory gaseous neurotransmission. Thus, TAS2R10 might represent a valuable therapeutic target in ED.

Utilizing the Off-Target Effects of T1R3 Antagonist Lactisole to Enhance Nitric Oxide Production in Basal Airway Epithelial Cells.

Human airway sweet (T1R2 + T1R3), umami (T1R1 + T1R3), and bitter taste receptors (T2Rs) are critical components of the innate immune system, acting as sensors to monitor pathogenic growth. T2Rs detect bacterial products or bitter compounds to drive nitric oxide (NO) production in both healthy and diseased epithelial cell models. The NO enhances ciliary beating and also directly kills pathogens. Both sweet and umami receptors have been characterized to repress bitter taste receptor signaling in healthy and disease models. We hypothesized that the sweet/umami T1R3 antagonist lactisole may be used to alleviate bitter taste receptor repression in airway basal epithelial cells and enhance NO production. Here, we show that lactisole activates cAMP generation, though this occurs through a pathway independent of T1R3. This cAMP most likely signals through EPAC to increase ER Ca2+ efflux. Stimulation with denatonium benzoate, a bitter taste receptor agonist which activates largely nuclear and mitochondrial Ca2+ responses, resulted in a dramatically increased cytosolic Ca2+ response in cells treated with lactisole. This cytosolic Ca2+ signaling activated NO production in the presence of lactisole. Thus, lactisole may be useful coupled with bitter compounds as a therapeutic nasal rinse or spray to enhance beneficial antibacterial NO production in patients suffering from chronic inflammatory diseases such as chronic rhinosinusitis.

Cilia Stimulatory and Antibacterial Activities of T2R Bitter Taste Receptor Agonist Diphenhydramine: Insights into Repurposing Bitter Drugs for Nasal Infections.

T2R bitter taste receptors in airway motile cilia increase ciliary beat frequency (CBF) and nitric oxide (NO) production. Polymorphisms in some T2Rs are linked to disease outcomes in chronic rhinosinusitis (CRS) and cystic fibrosis (CF). We examined the expression of cilia T2Rs during the differentiation of human nasal epithelial cells grown at air–liquid interface (ALI). The T2R expression increased with differentiation but did not vary between CF and non-CF cultures. Treatment with Pseudomonas aeruginosa flagellin decreased the expression of diphenhydramine-responsive T2R14 and 40, among others. Diphenhydramine increased both NO production, measured by fluorescent dye DAF-FM, and CBF, measured via high-speed imaging. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired the growth of lab and clinical strains of P. aeruginosa, a major pathogen in CF and CF-related CRS. Diphenhydramine impaired biofilm formation of P. aeruginosa, measured via crystal violet staining, as well as the surface attachment of P. aeruginosa to CF airway epithelial cells, measured using colony-forming unit counting. Because the T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, diphenhydramine-derived compounds may have potential clinical usefulness in CF-related CRS as a topical therapy. However, utilizing T2R agonists as therapeutics within the context of P. aeruginosa infection may require co-treatment with anti-inflammatories to enhance T2R expression.

Therapeutic effects of quinine in a mouse model of atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that seriously affects quality of life. Quinine is a bitter taste receptor agonist that exhibits antimalarial effects. The aim of the present study was to examine the therapeutic effects of quinine in AD-like mice. AD was induced with 2,4-dinitrochlorobenzene, and the mice were treated with 10 mg/kg quinine for 1, 4 and 7 days. A total of 60 BALB/c mice were divided into the following groups: Healthy, AD-like, AD-like + quinine and healthy + quinine, with 1, 4 and 7 days groups for each treatment. Blood was extracted from all mice and ELISA was performed to detect immunoglobulin E (IgE) levels. H&E-stained tissue sections were prepared from skin lesions on the backs of the mice and pathological changes were observed. Cytokines were detected via ELISA, and the filaggrin (FLG) and kallikrein-7 (KLK7) proteins were detected via western blotting and immunohistochemistry. IKKα and NF-κB mRNA were analyzed via reverse transcription-quantitative PCR. Quinine ameliorated skin damage in the AD-like mice, reduced IgE expression in the blood, inhibited expression of IKKα and NF-κB, reduced cytokine secretion, reduced KLK7 expression, reduced scratching frequency, increased FLG expression and repaired the skin barrier. These results suggested that quinine exhibited therapeutic effects in AD-like mice.

Chloroquine: Autophagy inhibitor, antimalarial, bitter taste receptor agonist in fight against COVID-19, a reality check?

The recent SARS-CoV-2 pandemic poses one of the greatest challenges to modern medicine. Therefore, identification of new therapeutic strategies seems essential either based on novel vaccines or drugs or simply repurposing existing drugs. Notably, due to their known safety profile, repurposing of existing drugs is the fastest and highly efficient approach to bring a therapeutic to a clinic for any new indication. One such drug that has been used extensively for decades is chloroquine (CQ, with its derivatives) either for malaria, lupus and rheumatoid arthritis. Accumulating body of evidence from experimental pharmacology suggests that CQ and related analogues also activate certain pathways that can potentially be exploited for therapeutic gain. For example, in the airways, this has opened an attractive avenue for developing novel bitter taste ligands as a new class of bronchodilators for asthma. While CQ and its derivatives have been proposed as a therapy in COVID-19, it remains to be seen whether it really work in the clinic? To this end, our perspective aims to provide a timely yet brief insights on the existing literature on CQ and the controversies surrounding its use in COVID-19. Further, we also highlight some of cell-based mechanism(s) that CQ and its derivatives affect in mediating variety of physiological responses in the cell. We believe, data emanating from the clinical studies and continual understanding of the fundamental mechanisms may potentially help in designing effective therapeutic strategies that meets both efficacy and safety criteria for COVID-19.

Bitter taste receptor agonists regulate epithelial two-pore potassium channels via cAMP signaling

BackgroundEpithelial solitary chemosensory cell (tuft cell) bitter taste signal transduction occurs through G protein coupled receptors and calcium-dependent signaling pathways. Type II taste cells, which utilize the same bitter taste signal transduction pathways, may also utilize cyclic adenosine monophosphate (cAMP) as an independent signaling messenger in addition to calcium.MethodsIn this work we utilized specific pharmacologic inhibitors to interrogate the short circuit current (Isc) of polarized nasal epithelial cells mounted in Ussing chambers to assess the electrophysiologic changes associated with bitter agonist (denatonium) treatment. We also assessed release of human β-defensin-2 from polarized nasal epithelial cultures following treatment with denatonium benzoate and/or potassium channel inhibitors.ResultsWe demonstrate that the bitter taste receptor agonist, denatonium, decreases human respiratory epithelial two-pore potassium (K2P) current in polarized nasal epithelial cells mounted in Ussing chambers. Our data further suggest that this occurs via a cAMP-dependent signaling pathway. We also demonstrate that this decrease in potassium current lowers the threshold for denatonium to stimulate human β-defensin-2 release.ConclusionsThese data thus demonstrate that, in addition to taste transducing calcium-dependent signaling, bitter taste receptor agonists can also activate cAMP-dependent respiratory epithelial signaling pathways to modulate K2P currents. Bitter-agonist regulation of potassium currents may therefore serve as a means of rapid regional epithelial signaling, and further study of these pathways may provide new insights into regulation of mucosal ionic composition and innate mechanisms of epithelial defense.

Bitter taste receptor agonists induced chronotropic effects on rat heart

BackgroundBitter taste receptors (Tas2rs), the members of the G‐protein‐coupled receptors, mediate the bitter taste and express in extra‐oral tissues. Previous studies have shown that Tas2r mRNAs are expressed in the whole heart and cultured cardiomyocytes of neonatal rat.AimThis study investigates the expression of Tas2rs in the sinoatrial node (SA node) and left ventricle of rat heart, and the mechanism of bitter compounds regulation of the heart rate.MethodsRats were excised by thoracotomy and the sinoatrial node and left ventricle tissues were isolated. These tissues were processed to study the mRNA expression levels of Tas2rs and their coupled G‐protein subunits by real time quantitative PCR (qPCR). Langendorff isolated heart models were used to record the heart rate and electrocardiogram. SA node beating recordings were used to measure the beating rate in combination with bitter compounds, quinine and chloroquine. The statistic dada are expressed as mean ± SEM.ResultsWe detected the mRNA expression of 7 subtypes of Tas2rs (Tas2r108, 120, 121, 126, 135, 137, 143) in the left ventricle and 8 subtypes in the SA node including Tas2r38 in addition to the 7 subtypes in the left ventricle, in which the mRNA level of Tas2r120 is the highest among them followed by Tas2r143 and 121. In addition, Tas2r coupled G‐protein subunits also expressed in the SA node and left ventricle.Tas2r agonists, quinine (1–50 μM) and chloroquine (1–20 μM), decreased the heart rate and increased the RR interval and QRS duration in Langendoff‐perfused isolated rat hearts; they reduced the spontaneous beating rate of isolated SA nodes with pEC50 values of 4.907 ± 0.045 and 4.968 ± 0.030, respectively.The blockade of Tas2r108 with abscisic acid (500 μM), the inhibition of PDEs with IBMX (50 μM), or the selective inhibition of PDE3 and PDE4 with a cocktail of cilostamide (0.5 μM) and rolipram (10 μM), attenuated the negative chronotropic effects of quinine and chloroquine on the SA node. Furthermore, quinine (20 μM) and chloroquine (10 μM) suppressed the tachycardia effect of isoprenaline on the SA node and shifted the concentration‐response curve of isoprenaline rightward.ConclusionTas2r agonists, quinine and chloroquine, induce negative ionotropic effects in rat hearts. Our data provide mechanistic evidence that Tas2r agonists decreased the heart rate by prolonging ventricular depolarization, and by attenuating the SA node pace in a PDE‐dependent manner. Tas2r agonists can counteract with β‐adrenergic receptor activation and eliminate isoprenaline‐induced tachycardia, suggesting Tas2rs could be the potential targets of drugs for treating sinus or ventricular tachycardia and elevated heart rate related to heart failure.Support or Funding InformationThis work was supported by Funding from Southwest University [Grant number SWU 115086, 104290/22300503] and Administration of Foreign Experts Affairs of China [Grant number BC2018042].

Denatonium as a bitter taste receptor agonist damages jejunal epithelial cells of yellow-feathered chickens via inducing apoptosis

The sense of bitter taste is critical for chickens to acquire and select feeds. It is important to understand the roles and mechanisms of bitter taste transduction in chickens. Denatonium is extensively used as a bitter taste receptor agonist to activate bitter taste receptors in recent studies. The objective of this study was to investigate the physiological effects and the potential molecular mechanisms of dietary exposure to a strong bitter taste receptor agonist on the jejunal epithelial cells of yellow-feathered chickens. A total of 240 yellow-feathered chickens were divided into four treatments receiving a normal diet (Control), a low-dose denatonium treatment (Control + 5 mg/kg denatonium), a middle-dose denatonium treatment (Control + 20 mg/kg denatonium) and a high-dose denatonium treatment (Control + 100 mg/kg denatonium) for 56 days, respectively. The results showed that dietary denatonium reduced (P < 0.05) the growth performance of chickens. High-dose denatonium damaged the morphology of the jejunal epithelium and decreased (P < 0.05) the activities of Ca2+-ATPase, sucrase and maltase after 56 days of exposure. Meanwhile, high-dose denatonium increased (P < 0.05) mRNA expressions of bitter taste receptors, which resulted in enhanced apoptosis in jejunal epithelial cells after 56 days of exposure. Furthermore, middle-dose and high-dose denatonium exhibited increased (P < 0.05) mRNA level of claudin 2 and decreased (P < 0.05) mRNA level of occludin after 28 days of exposure. Only high-dose denatonium decreased (P < 0.05) mRNA level of occludin after 56 days of exposure. In conclusion, denatonium manifested deleterious effects on the jejunum of chickens in a dose–effect manner via damaging the morphology of the jejunal epithelium, and inducing apoptosis associated with bitter taste receptors. Our data suggest that bitter-tasting feed additives may have side effects on the growth and development of intestines in chickens.

Bitter taste receptor agonists to abate the bitterness of genital infections: A hype or hope?

Bitter taste receptor agonists to abate the bitterness of genital infections: A hype or hope?

The future of bronchodilation: looking for new classes of bronchodilators.

Available bronchodilators can satisfy many of the needs of patients suffering from airway disorders, but they often do not relieve symptoms and their long-term use raises safety concerns. Therefore, there is interest in developing new classes that could help to overcome the limits that characterise the existing classes.At least nine potential new classes of bronchodilators have been identified: 1) selective phosphodiesterase inhibitors; 2) bitter-taste receptor agonists; 3) E-prostanoid receptor 4 agonists; 4) Rho kinase inhibitors; 5) calcilytics; 6) agonists of peroxisome proliferator-activated receptor-γ; 7) agonists of relaxin receptor 1; 8) soluble guanylyl cyclase activators; and 9) pepducins. They are under consideration, but they are mostly in a preclinical phase and, consequently, we still do not know which classes will actually be developed for clinical use and whether it will be proven that a possible clinical benefit outweighs the impact of any adverse effect.It is likely that if developed, these new classes may be a useful addition to, rather than a substitution of, the bronchodilator therapy currently used, in order to achieve further optimisation of bronchodilation.

Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions.

Simple SummaryDenatonium benzoate is a strong bitter taste receptor agonist, extensively used for its activation of different cell pathways. Taste signals have been associated to food recognition and avoidance, and bitter taste provokes an aversive reaction and is assumed to protect chickens from consuming poisons and harmful toxic substances. The results of the study revealed that dietary supplementation with medium and high doses of denatonium benzoate damaged the epithelial cells of the heart and kidneys by inducing apoptosis and autophagy and reduced the growth of chickens, respectively. However, mRNA expressions of bitter taste receptors, downstream signaling effector genes, apoptosis-, autophagy- and antioxidant-related genes were higher on day 7, while these expressions were subsequently decreased on day-28 in the heart and kidney of Chinese Fast Yellow chickens in a dose-response manner.The sense of taste which tells us which prospective foods are nutritious, poisonous and harmful is essential for the life of the organisms. Denatonium benzoate (DB) is a bitter taste agonist known for its activation of bitter taste receptors in different cells. The aim of the current study was to investigate the mRNA expressions of bitter taste, downstream signaling effectors, apoptosis-, autophagy- and antioxidant-related genes and effector signaling pathways in the heart/kidney of chickens after DB dietary exposure. We randomly assigned 240, 1-day-old Chinese Fast Yellow chicks into four groups with five replicates of 12 chicks and studied them for 28 consecutive days. The dietary treatments consisted of basal diet and feed containing DB (5, 20 and 100 mg/kg). The results revealed that dietary DB impaired (p < 0.05) the growth performance of the chickens. Haemotoxylin and eosin staining and TUNEL assays confirmed that medium and high doses of DB damaged the epithelial cells of heart/kidney and induced apoptosis and autophagy. Remarkably, the results of RT-PCR and qRT-PCR indicated that different doses of DB gradually increased (p < 0.05) mRNA expressions of bitter taste, signaling effectors, apoptosis-, autophagy- and antioxidant- related genes on day 7 in a dose-response manner, while, these expressions were decreased (p < 0.05) subsequently by day-28 but exceptional higher (P < 0.05) expressions were observed in the high-dose DB groups of chickens. In conclusion, DB exerts adverse effects on the heart/kidney of chickens in a dose-response manner via damaging the epithelium of the heart/kidney by inducing apoptosis, autophagy associated with bitter taste and effector gene expressions. Correlation analyses for apoptosis/autophagy showed agonistic relationships. Our data provide a novel perspective for understanding the interaction of bitter taste, apoptosis, autophagy and antioxidative genes with bitter taste strong activators in the heart/kidney of chicken. These insights might help the feed industries and pave the way toward innovative directions in chicken husbandry.

Responsiveness Expressions of Bitter Taste Receptors Against Denatonium Benzoate and Genistein in the Heart, Spleen, Lung, Kidney, and Bursa Fabricius of Chinese Fast Yellow Chicken.

Simple SummaryIn chickens, bitter taste is the most significant biological taste disrupter; it is believed to protect chickens against consuming poisonous/toxic materials and considered a warning signal prior to ingestion. The bitter taste receptors’ extraoral expression information is deficient in chicken, and denatonium benzoate is extensively used as a bitter taste receptor agonist in different cells. Our results found that qRT-PCR showed a high level of dose-dependent expressions of ggTas2Rs in the starter and grower stages in the heart, spleen, lungs, and kidneys, while the dose-dependent expressions were lower in the bursa Fabricius. The growth performance of the selected organs significantly (and unexpectedly) improved upon the administration of denatonium benzoate 5 mg/kg and genistein 25 mg/kg treatments, while the gains in organ weights were impaired in the groups given denatonium benzoate 20 mg/kg and 100 mg/kg, respectively.The present study was conducted to investigate the responsiveness expressions of ggTas2Rs against denatonium benzoate (DB) and genistein (GEN) in several organs of the Chinese Fast Yellow Chicken. A total of 300 one-day-old chicks that weighed an average of 32 g were randomly allocated into five groups with five replicates for 56 consecutive days. The dietary treatments consisted of basal diet, denatonium benzoate (5 mg/kg, 20 mg/kg, and 100 mg/kg), and genistein 25 mg/kg. The results of qRT-PCR indicated significantly (p < 0.05) high-level expressions in the heart, spleen, and lungs in the starter and grower stages except for in bursa Fabricius. The responsiveness expressions of ggTas2Rs against DB 100 mg/kg and GEN 25 mg/kg were highly dose-dependent in the heart, spleen, lungs, and kidneys in the starter and grower stages, but dose-independent in the bursa Fabricius in the finisher stage. The ggTas2Rs were highly expressed in lungs and the spleen, but lower in the bursa Fabricius among the organs. However, the organ growth performance significantly (p < 0.05) increased in the groups administered DB 5 mg/kg and GEN 25 mg/kg; meanwhile, the DB 20 mg/kg and DB 100 mg/kg treatments significantly reduced the growth of all the organs, respectively. These findings indicate that responsiveness expressions are dose-dependent, and bitterness sensitivity consequently decreases in aged chickens. Therefore, these findings may improve the production of new feedstuffs for chickens according to their growing stages.

Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2+–shuttling event

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 μm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3β-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the β2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.

Biased TAS2R Bronchodilators Inhibit Airway Smooth Muscle Growth by Downregulating Phosphorylated Extracellular Signal-regulated Kinase 1/2.

Bitter taste receptor (TAS2R) agonists dilate airways by receptor-dependent smooth muscle relaxation. Besides their coupling to relaxation, we have found that human airway smooth muscle (HASM) cell TAS2Rs activate (phosphorylate) extracellular signal-related kinase 1/2 (ERK1/2), but the cellular effects are not known. In the present study, we show in HASM cells that TAS2R agonists initially stimulate phosphorylated ERK1/2 (pERK1/2) but by 24 hours cause a marked (50-70%) downregulation of pERK1/2 without a change in total ERK1/2. It was hypothesized that TAS2R agonists suppress cell growth through this pERK1/2 downregulation. Agonist-dependent inhibition of cell proliferation was indeed found in HASM cells derived from normal and asthmatic human lungs, as well as in an immortalized HASM cell line. pERK1/2 downregulation was linked to downregulation of the upstream kinase MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase). Various structurally diverse TAS2R agonists evoked a range of inhibition of HASM proliferation, the magnitude of which directly correlated with the downregulation of pERK1/2 (R2 = 0.86). Some TAS2R agonists were as effective as pharmacological inhibitors of Raf1 and MEK1/2 in suppressing growth. siRNA silencing of TAS2Rs (subtypes 10, 14, and 31) ablated the pERK1/2 and growth-inhibitory effects of TAS2R agonists. These phenotypes were attenuated by inhibiting the TAS2R G protein Gαi and by knocking down β-arrestin 1/2, indicating a dual pathway, although there may be additional mechanisms involved in this HASM TAS2R multidimensional signaling. Thus, TAS2R agonist structure can be manipulated to maintain the relaxation response and can be biased toward suppression of HASM growth. The latter response is of potential therapeutic benefit in asthma, in which an increase in smooth muscle mass contributes to airway obstruction.

Artesunate attenuates airway resistance in vivo and relaxes airway smooth muscle cells in vitro via bitter taste receptor-dependent calcium signalling.

What is the central question of this study? The aim of this study was to evaluate artesunate for its use as a bronchodilator in asthma treatment. What is the main finding and its importance? We found that artesunate reduces airway resistance in both normal and ovalbumin-treated Balb/c mice in vivo. Artesunate reduces traction force while inducing Ca2+ influx into cultured airway smooth muscle cells in vitro, most probably via the bitter taste receptor. These findings provide important evidence at both animal and cellular levels that artesunate might potentially be used as a bronchodilator for treating obstructive airway diseases, such as asthma. Following the surprising discovery that bitter taste receptors (TAS2Rs) expressed in the lung and can be stimulated to relax airway smooth muscle cells (ASMCs), there is great interest in searching for a bitter taste receptor agonist as a new bronchodilator for asthma therapy. Among the great many other natural bitter substances, artesunate is of special interest to be evaluated for this purpose because of its pharmacological value as a derivative from the well-known anti-malarial, artemisinin. Therefore, in this study we treated either normal or ovalbumin (OVA)-induced asthmatic Balb/c mice in vivo with artesunate (30, 60 or 120μg) via aerosol inhalation. Subsequently, we measured the airway resistance of the mice in the presence or absence of artesunate. In addition, we treated either mouse or human ASMCs cultured in vitro with artesunate (0.25-2.0mM) and then measured the traction force and [Ca2+ ]i flux of the cells in the presence or absence of artesunate. The results demonstrate that artesunate attenuated airway resistance in a dose-dependent manner in both the normal and the OVA-treated mice, but more potently in the latter. The in vivo efficacy of artesunate at 120μg was comparable to that of the conventional bronchodilator, salbutamol, at 3μg in terms of the reduction in airway resistance. Artesunate also reduced traction force and induced an increase in [Ca2+ ]i in the cultured ASMCs, which was mediated, at least in part, by TAS2R signalling in the human ASMCs. These results together suggest that artesunate might potentially be a cheap and safe bronchodilator to complement the current therapy of asthma.