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Cellular and Molecular Mechanisms of bitter tastant-induced bronchodilation

苦味剂诱导支气管扩张的细胞和分子机制

基本信息

批准号：
8578052
负责人：
Ronghua ZhuGe
金额：
$ 39.63万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8578052
关键词：
Acute Address Adverse effects Affect Agonist Allergens American Asthma Biological Process Biology Biophysics Breathing Bronchoconstrictor Agents Bronchodilation Bronchodilator Agents Cause of Death Cell Line Cells Chronic Chronic Disease Chronic Obstructive Airway Disease Contracts Cycloheximide Data Development Disease Dose Effectiveness Event Genetic Heterotrimeric GTP-Binding Proteins Hormones Human Image Immunology In Vitro Inflammatory Inhalators Interphase Cell Ion Channel Knockout Mice Knowledge Length Ligands Link Lung Lung diseases Molecular Molecular Mechanisms of Action Mus Muscle Muscle Contraction Muscle relaxation phase Neurotransmitters Obstruction Oral Pathway interactions Patients Pertussis Toxin Pharmacology Proteins Regulation Relaxation Reporting Scientist Shortness of Breath Signal Pathway Signal Transduction Smooth Muscle Myocytes Specimen System Taste Buds Taste Perception Therapeutic Tissues Tongue Translating United States Wheezing Work airway obstruction alpha-gustducin base experience in vivo inhibitor/antagonist mouse genome mouse model novel therapeutics patch clamp prevent public health relevance rat Gnat3 protein receptor research study respiratory smooth muscle response

项目摘要

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.

描述（由申请人提供）：苦味促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，苦味促味剂作为支气管扩张剂前景广阔，是全球3亿哮喘和慢性阻塞性肺病患者不可或缺的药物。我们试图解决两个基本的问题，这种放松：在ASM中的促苦味剂的作用的细胞和分子机制，和慢性哮喘的促苦味剂的有效性。苦味促味剂激活味觉细胞和一些口腔外细胞中的2型味觉受体（TAS 2 R）-味蛋白-PLC β 2途径。然而，我们的初步数据表明，TAS 2 Rs和gustducin，而不是PLC β 2，是必不可少的苦味剂诱导的支气管扩张。为了坚定地建立这一观点，我们将研究删除TAS 2 R105、α-味蛋白或PLC β 2对苦味素诱导的ASM中[Ca 2 +]i、细胞长度和张力变化的影响（目的1）。Tas 2 r105-/-小鼠的独特之处在于，可以用放线菌酮（一种对TAS 2 R105具有特异性的配体）可靠地评估所产生的功能变化。另一方面，α-味蛋白-/-小鼠的优势在于，尽管小鼠基因组含有35个TAS 2 R，但它们都与味蛋白偶联。因此，味觉蛋白缺失可以阻断支气管扩张对广谱苦味促味剂的反应。我们的初步数据进一步揭示，L型CaV1.2通道的抑制是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PLC β。利用膜片钳、药理学、基因敲除小鼠和异源表达系统，我们将揭示促苦味剂抑制该通道的分子机制（目的2）。最后，促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，必须确定促苦味剂在慢性哮喘中的有效性。哮喘我们的初步研究表明，促苦味剂逆转了两种慢性哮喘小鼠模型的气道收缩，其中一种由烟曲霉粗蛋白提取物（一种常见的人类哮喘过敏原）诱导。在目标3中，我们将在这些小鼠中系统地表征这种体外和体内效应，并使用过敏原致敏的Tas 2 r105-/-或α-gustducin-/-小鼠确定其分子基础。为了将我们在小鼠中的发现转化为人类，我们将使用人类肺标本揭示苦味剂诱导支气管扩张的机制。这项工作不仅要确定促苦味剂如何引起小鼠和人的支气管扩张，而且要确定它们在治疗慢性哮喘小鼠模型气道疾病中的有用性。这些进展将加深我们对ASM生物学的理解，并促进苦味促味剂作为新的支气管扩张剂的发展。