Cellular and Molecular Mechanisms of bitter tastant-induced bronchodilation

描述（申请人提供）：苦味剂诱导的支气管扩张是一种新发现的气道平滑肌（ASM）松弛形式，苦味剂作为支气管扩张剂有很大的前景，对全球3亿哮喘和慢性阻塞性肺疾病患者是必不可少的。我们试图解决与这种放松相关的两个基本问题：苦味剂在ASM中的作用的细胞和分子机制，以及苦味剂在慢性哮喘中的有效性。苦味物质激活味觉细胞和一些口腔外细胞中的2型味觉受体(TAS2R)-gustducin-PLCbeta2通路。然而，我们的初步数据表明，TAS2Rs和gustducin，而不是PLCbeta2，是苦味诱导的支气管扩张所必需的。为了巩固这一观点，我们将研究删除TAS2R105、α -gustducin或PLCbeta2对苦味剂诱导的ASM中[Ca2+]i、细胞长度和张力变化的影响（Aim 1）。Tas2r105-/-小鼠的独特之处在于，由此产生的功能变化可以用环己亚胺（Tas2r105特异性配体）可靠地评估。另一方面，α -gustducin-/-小鼠的优势在于，尽管小鼠基因组包含35个TAS2Rs，但它们都与gustducin偶联。因此，gustducin的缺失可能会阻止支气管扩张，以应对广泛的苦味。我们的初步数据进一步表明，抑制l型CaV1.2通道是导致苦味剂诱导的支气管扩张的关键分子事件，这种抑制依赖于百日咳毒素敏感的gustducin，而不是PLCbeta。通过膜片钳、药理学、基因敲除小鼠和异种表达系统，我们将揭示苦味剂抑制这一通道的分子机制（Aim 2）。最后，苦味剂在急性哮喘小鼠模型中是有效的支气管扩张剂，这意味着它们在这种疾病中具有巨大的治疗潜力。然而，哮喘是一种慢性疾病；因此，有必要确定苦味剂在慢性疾病中的有效性

DESCRIPTION (provided by applicant): Bitter tastant-induced bronchodilation is a newly discovered form of airway smooth muscle (ASM) relaxation, and bitter tastants hold great promise as bronchodilators, which are indispensable for 300 million patients worldwide with asthma and chronic obstructive pulmonary disease. We seek to address two fundamental issues related to this relaxation: the cellular and molecular mechanisms of action of bitter tastants in ASM, and the effectiveness of bitter tastants in chronic asthma. Bitter tastants activate the type 2 taste receptor (TAS2R)-gustducin-PLCbeta2 pathway in taste cells and some extra-oral cells. Our preliminary data, however, suggest that TAS2Rs and gustducin, but not PLCbeta2, are essential for bitter tastant-induced bronchodilation. To firmly establish this view, we will study the effects of deleting TAS2R105, alpha-gustducin or PLCbeta2 on bitter tastant-induced changes in [Ca2+]i, cell length and tension in ASM (Aim 1). The uniqueness of Tas2r105-/- mice is that the resultant function changes can be assessed reliably with cycloheximide, a ligand specific to TAS2R105. On the other hand, the advantage of alpha-gustducin-/- mice is that although the mouse genome contains 35 TAS2Rs, all of them couple with gustducin. As a result, gustducin deletion could block bronchodilation in response to a broad spectrum of bitter tastants. Our preliminary data further revealed that inhibition of L-type CaV1.2 channels is the key molecular event responsible for bitter tastant-induced bronchodilation, and this inhibition depends on pertussis toxin sensitive gustducin but not PLCbeta. Using patch clamp, pharmacology, genetic knockout mice and heterologous expression systems, we will uncover the molecular mechanism by which bitter tastants inhibit this channel (Aim 2). Finally, bitter tastants are effective bronchodilators in a mouse model of acute asthma, implying their tremendous therapeutic potential in this disorder. Yet, asthma is a chronic disease; it is thus imperative to establish the effectiveness of bitter tastants in chronic asthma. Our preliminary study revealed that bitter tastants reverse the contraction of airways from two mouse models of chronic asthma including one induced by Asperigillus fumigates crude protein extract, a common allergen of human asthmatics. In Aim 3, we will systematically characterize this effect in vitro and in vivo in these mice, and determine its molecular basis using allergen sensitized Tas2r105-/- or alpha-gustducin-/- mice. To translate our findings in mice to human, we will uncover the mechanisms of bitter tastant-induced bronchodilation using human lung specimens. This work should establish not only how bitter tastants cause bronchodilation in mouse and human, but also their usefulness in treating airway diseases in mouse models of chronic asthma. Such advances will deepen our understanding of ASM biology and facilitate the development of bitter tastants as new bronchodilators.