The FASEB Journal • Research Communication Bitter taste receptor agonists elicit G-protein-dependent negative inotropy in the murine heart Simon R. Foster,* Kristina Blank,† Louise E. See Hoe,‡ Maik Behrens,† Wolfgang Meyerhof,† Jason N. Peart,‡ and Walter G. Thomas*,1 *School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia; †Department of Molecular Genetics, German Institute of Human Nutrition (DIfE) Potsdam- Rehbrücke, Nuthetal, Germany; and ‡Griffith Health Institute, Griffith University, Gold Coast, Queensland, Australia ABSTRACT G-protein-coupled receptors (GPCRs) The activation of G-protein-coupled receptors are key mediators in cardiovascular physiology, yet (GPCRs) converts extracellular stimuli into intracellu- frontline therapies for heart disease target only a small lar signaling to mediate a broad range of cellular and fraction of the cardiac GPCR repertoire. Moreover, physiological responses, as well as the senses of smell, there is emerging evidence that GPCRs implicated in taste, and vision. GPCRs represent the largest receptor taste (Tas1r and Tas2rs) have specific functions beyond superfamily in the human genome (1), and on the the oral cavity. Our recent description of these recep- strength of more than 4 decades of research, continue tors in heart tissue foreshadows a potential novel role to be prime therapeutic targets. The effect of GPCR- in cardiac cells. In this study, we identified novel targeted drugs is particularly evident in the treatment agonist ligands for cardiac-Tas2rs to enable the func- of cardiovascular disease, in which GPCR signaling is tional investigation of these receptors in heart tissue. modulated to control hypertension, stimulate inotropy Five Tas2rs cloned from heart tissue were screened in heart failure, and prevent thrombosis (2). Nonethe- less, current cardiovascular therapeutics target only a against a panel of 102 natural or synthetic bitter com- small fraction of the cardiac GPCR repertoire, predom- pounds in a heterologous expression system. We iden- inantly focusing on the adrenergic and angiotensin tified agonists for Tas2r108, Tas2r137, and Tas2r143 receptor systems. that were then tested in Langendorff-perfused mouse GPCR expression profiling analyses in human and hearts (from 8-wk-old male C57BL/6 mice). All Tas2r mouse tissues suggest that the heart expresses Ͼ150 agonists tested exhibited concentration-dependent ef- different GPCRs (3–5), reinforcing the notion that there fects, with agonists for Tas2r108 and Tas2r137, leading are many as yet unexplored, potential GPCR targets for ϳ to a 40% decrease in left ventricular developed the modulation of the heart and cardiovascular physiol- pressure and an increase in aortic pressure, respec- ogy. Moreover, despite representing more than half of the tively. These responses were abrogated in the presence GPCR repertoire, taste and odorant receptor genes have ␣ ␤␥ of G i and G inhibitors (pertussis toxin and gallein). been excluded from these previous analyses, but are This study represents the first demonstration of pro- attracting more attention, as they are increasingly being found Tas2r agonist-induced, G protein-dependent ef- identified in cells and tissues outside those of the mouth fects on mouse heart function.—Foster, S. R., Blank, and nose (reviewed in ref. 6). For example, the type 1 K., See Hoe, L. E., Behrens, M., Meyerhof, W., Peart, taste receptors (Tas1rs) are family C GPCRs that respond J. N., Thomas, W. G. Bitter taste receptor agonists elicit to sugars and amino acids and have been implicated in G-protein-dependent negative inotropy in the murine nutrient sensing and hormone secretion in the brain, gut, heart. FASEB J. 28, 4497–4508 (2014). www.fasebj.org and skeletal muscle (7–11). Type 2 taste receptors (Tas2rs) are a family of ϳ30 highly variable, single-exon genes that mediate bitter taste. Tas2rs also have a broad Key Words: Tas2r ⅐ GPCR ⅐ Langendorff ⅐ cardiovascular tissue expression profile beyond the gustatory system, system most notably throughout the gastrointestinal tract and in the airways (12–16). We have recently provided the first evidence that members of both the Tas1r and Tas2r gene Abbreviations: AoP, aortic pressure; DMEM, Dulbecco’s families are expressed in rodent and human heart tissue, modified Eagle’s medium; DMSO, dimethyl sulfoxide; EDP, with a subset of the Tas2rs up-regulated in the heart end diastolic pressure; FBS, fetal bovine serum; FLIPR, fluo- rometric imaging plate reader; GPCR, G-protein-coupled receptor; HEK293T, human embryonic kidney 293T; HSV, 1 Correspondence: School of Biomedical Sciences, Univer- herpes simplex virus; LVDP, left ventricular developed pres- sity of Queensland, St. Lucia, QLD 4072, Australia. E-mail: sure; PTX, pertussis toxin; SST, somatostatin; SysP, systolic [email protected] pressure; Tas1r, type 1 taste receptor; Tas2r, type 2 taste doi: 10.1096/fj.14-256305 receptor; TRPM5, transient receptor potential cation chan- This article includes supplemental data. Please visit http:// nel, subfamily M, member 5 www.fasebj.org to obtain this information. 0892-6638/14/0028-4497 © FASEB 4497 Downloaded from www.fasebj.org by (2001:388:608c:4800:e578:c55:a347:3276) on September 12, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4497-4508. during starvation (17). These initial findings led us to cloned into pcDNA5/FRT (Invitrogen, Carlsbad, CA, USA) in speculate that cardiac-expressed Tas2rs respond to local a cassette containing the first 45 aa of the rat somatostatin nutrient status in the heart, although the precise func- type 3 receptor as an amino terminus cell membrane– tional role of these receptors in the cardiac setting is yet to targeting signal (24) and a carboxyl-terminal herpes simplex be thoroughly investigated. virus (HSV) glycoprotein D epitope, which does not interfere with receptor signaling and can be used for immunocyto- A major challenge to the successful broader physio- chemistry (25). The coding regions of the remaining cardiac- logical and pharmacological characterization of these expressed Tas2rs (identified in (17) were cloned from neo- GPCRs is the paucity of experimental tools for the natal rat heart cDNA, using gene-specific primers including rodent Tas2rs—most important among them, the lack EcoRI and NotI restriction sites into the pcDNA5/FRT soma- of selective agonist and antagonist ligands. Understand- tostatin (SST) HSV vector backbone (Table 1). Cloned full- ably, most studies directed at agonist identification length Tas2rs were confirmed by sequencing. have focused on the human TAS2Rs (18–20). Culmi- Human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection, Manassas, VA, USA) were maintained nating with comprehensive compound screening with ϩ heterologous expression systems, almost all of the hu- in Dulbecco’s modified Eagle’s medium (DMEM) 10% fetal bovine serum (FBS). HEK293T cells that were modified to man TAS2R repertoire has now been effectively “deor- ␣ stably express the chimeric G-protein subunit G 16/gust44 (26) phanized” (21). Incidentally, the only definitive agon- were used for compound screening experiments. Cells were ist–receptor pairings for rodent Tas2rs are denatonium grown in a humidified incubator in 95% O2 and 5% CO2 at benzoate, with the mouse receptor Tas2r108 (previ- 37°C. ously designated mT2R8), and cycloheximide, with All compounds used in ligand screening were designated mouse Tas2r105 (mT2R5) (22) and its rat homologue as bitter-tasting in human psychophysical tests or were se- Tas2r105 (previously rT2R9) (20). The lack of rodent lected based on existing knowledge (21). A complete com- Tas2r ligand data is an important consideration, given pound list, including the concentrations tested in the initial that there is often substantial amino acid sequence screening, is provided in Supplemental Table S1. Analytical divergence between homologous human and rodent grade compounds were purchased from Sigma-Aldrich (St. bitter taste receptor genes, which may result in func- Louis, MO, USA), except for amarogentin (Chromadex, Irvine, CA, USA), or were isolated (19, 21). The compounds tionally distinct receptors. There are precedents in the were dissolved in Ca2ϩ assay buffer (10 mM d-glucose, 130 taste receptor literature where bitter ligand-mediated mM NaCl, 5 mM KCl, 10 mM HEPES, and 2 mM CaCl2,pH effects have been attributed to Tas2r function, most 7.4) or in dimethyl sulfoxide (DMSO), with the final DMSO prominently in the airways and gut (12, 16, 23). How- concentration not exceeding 0.1% (v/v), to minimize cellular ever, without specific pharmacological tools or appro- toxicity. Many of the bitter compounds are pharmacologically priate genetically modified mouse models, there re- active or are hydrophobic or amphiphilic and therefore could mains some controversy around the Tas2r dependence interfere with cellular calcium responses. Accordingly, pilot experiments were performed before screening in untrans- of the observed effects. ␣ In this study, we used an agonist screen, with the fected HEK293T G 16/gust44 cells, to ensure that the concen- specific purpose of identifying novel activating agonists trations tested were below those that elicited nonspecific calcium responses (21). for the Tas2rs that are expressed in cardiac tissue. We For Langendorff perfusion experiments, pertussis toxin then characterized the effect of putative Tas2r ligands on (PTX) was purchased from List Biological Laboratories the spectrum