[Show abstract][Hide abstract] ABSTRACT: Nearly full-length RNA genome sequences for 39 rhinovirus B isolates (RV-B), representing 13 different genotypes, were resolved as part of ongoing studies at the University of Wisconsin that attempt to link rhinovirus (RV) diversity and respiratory disease in infants.
[Show abstract][Hide abstract] ABSTRACT: Human rhinovirus (RV) isolates from the RV-C species are recently discovered infectious agents that are closely linked to asthma and wheezing etiologies in infants. Clinical study samples collected at the University of Wisconsin-Madison describe 41 nearly complete genome sequences representing 21 RV-C genotypes.
[Show abstract][Hide abstract] ABSTRACT: Full-length or nearly full-length RNA genome sequences for 98 rhinovirus (RV) A isolates (from the Enterovirus genus of the Picornaviridae family), representing 43 different genotypes, were resolved as part of ongoing studies to define RV genetic diversity and its potential link to respiratory disease.
[Show abstract][Hide abstract] ABSTRACT: There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (β-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than β-agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca(2+)-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by β-agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.
Transactions of the American Clinical and Climatological Association 01/2014; 125:64-75.
[Show abstract][Hide abstract] ABSTRACT: Bitter taste receptors (TAS2Rs) have recently been found to be expressed on human airway smooth muscle (HASM) and their activation results in marked relaxation. These agents have been proposed as a new class of bronchodilators in the treatment of obstructive lung diseases since they act via a different mechanism than -agonists. The TAS2R signal transduction pathway in HASM has multiple elements that are potentially subject to regulation by inflammatory, genetic, and epigenetic mechanisms associated with asthma. To address this, expression, signaling and physiologic functions of the three major TAS2Rs (subtypes 10, 14, 31) on HASM were studied. Transcript expression of these TAS2Rs was not decreased in HASM derived from asthmatic donors compared to nonasthmatics (n=6 from each group). In addition, [Ca2+]i signaling using TAS2R subtype specific agonists (diphenhydramine, chloroquine, saccharin, and flufenamic acid) was not impaired in the asthmatic-derived cells, nor was the response to quinine which activates all three subtypes. HASM cell mechanics measured by magnetic twisting cytometry revealed equivalent TAS2R-mediated relaxation of methacholine treated cells between the two groups. Human precision cut lung slices (PCLS) treated with IL-13 caused a decrease in -agonist (formoterol) mediated relaxation of carbachol contracted airways compared to control slices. In contrast, TAS2R-mediated relaxation was unaffected by IL-13. Taken together, we conclude that TAS2R expression or function is unaffected in HASM derived from asthmatics or the IL-13 inflammatory environment.
American Journal of Respiratory Cell and Molecular Biology 11/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study assessed the impact of bucindolol, a beta-blocker/sympatholytic agent, on the development of atrial fibrillation (AF) in advanced chronic heart failure with reduced left ventricular ejection fraction (HFREF) patients enrolled in the BEST (Beta-Blocker Evaluation of Survival Trial).
β-Blockers have modest efficacy for AF prevention in HFREF patients. Bucindolol's effects on HF and ventricular arrhythmic endpoints are genetically modulated by β1- and α2c-adrenergic receptor (AR) polymorphisms that can be used to subdivide HFREF populations into those with bucindolol effectiveness levels that are enhanced, unchanged, or lost.
BEST enrolled 2,708 New York Heart Association (NYHA) class III to IV HFREF patients. A substudy in which 1,040 patients' DNA was genotyped for the β1-AR position 389 Arg/Gly and the α2c322-325 wild type (Wt)/deletion (Del) polymorphisms, and new-onset AF was assessed from adverse event case report forms or electrocardiograms at baseline and at 3 and 12 months.
In the entire cohort, bucindolol reduced the rate of new-onset AF compared to placebo by 41% (hazard ratio [HR]: 0.59 [95% confidence interval (CI): 0.44 to 0.79], p = 0.0004). In the 493 β1389 arginine homozygotes (Arg/Arg) in the DNA substudy, bucindolol reduced new-onset AF by 74% (HR: 0.26 [95% CI: 0.12 to 0.57]), with no effect in β1389 Gly carriers (HR: 1.01 [95% CI: 0.56 to 1.84], interaction test = 0.008). When β1389 Gly carriers were subdivided by α2c Wt homozygotes (n = 413, HR: 0.94 [95% CI: 0.48 to 1.82], p = 0.84) or Del variant carriers (n = 134, HR: 1.33 [95% CI: 0.32 to 5.64], p = 0.70), there was a positive interaction test (p = 0.016) when analyzed with β1389 Arg homozygotes.
Bucindolol prevented new-onset AF; β1 and α2c polymorphisms predicted therapeutic response; and the 47% of patients who were β1389 Arg homozygotes had an enhanced effect size of 74%. (Beta-Blocker Evaluation in Survival Trial [BEST]; NCT00000560).
[Show abstract][Hide abstract] ABSTRACT: Introduction: There is an unmet need for a new class of direct bronchodilators for the treatment of asthma and chronic obstructive lung disease. Unexpectedly, bitter taste receptors (TAS2Rs) have been localized on airway smooth muscle and when activated cause marked smooth muscle relaxation through a mechanism that is distinct from β2-adrenegic receptors. Thus TAS2R agonists have emerged as a novel class of bronchodilator. Areas covered: A synopsis of the TAS2R family and its biology for bitter taste perception on the tongue is provided, followed by a review of the identification and molecular and physiological characterization of TAS2R subtypes on human and mouse airway smooth muscle. The proposed molecular mechanisms leading to the relaxation response are provided, along with gaps in our understanding at certain points in the signaling cascade. Unresolved issues that may need to be considered for drug development are discussed. Expert opinion: TAS2R agonists show promise as a new class of highly efficacious bronchodilators for treatment of obstructive lung disease. With tens of thousands of known natural and synthetic bitter compounds, there is substantial diversity within the known agonists, and, a ready source of agents for screening and further development of an inhaled TAS2R agonist for therapeutic purposes.
Expert Opinion on Therapeutic Targets 04/2013; · 4.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: -In patients with chronic heart failure and reduced left ventricular ejection fraction (HFREF) β-blockers prevent cardiac arrhythmias, including ventricular tachycardia/fibrillation (VT/VF). We hypothesized that prevention of ventricular arrhythmias by the β-blocker/sympatholytic agent bucindolol is influenced by genetic variation in adrenergic receptors (ARs). METHODS AND RESULTS: -From a substudy of the β-Blocker Evaluation of Survival Trial (BEST, n = 1040), we identified those with the high functioning 389Arg vs. the lower function 389Gly β(1) adrenergic receptor (AR) variant, and the loss of function (α2C)322-325 AR deletion vs. the 322-325 wild type (Wt)/deletion (Del) variant. VT/VF was recorded on case report forms as an adverse event. There were 493 Arg 389 β(1) receptor homozygotes (β(1)389 Arg/Arg) vs. 547 Gly389 carriers and 207 (α2C)322-325 Del carriers vs. 833 homozygous Wts ((α2C)322-325 Wt/Wt). In all genotypes bucindolol was associated with a lower incidence of VT/VF (subhazard ratio (SHR) 0.42 (0.27, 0.64), P=0.00006). Bucindolol reduced VT/VF in β(1)389 Arg homozygotes (SHR 0.26 (0.14, 0.50), P=0.00005) but not in β(1)389 Gly carriers (SHR 0.60 (0.34, 1.07), P=0.09). For genotype combinations, the (α2C)322-325 polymorphism altered the VT/VF bucindolol response in β(1)389 Gly carriers, with (α2C) Del genotypes associated with complete efficacy loss. A test of interaction was statistically significant (P=0.028) for treatment group and a β(1)389/(α2C)322-325 three genotype construct, effectively identifying patients who exhibited enhanced response, no substantial response modification and loss of response. CONCLUSIONS: -Bucindolol prevents VT/VF in HFREF subjects, and this effect is modulated by β(1)389 Arg/Gly and (α2C)322-325 Wt/Del AR polymorphisms.
Circulation Arrhythmia and Electrophysiology 12/2012; · 5.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a β-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (β1 adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α2C AR Ins [wild-type (Wt)] 322–325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology.
In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of β1389 and α2C322–325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for β1389 AR variants was measured in human explanted left ventricles.
The combination of β1389 Arg+α2C322–325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in β1389 Arg homozygotes+α2C322–325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in β1389 Arg vs. Gly ARs, which converts α2CDel minor allele-associated NE lowering from a therapeutic liability to a benefit.
On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (β1389 Arg homozygotes), intermediate (β1389 Gly carriers+α2C322–325 Wt homozygotes), and no (β1389 Gly carriers+α2C322–325 Del carriers) efficacy.
[Show abstract][Hide abstract] ABSTRACT: We present a novel hypothesis that could explain many off-target effects of diverse pharmaceuticals. Specifically, we propose that any drug with a bitter taste could have unintended actions in the body through stimulation of extraoral type 2 taste receptors (T2Rs). T2Rs were first identified in the oral cavity, where they function as bitter taste receptors. However, recent findings indicate that they are also expressed outside the gustatory system, including in the gastrointestinal and respiratory systems. T2R ligands include a diverse array of natural and synthetic compounds, many of which are toxins. Notably, many pharmaceuticals taste bitter, with compounds such as chloroquine, haloperidol, erythromycin, procainamide, and ofloxacin known to activate T2Rs. Bitter-tasting compounds can have specific physiological effects in T2R-expressing cells. For example, T2Rs are found in some gastrointestinal endocrine cells, including those that secrete the peptide hormones (e.g., ghrelin and glucagon-like peptide-1) in response to stimulation by bitter-tasting compounds. In the respiratory system, stimulation of T2Rs expressed in respiratory epithelia and smooth muscle has been implicated in protective airway reflexes, ciliary beating, and bronchodilation. If our hypothesis is confirmed, it would offer a new paradigm for understanding the off-target actions of diverse drugs and could reveal potential new therapeutic targets.-Clark, A. A., Liggett, S. B., Munger, S. D. Extraoral bitter taste receptors as mediators of off-target drug effects.
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized that 3 nonsynonymous GRK4 single-nucleotide polymorphisms, R65L (rs2960306), A142V (rs1024323), and A486V (rs1801058), would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long-term cardiovascular outcomes (all-cause death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 single-nucleotide polymorphisms were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In whites and blacks, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (-9.1±6.8 versus -6.8±7.1 versus -5.3±6.4 mm Hg in participants with 0, 1, and 2 copies of 65L-142V, respectively; P=0.0088). One thousand four hundred sixty participants with hypertension and coronary artery disease from the INternational VErapamil SR/Trandolapril STudy (INVEST) were genotyped, and variant alleles of all 3 GRK4 single-nucleotide polymorphisms were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (odds ratio, 2.29 [1.48-3.55]; P=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest that the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R homozygotes, suggesting a potential interaction between these 2 genes.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A pro-asthmatic culture milieu and β(2)-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single-nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing P-values <0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5' of the thyroid hormone receptor-β gene was associated with BDR in the childhood population and two adult populations (P-value=0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.The Pharmacogenomics Journal advance online publication, 3 January 2012; doi:10.1038/tpj.2011.56.
The Pharmacogenomics Journal 01/2012; · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several studies in humans or transgenic animals have reported that the 389 Arg or Gly polymorphic variation of the β1-adrenergic receptor (AR) is associated with differential responses to beta-blocker therapy and/or myocardial disease progression. Analysis of changes in gene expression is an important means of defining molecular differences associated with structural or functional phenotypic variations. To determine if structural and functional myocardial phenotypic differences between β1389 Arg vs. Gly transgenic overexpressors are associated with qualitative and/or quantitative differences in gene expression, a comprehensive analysis of mRNAs and miRNAs expressed in the hearts of 3 and 6-8 mo old β1-Arg389 and β1-Gly389 overexpressor transgenic mice was performed. Changes in mRNA and miRNA expression were analyzed by arrays and partially confirmed by RT-qPCR. Bioinformatic analysis demonstrated that several genes, including those involved in PKA and CaMK signaling pathways, are regulated in a temporal- or phenotype-specific manner. Furthermore, expression signature analyses indicated that miRNAs have the potential to target expression of a number of genes involved in multiple cardiomyopathy-related pathways, and changes in miRNA expression can precede the onset of disease. Differences in gene expression between β1-Arg389 and β1-Gly389 transgenic mice are largely quantitative rather than qualitative and are associated with the development of cardiomyopathy in a time-dependent manner. Chronic β1-AR overdrive results in increased expression of components of the CaMK pathway, with correspondingly decreased levels of components of the PKA pathway. Based on the temporal and genotype-specific pattern of miRNA expression, miRNAs are likely to be important predictors of disease states, especially when miRNA expression is paired with mRNA expression, and that miRNA/mRNA expression signatures have the potential to be useful in determining the underlying risk associated with cardiac disease progression.
[Show abstract][Hide abstract] ABSTRACT: Perioperative myocardial infarction is a common and potentially fatal complication after noncardiac surgery, particular among patients with cardiovascular risk factors. β-blockers have been considered a mainstay in prevention and treatment of perioperative myocardial infarction, yet recent evidence suggests that β-blockers may have an unfavorable risk profile in this setting, and the use has become controversial. What seems conspicuously absent from the current discussion is the appreciation of how much interindividual genetic variation influences the clinical response to β-blocker therapy. Genetic variation in the adrenergic signaling pathway is common, and has a major impact on adrenergic receptor function and β-blocker efficacy in other cardiovascular diseases, such as heart failure and hypertension. Genetic variation in the cytochrome P450 2D6, or CYP2D6, enzyme, which is responsible for the metabolism of most β-blockers, is also important and can lead to poor metabolizing of β-blockers (potential toxicity) or their ultra-rapid degradation (decreased efficacy). Here, we review the molecular, cellular, and physiologic consequences of polymorphisms in the adrenergic signaling pathway and CYP2D6 gene, and show that these are likely relevant factors influencing efficacy, safety, and toxicity of β-blocker therapy in prevention and treatment of perioperative myocardial infarction.
[Show abstract][Hide abstract] ABSTRACT: A unifying mechanism by which G protein-coupled receptors (GPCRs) signal in cell type-dependent and G protein-independent ways has developed over the past decade. GPCR kinases (GRKs) are mediators of homologous desensitization: GRK phosphorylation of the receptors leads to the subsequent binding of β-arrestins, which partially quenches receptor coupling to G proteins. For some receptors, this GRK-mediated phosphorylation stimulates additional signaling through the scaffolding action of β-arrestin. These downstream signals are configured by β-arrestin conformation, which is dictated by the GRK phosphoacceptors on the receptors in a barcode-like fashion. Furthermore, each of the GRKs can potentially phosphorylate different serine and threonine residues on a given receptor, and the phosphorylation pattern can be biased by the receptor conformation established by bound ligand. Finally, the arrangement of potential GRK phosphorylation sites-and thus the conformation of β-arrestin and its effect on downstream signaling-can differ substantially between even closely related GPCRs stimulated by the same agonist. The diversity of the barcoding to flexible β-arrestin explains the multidimensional nature of signaling in the superfamily and represents new opportunities for drug discovery.
[Show abstract][Hide abstract] ABSTRACT: Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca(2+)](i) release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein-coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca(2+)](i) response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca(2+)](i) response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca(2+)](i) response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin-mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.
American Journal of Respiratory Cell and Molecular Biology 06/2011; 45(5):1069-74. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.
Nature medicine 05/2011; 17(5):627-32. · 27.14 Impact Factor