TRPV4 in Regulation of Lung Endothelial Permeability

TRPV4 调节肺内皮通透性

• 批准号: 6976874
• 负责人: MARY I TOWNSLEY
• 金额: $ 36.2万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6976874
• 关键词: biological signal transduction; calcium channel; calcium flux; disease /disorder model; electroporation; heart failure; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rat; lung alveolus; lung injury; membrane activity; membrane permeability; polymerase chain reaction; receptor expression; respiratory epithelium; small interfering RNA; transmission electron microscopy; vascular endothelium permeability

DESCRIPTION (provided by applicant): We propose that acute lung injury paradigms are likely to be characterized by unique endothelial injury "finger prints" based on the Ca2+ entry pathways they target and the expression pattern of these channel proteins. The consensus of data suggests that members of the canonical subfamily of transient receptor potential (TRP) proteins comprise subunits of store-operated Ca2+ channels and participate in Ca2+ entry-dependent regulation of lung endothelial permeability in extra-alveolar vessels. Our observation that heart failure leads to loss of the permeability response to store depletion but not that to 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid that promotes Ca2+ entry-dependent acute lung injury only in alveolar septal capillaries, suggests that 14,15-EET targets a distinct channel expressed in septal endothelium. Our preliminary data suggests a novel candidate -TRPV4-a member of the vanilloid subfamily of TRP proteins that is diversely regulated by endocannabinoids, arachidonic acid, EETs, heat, and mechanical perturbation. Our hypothesis is that regulation of TRPV4 channels expressed in alveolar septal endothelium integrates the Ca2+- entry-dependent permeability response to diverse stimuli, including endocannabinoids, EETs, high vascular pressure, hypotonicity, and heat. To address our hypothesis, we have devised 2 specific aims. AIM 1 will determine whether TRPV4 is a common target by which endocannabinoids, EETs, mechanical perturbation, hypotonicity and heat promote Ca2+ influx required for increased endothelial permeability in the alveolar septal compartment of the lung. AIM 2 will reveal whether acute chronic heart failure leads to a selective susceptibility to acute lung injury due to retention of responsiveness to activation of TRPV4 channels but loss of responsiveness to store depletion. We will address these aims using specific measures of permeability and Ca2 + entry in the intact lung endothelium, microscopy of lung and vascular corrosion casts to map spatial heterogeneity in the permeability response, and pharmacological tools to manipulate signaling pathways implicated in gating of TRPV4. The impact of TRPV4 down-regulation in rat lung by siRNA will be compared to outcomes in TRPV4-/ mice, and to that in chronic heart failure where store-operated TRP channels alone appear to be down regulated. This work will provide the first rigorous analysis of compartmentalized TRPV4 channel expression linked to compartmentalized regulation of endothelial permeability in the intact lung.

描述（由申请人提供）：我们提出，急性肺损伤范例可能以独特的内皮损伤“指纹”为特征，基于它们靶向的 Ca2+ 进入途径和这些通道蛋白的表达模式。数据一致表明，瞬时受体电位 (TRP) 蛋白典型亚家族的成员包含钙池操纵的 Ca2+ 通道亚基，并参与肺泡外血管中肺内皮通透性的 Ca2+ 进入依赖性调节。我们观察到心力衰竭会导致对储备耗尽的通透性反应丧失，但不会导致对 14,15-环氧二十碳三烯酸 (14,15-EET) 的通透性反应丧失，14,15-环氧二十碳三烯酸 (14,15-EET) 是一种仅在肺泡间隔毛细血管中促进 Ca2+ 进入依赖性急性肺损伤的脂质，这表明 14,15-EET 靶向在间隔内皮中表达的独特通道。我们的初步数据表明了一种新的候选者 -TRPV4 - TRP 蛋白香草酸亚家族的成员，它受到内源性大麻素、花生四烯酸、EET、热和机械扰动的多种调节。我们的假设是，肺泡间隔内皮表达的TRPV4通道的调节整合了Ca2+-进入依赖性通透性对不同刺激的反应，包括内源性大麻素、EET、高血管压力、低渗性和热。为了解决我们的假设，我们设计了两个具体目标。 AIM 1 将确定 TRPV4 是否是内源性大麻素、EET、机械扰动、低渗性和热量促进肺泡间隔室内皮通透性增加所需的 Ca2+ 流入的共同目标。 AIM 2 将揭示急性慢性心力衰竭是否会由于保留对 TRPV4 通道激活的反应性但丧失对储备耗尽的反应性而导致对急性肺损伤的选择性易感性。我们将使用完整肺内皮细胞渗透性和 Ca2+ 进入的具体测量、肺和血管腐蚀铸型的显微镜检查来绘制渗透性反应的空间异质性，以及操纵与 TRPV4 门控相关的信号通路的药理学工具来实现这些目标。将 siRNA 对大鼠肺中 TRPV4 下调的影响与 TRPV4-/ 小鼠的结果进行比较，并与慢性心力衰竭的结果进行比较，在慢性心力衰竭中，仅存储操作的 TRP 通道似乎被下调。这项工作将首次对与完整肺内皮通透性的分区调节相关的分区 TRPV4 通道表达进行严格分析。