Signaling and Crosstalk by Airway Prostanoid Receptors

描述（由申请人提供）：前列腺素类药物与支气管收缩和气道高反应性（AHR）的发病机制有关，因为它们能有效收缩气道平滑肌（ASM），并且它们的水平在哮喘肺中升高。血栓素、PGD2和PGF2alpha通过激活血栓素类前列腺素（TP）受体收缩ASM。即使是通常被认为是支气管扩张剂的PGE2，也有能力通过受体亚型之一（EP1）传导收缩信号，它是其同源配体。然而，尽管它们对ASM有强大的作用，但由于相同的酶促途径同时产生收缩性和松弛性前列腺素，多种配体激活受体，以及存在具有不同信号转导特性的多种受体异构体，因此很难明确地确定这些受体的病理生理相关性。我们相信这些限制可以通过使用转基因和基因靶向小鼠来选择性地调节特定前列腺素受体的信号转导来克服。在本提案中，我们将验证当支气管收缩前列腺素受体的激活优于那些促进支气管扩张的受体时，支气管收缩和AHR发生的整体假设。在Specific Aim 1中，我们将使用过表达TP受体的小鼠与TP受体缺陷基因靶向小鼠联合来确定TP受体是否促进支气管收缩和AHR。我们将使用从基因工程小鼠中分离的原代ASM细胞在体外解剖介导TP受体依赖性收缩的信号通路，并在气管环和完整小鼠中确定其生理意义。在特异性目标2中，我们将确定EPt受体是否是支气管收缩和AHR的介质。为这些实验开发的转基因模型将使我们能够区分EP1受体激活和其他EP受体亚型的影响。由于人们普遍认为一个g蛋白偶联受体的主要生理作用经常被另一个g蛋白偶联受体调节，因此特异性靶3的实验将验证TP和EP1受体的受体互扰是ASM中肾上腺素能受体功能障碍的机制。初步数据显示TP和EP1受体激动剂减弱异丙肾上腺素介导的气管环松弛，这一假设得到了支持。这些目标将融合细胞、组织和整个动物的生化、药理学和生理学研究，以便体外信号事件可以直接与体内生理功能相关。完成这些目标可能会发现与哮喘和其他阻塞性肺疾病具有治疗相关性的新信号事件。

DESCRIPTION (provided by applicant): Prostanoids have been implicated in the pathogenesis of bronchoconstriction and airway hyperreactivity (AHR) because they potently contract airway smooth muscle (ASM), and their levels, are elevated in asthmatic lung. Thromboxane, PGD2 and PGF2alpha contract ASM via activation of thromboxane prostanoid (TP) receptors. Even PGE2, which is generally considered a bronchodilator, has the capacity to transduce a constrictor signal via one of the receptor subtypes (EP1) for which it is the cognate ligand. However, despite their potent effects on ASM, it has been difficult to unambiguously assign pathophysiological relevance to these receptors due to pleiotropic responses resulting from the simultaneous production of constrictor and relaxant prostanoids by the same general enzymatic pathway, receptor activation by multiple ligands, and the existence of multiple receptor isoforms with different signal transduction properties. We believe these limitations can be overcome by using transgenic and gene-targeted mice to selectively modulate signal transduction by specific prostanoid receptors. In this proposal, we will test the overall hypothesis that bronchoconstriction and AHR occur when the activation of bronchoconstrictor prostanoid receptors is favored over those that promote bronchodilation. In Specific Aim 1, we will use mice that overexpress the TP receptor in combination with TP receptor deficient gene-targeted mice to determine whether TP receptors promote bronchoconstriction and AHR. The signaling pathways, which mediate TP receptor-dependent contraction will be dissected in vitro using primary ASM cells, isolated from the genetically engineered mice, and their physiologic significance determined in tracheal rings and intact mice. In Specific Aim 2, we will determine whether the EPt receptor is a mediator of bronchoconstriction and AHR. The transgenic models developed for these experiments will enable us to discriminate between the effects of EP1 receptor activation and those of the other EP receptor subtypes. Since it widely recognized that the primary physiologic actions of one G-protein-coupled receptor are frequently modulated by another, experiments in Specific Aim 3 will test the hypothesis that receptor cross talk by TP and EP1 receptors is a mechanism of (-adrenergic receptor dysfunction in ASM. This hypothesis is supported by preliminary data that show TP and EP1 receptor agonists attenuate isoproterenol-mediated relaxation of tracheal rings. Each of these aims will merge biochemical, pharmacologic and physiologic studies from cells, tissues, and whole animals so that in vitro signaling events can be directly correlated to in vivo physiological function. Completion of these aims may identify novel signaling events that have therapeutic relevance to asthma and other obstructive lung diseases.