Calcium microdomains regulating pulmonary endothelial permeability

项目总结 我们先前已经确定了肺泡间隔内皮细胞激活后不同的功能结果。 1G-T型电压门控性钙通道 2+2+ 频道。尽管在极薄的间隔内皮细胞中存在等量的全细胞钙瞬变，但仅TRPV4 增加内皮通透性，而T通道仅增加内皮细胞表面P- 选择素。因此，我们提出了一个关键的范式转变，从钙依赖的全球角度来看 向线粒体依赖的钙缓冲所协调的钙微域发出信号 组织以在肺微血管内皮细胞内产生离散的功能结果。我们的预赛 数据表明，这些钙通道的离散定位和线粒体的分布甚至进入了 原位肺微血管内皮细胞周边变薄。此外，我们还记录了 线粒体生物能功能障碍导致结构域限制的丧失，导致更大的传播和持续时间 TRPV4介导的肺微血管内皮细胞钙瞬变和内皮通透性增加。 总而言之，这些观察结果让我们得出了这样的假设：在肺微血管内皮细胞中， 线粒体钙缓冲限制通过TRPV4或T型通道的钙内流在空间上限定 胞质微域产生功能结果的特异性，与线粒体失去约束 功能障碍。我们的具体目标是：1)确定线粒体对缓冲细胞的作用 钙信号在TRPV4或T型钙激活中的空间分布、动力学和功能特异性 通道，以及2)确定线粒体生物能量障碍降低阈值的程度 TRPV4或T型钙通道激活的功能结果及其特异性。我们将利用 创新的高速高光谱激发扫描成像和新的分析工具，以检测和 以高空间和时间分辨率解释信号动力学。这些数据将在以下背景下进行解释 线粒体破裂后，在单纯的内皮中，在内皮中的局部功能结果- 依赖缓冲和启动线粒体生物能量障碍后的完整肺 高氧和铜绿假单胞菌致脓毒症。我们预测，这种功能障碍将导致模糊 钙信号的特异性，改变肺内皮细胞解释钙信号的设定点 机械应力。这项工作将首次深入了解钙离子微域的潜在机制 肺微血管内皮细胞。为了实现这一目标，我们组建了一支拥有专业知识的优秀团队。 从内皮通透性的结构和功能决定因素出发，开发和使用新的 工具，以及生物能量学和脓毒症的信号域/网络建模。

PROJECT SUMMARY We have previously identified distinct functional outcomes in lung alveolar septal endothelium on activation of TRPV4, a Ca channel in the vanilloid transient receptor potential family, or the 1G T-type voltage-gated Ca 2+ 2+ channel. Despite equivalent whole-cell Ca2+ transients in the extremely thin septal endothelium, TRPV4 only increases endothelial permeability while the T-channel only increases endothelial surface expression of P- selectin. As a result, we propose a critical paradigm shift, from a global perspective of Ca2+-dependent signaling to one where Ca2+ microdomains, orchestrated by mitochondrial-dependent Ca2+ buffering, are organized to yield discrete functional outcomes within lung microvascular endothelial cells. Our preliminary data suggest discrete localization of these Ca2+channels and distribution of mitochondria even into the attenuated cell periphery in lung microvascular endothelium in situ. Further, we have documented that mitochondrial bioenergetic dysfunction leads to loss of domain constraints with greater spread and duration of TRPV4-mediated Ca2+ transients and increased endothelial permeability in lung microvascular endothelium. Collectively, these observations led us to the HYPOTHESIS that in lung microvascular endothelium, mitochondrial Ca2+ buffering constrains Ca2+ influx via TRPV4 or the T-type channel to spatially delimited cytosolic microdomains yielding specificity of functional outcomes, constraints lost with mitochondrial dysfunction. Our SPECIFIC AIMS are to: 1) determine the contribution of mitochondria to buffering of the spatial spread, dynamics and functional specificity of Ca2+ signals on activation of TRPV4 or T-type Ca2+ channels, and 2) determine the extent to which mitochondrial bioenergetic dysfunction decreases the threshold for and specificity of functional outcomes on activation of TRPV4 or T-type Ca2+ channels. We will utilize innovative high-speed hyperspectral excitation scanning imaging and novel analytical tools to detect and interpret signal dynamics with high spatial and temporal resolution. These data will be interpreted in context of localized functional outcomes, in naïve endothelium, in endothelium after disruption of mitochondrial- dependent buffering and after initiation of mitochondrial bioenergetic dysfunction in the intact lung with hyperoxia and Pseudomonas aeruginosa-induced sepsis. We predict that such dysfunction will lead to blurring of specificity for Ca2+ signaling, altering the set point from which lung endothelium interprets Ca2+ signaling with mechanical stress. This work will provide the first insight into mechanisms underlying Ca2+ microdomains in lung microvascular endothelium. To accomplish this, we have assembled an outstanding team with expertise spanning from structural and functional determinants of endothelial permeability, development and use of novel tools, and modeling of signaling domains/networks to bioenergetics and sepsis.