Airway Smooth Muscle Bitter Taste Receptors as Targets for Novel Bronchodilators

项目总结（见说明书）：支气管扩张剂用于治疗哮喘，以急性缓解支气管痉挛和长期控制。目前，只有一类直接支气管扩张剂，作用于气道平滑肌P2-肾上腺素能受体的β-激动剂在使用中。由于许多哮喘患者没有达到充分的疾病控制，因此需要额外的治疗方法，包括通过新机制发挥作用的直接支气管扩张剂。我们在人气道平滑肌上发现了多种苦味受体（TAS 2 R）亚型。苦味配体激活TAS 2 R在体外和体内以及各种哮喘模型中引起显著的气道平滑肌松弛。TAS 2 R介导的支气管扩张与β-激动剂一样有效，并且是通过涉及专门的Ca 2+信号传导的完全不同的机制。有数千种潜在的TAS 2 R激动剂来源于植物或出于其他目的合成，但在我们的发现之前，这些激动剂未被考虑用于哮喘治疗。在这个项目中，我们将研究HASM TAS 2 Rs的性质，着眼于激动剂成为治疗哮喘的一类新的支气管扩张剂。在目标1中，我们将重点关注急性脱敏的可能性，这可能会限制治疗效果。将在HASM细胞、来自精确切割的人肺切片的支气管和重组表达模型细胞中检查激动剂促进的3种最丰富的HASM TAS 2 R的脱敏。将建立G蛋白偶联、磷酸化、内化、B抑制蛋白相互作用和偏向配体性质。还将使用2个模型确定哮喘素质对这些特性的影响，从而可以确定哮喘治疗的适用性。目的二是通过定点突变和重组表达的方法，建立HASM TAS 2 RS的GRK磷酸化位点，从而确定其信号猝灭的确切机制。当长期暴露于激动剂时，一些TAS 2 R似乎经历上调，这是一种与长期β-激动剂治疗后下调的β 2-肾上腺素能受体相反的表型。TAS 2 R上调将是一个非常有利的概况，起到限制快速耐受的作用。目的3将确定哪些HASM TAS 2 Rs显示上调，该过程的分子机制，以及偏向配体促进效应的基础。总的来说，这些研究将确定HASM TAS 2 R的分子和药理学特性，这些特性是未来开发治疗哮喘的新型支气管扩张剂所必需的。

PROJECT SUMMARY (See Instructions): Bronchodilators are used in the treatment of asthma for acute relief of bronchospasm and for long-term control. Currently, only one class of direct bronchodilators, p-agonists acting at airway smooth muscle P2- adrenergic receptors, are in use. With many asthmatics not achieving adequate disease control, there is a need for additional therapeutics, including direct bronchodilators acting by novel mechanisms. We have discovered multiple bitter taste receptor (TAS2R) subtypes on human airway smooth muscle (HASM). Activation of TAS2Rs by bitter ligands causes marked airway smooth muscle relaxation in vitro and in vivo, and in various models of asthma. TAS2R-mediated bronchodilation is as efficacious as p-agonists, and is via a completely different mechanism involving specialized Ca^"^ signaling. There are thousands of potential TAS2R agonists derived from plants or synthesized for other purposes, yet prior to our findings these were not considered for asthma therapy. In this Project, we will examine the properties of HASM TAS2Rs with an eye towards agonists being a new class of bronchodilators for treating asthma. In Aim 1, we will focus on the potential for acute desensitization, which can limit therapeutic efficacy. Agonist-promoted desensitization of the 3 most abundant HASM TAS2Rs will be examined in HASM cells, bronchi from precision-cut human lung slices and recombinantly expressing model cells. G-protein coupling, phosphorylation, internalization, B arrestin interactions, and biased ligand properties will be established. The influence ofthe asthma diathesis on these properties will also be determined using 2 models, so that applicability to asthma treatment can be ascertained. In Aim 2, we will establish the specific GRK phosphorylation sites for the relevant HASM TAS2RS, using site-directed mutagenesis and recombinant expression, so that a precise mechanism ofthe signal quenching is determined. Some TAS2Rs appear to undergo upregulation when exposed chronically to agonists, a phenotype that is opposite to P2-adrenergic receptors which downregulate upon chronic p-agonist treatment. TAS2R upregulation would be an extremely favorable profile, acting to limit tachyphylaxis. Aim 3 will determine which ofthe HASM TAS2Rs display upregulation, the molecular mechanism ofthe process, and the basis for biased ligands to promote the effect. Collectively, these studies will define the molecular and pharmacologic properties of HASM TAS2Rs required for future development of a novel class of bronchodilators for treating asthma.