Cellular and Molecular Mechanisms of bitter tastant-induced bronchodilation

描述(申请人提供)：苦味诱导的支气管扩张是一种新发现的呼吸道平滑肌(ASM)松弛形式，苦味作为支气管扩张剂具有很大的前景，对全球3亿哮喘和慢性阻塞性肺疾病患者来说是不可或缺的。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中的细胞和分子作用机制，以及苦味剂在慢性哮喘中的有效性。苦味者在味觉细胞和一些口腔外细胞中激活2型味觉受体(TAS2R)-Gustducin-PLCbeta2途径。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。为了坚定这一观点，我们将研究缺失TAS2R105、α-Gustducin或PLCbeta2对苦味诱导的ASM[Ca~(2+)]i、细胞长度和张力变化的影响(目标1)。Tas2R105-/-小鼠的独特之处在于，TAS2R105的特异性配体放线菌亚胺可以可靠地评估所产生的功能变化。另一方面，α-Gustducin-/-小鼠的优势在于，尽管小鼠基因组包含35个TAS2R，但它们都与Gustducin偶联。因此，Gustducin的缺失可能会阻止支气管扩张，以回应广泛的苦味。我们的初步数据进一步表明，抑制L型CaV1.2通道是苦味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PLCβ。利用膜片钳、药理学、基因敲除小鼠和异源表达系统，我们将揭示苦味抑制这一通道的分子机制(目标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，确定苦味对慢性哮喘的有效性是当务之急。 哮喘。我们的初步研究显示，苦味逆转了两种慢性哮喘小鼠模型的呼吸道收缩，其中一种是由烟熏曲霉粗蛋白提取物诱导的，这是一种人类哮喘患者的常见过敏原。在目标3中，我们将在这些小鼠体内和体外系统地表征这种效应，并使用过敏原致敏的Tas2r105-/-或α-Gustducin-/-小鼠来确定其分子基础。为了将我们在老鼠身上的发现转化为人类，我们将使用人类肺标本来揭示苦味诱导的支气管扩张的机制。这项工作不仅应该确定苦味如何导致小鼠和人类的支气管扩张，而且应该确定它们在治疗慢性哮喘小鼠模型中的呼吸道疾病方面的有效性。这些进展将加深我们对ASM生物学的理解，并促进苦味剂作为新的支气管扩张剂的发展。

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.