Activation of airway epithelial bitter taste receptors by Pseudomonas aeruginosa quinolones modulates calcium, cyclic-AMP, and nitric oxide signaling.

Bitter taste receptors (taste family 2 bitter receptor proteins; T2Rs), discovered in many tissues outside the tongue, have recently become potential therapeutic targets. We have shown previously that airway epithelial cells express several T2Rs that activate innate immune responses that may be important for treatment of airway diseases such as chronic rhinosinusitis. It is imperative to more clearly understand what compounds activate airway T2Rs as well as their full range of functions. T2R isoforms in airway motile cilia (T2R4, -14, -16, and -38) produce bactericidal levels of nitric oxide (NO) that also increase ciliary beating, promoting clearance of mucus and trapped pathogens. Bacterial quorum-sensing acyl-homoserine lactones activate T2Rs and stimulate these responses in primary airway cells. Quinolones are another type of quorum-sensing molecule used by Pseudomonas aeruginosa To elucidate whether bacterial quinolones activate airway T2Rs, we analyzed calcium, cAMP, and NO dynamics using a combination of fluorescent indicator dyes and FRET-based protein biosensors. T2R-transfected HEK293T cells, several lung epithelial cell lines, and primary sinonasal cells grown and differentiated at the air-liquid interface were tested with 2-heptyl-3-hydroxy-4-quinolone (known as Pseudomonas quinolone signal; PQS), 2,4-dihydroxyquinolone, and 4-hydroxy-2-heptylquinolone (HHQ). In HEK293T cells, PQS activated T2R4, -16, and -38, whereas HHQ activated T2R14. 2,4-Dihydroxyquinolone had no effect. PQS and HHQ increased calcium and decreased both baseline and stimulated cAMP levels in cultured and primary airway cells. In primary cells, PQS and HHQ activated levels of NO synthesis previously shown to be bactericidal. This study suggests that airway T2R-mediated immune responses are activated by bacterial quinolones as well as acyl-homoserine lactones.

Tissue-Dependent Expression of Bitter Receptor TAS2R38 mRNA.

TAS2R38 is a human bitter receptor gene with a common but inactive allele; people homozygous for the inactive form cannot perceive low concentrations of certain bitter compounds. The frequency of the inactive and active forms of this receptor is nearly equal in many human populations, and heterozygotes with 1 copy of the active form and 1 copy of the inactive form have the most common diplotype. However, even though they have the same genotype, heterozygotes differ markedly in their perception of bitterness, perhaps in part because of differences in TAS2R38 mRNA expression. Other tissues express this receptor too, including the nasal sinuses, where it contributes to pathogen defense. We, therefore, wondered whether heterozygous people had a similar wide range of TAS2R38 mRNA in sinonasal tissue and whether those with higher TAS2R38 mRNA expression in taste tissue were similarly high expressers in nasal tissue. To that end, we measured gene expression by quantitative PCR in taste and sinonasal tissue and found that expression abundance in one tissue was not related to the other. We confirmed the independence of expression in other tissue pairs expressing TAS2R38 mRNA, such as pancreas and small intestine, using autopsy data from the Genotype-Tissue Expression project (although people with high expression of TAS2R38 mRNA in colon also tended to have higher expression in the small intestine). Thus, taste tissue TAS2R38 mRNA expression among heterozygotes is unlikely to predict expression in other tissues, perhaps reflecting tissue-dependent function, and hence regulation, of this protein.

Flavones modulate respiratory epithelial innate immunity: Anti-inflammatory effects and activation of the T2R14 receptor.

Chronic rhinosinusitis has a significant impact on patient quality of life, creates billions of dollars of annual healthcare costs, and accounts for ∼20% of adult antibiotic prescriptions in the United States. Because of the rise of resistant microorganisms, there is a critical need to better understand how to stimulate and/or enhance innate immune responses as a therapeutic modality to treat respiratory infections. We recently identified bitter taste receptors (taste family type 2 receptors, or T2Rs) as important regulators of sinonasal immune responses and potentially important therapeutic targets. Here, we examined the immunomodulatory potential of flavones, a class of flavonoids previously demonstrated to have antibacterial and anti-inflammatory effects. Some flavones are also T2R agonists. We found that several flavones inhibit Muc5AC and inducible NOS up-regulation as well as cytokine release in primary and cultured airway cells in response to several inflammatory stimuli. This occurs at least partly through inhibition of protein kinase C and receptor tyrosine kinase activity. We also demonstrate that sinonasal ciliated epithelial cells express T2R14, which closely co-localizes (<7 nm) with the T2R38 isoform. Heterologously expressed T2R14 responds to multiple flavones. These flavones also activate T2R14-driven calcium signals in primary cells that activate nitric oxide production to increase ciliary beating and mucociliary clearance. TAS2R38 polymorphisms encode functional (PAV: proline, alanine, and valine at positions 49, 262, and 296, respectively) or non-functional (AVI: alanine, valine, isoleucine at positions 49, 262, and 296, respectively) T2R38. Our data demonstrate that T2R14 in sinonasal cilia is a potential therapeutic target for upper respiratory infections and that flavones may have clinical potential as topical therapeutics, particularly in T2R38 AVI/AVI individuals.

Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue.

We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual's perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays ( TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception.

Circulating Bmp10 acts through endothelial Alk1 to mediate flow-dependent arterial quiescence.

Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFß family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.

"A spoonful of sugar helps the medicine go down": bitter masking by sucrose among children and adults.

Sweeteners are often added to liquid formulations of drugs but whether they merely make them better tasting or actually reduce the perception of bitterness remains unknown. In a group of children and adults, we determined whether adding sucrose to urea, caffeine, denatonium benzoate, propylthiouracil (PROP), and quinine would reduce their bitterness using a forced-choice method of paired comparisons. To better understand individual differences, adults also rated each solution using a more complex test (general Labeled Magnitude Scale [gLMS]) and were genotyped for the sweet taste receptor gene TAS1R3 and the bitter receptor TAS2R38. Sucrose suppressed the bitterness of each agent in children and adults. In adults, sucrose was effective in reducing the bitterness ratings from moderate to weak for all compounds tested, but those with the sensitive form of the sweet receptor reported greater reduction for caffeine and quinine. For PROP, sucrose was most effective for those who were genetically the most sensitive, although this did not attain statistical significance. Not only is the paired comparison method a valid tool to study how sucrose improves the taste of pediatric medicines among children but knowledge gleaned from basic research in bitter taste and how to alleviate it remains an important public health priority.

Ethnic/racial and genetic influences on cerumen odorant profiles.

This report describes the volatile organic compounds (VOCs) associated with human cerumen (earwax) and the effects of ethnicity/race and variation on the ATP-binding cassette, sub-family C, member 11 gene (ABCC11). A single nucleotide polymorphism (SNP) in ABCC11 affects the cerumen VOC profiles of individuals from African, Caucasian, and Asian descent. Employing gas chromatography/mass spectrometry (GC/MS) we have identified the nature and relative abundance of cerumen VOCs from 32 male donors. Our results show that cerumen contains a complex mixture of VOCs and that the amounts of these compounds vary across individuals as well as across ethnic/racial groups. In six of the seven compounds whose detected concentrations were found to be statistically different across groups, individuals of African descent (AfD) > Caucasian descent (CaD) > Asians descent (AsD). Our findings also reveal that ABCC11 genotype alone does not predict the type and relative levels of volatiles found in human cerumen, and suggest that other biochemical pathways must be involved. Examination of the composition and diversity of external auditory canal microbiota in a small subset of our subject population revealed that the ear microbiota may not be directly correlated with either ethnic group membership or ABCC11 genotype.

Taste Exam: A Brief and Validated Test.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

Age-related differences in bitter taste and efficacy of bitter blockers.

BACKGROUND: Bitter taste is the primary culprit for rejection of pediatric liquid medications. We probed the underlying biology of bitter sensing and the efficacy of two known bitter blockers in children and adults. METHODS: A racially diverse group of 154 children (3-10 years old) and their mothers (N = 118) evaluated the effectiveness of two bitter blockers, sodium gluconate (NaG) and monosodium glutamate (MSG), for five food-grade bitter compounds (quinine, denatonium benzoate, caffeine, propylthiouracil (PROP), urea) using a forced-choice method of paired comparisons. The trial was registered at clinicaltrials.gov (NCT01407939). RESULTS: The blockers reduced bitterness in 7 of 10 bitter-blocker combinations for adults but only 3 of 10 for children, suggesting that efficacy depends on age and is also specific to each bitter-blocker combination. Only the bitterness of urea was reduced by both blockers in both age groups, whereas the bitterness of PROP was not reduced by either blocker in either age group regardless of TAS2R38 genotype. Children liked the salty taste of the blocker NaG more than did adults, but both groups liked the savory taste of MSG equally. CONCLUSIONS AND RELEVANCE: Bitter blocking was less effective in children, and the efficacy of blocking was both age and compound specific. This knowledge will pave the way for evidence-based strategies to help develop better-tasting medicines and highlights the conclusion that adult panelists and genotyping alone may not always be appropriate in evaluating the taste of a drug geared for children.