Polarity of Redox Control and Risk of Injury in the Alveolar Epithelium

描述（由申请人提供）：上皮衬里液（ELF）和血浆中还原性和氧化性谷胱甘肽（GSH/GSSG）、半胱氨酸和胱氨酸（Cys/CySS）的浓度和氧化还原电位存在显著的极性。此外，这些硫醇对在任何一个隔室中都不是平衡的。利用我们开发的方法，我们证明了线粒体、细胞核、细胞质和分泌途径之间的硫/二硫氧化还原梯度不平衡，这些区室内的扰动调节氧化还原敏感蛋白的功能。肺室间稳态氧化还原状态的显著差异以及这些氧化还原状态的变化与肺损伤风险相关，使我们假设肺泡间隙和血浆之间的细胞外氧化还原回路的破坏将1)改变细胞器氧化还原状态，2)激活氧化还原敏感信号，3)改变对肺损伤的易感性。同样重要的是，我们假设细胞外氧化还原状态的恢复将使细胞器氧化还原状态正常化并降低损伤的风险。本应用的重点是证明顶端或基底侧表面氧化还原电路的扰动并不仅仅反映氧化应激。相反，这些细胞外的变化影响不同细胞器的氧化还原状态，并启动独立于活性氧产生的细胞信号传导。利用肺泡I型（AT1）细胞和II型（AT2）细胞系以及原代AT2细胞单层，我们将确定顶端肺泡表面的GSH/GSSG或Cys/CySS氧化还原梯度是否会破坏1)基底外侧表面的氧化还原梯度，2)线粒体、细胞质、细胞核的氧化还原梯度以及AT1和AT2细胞的分泌途径，以及3)氧化还原信号（Aim 1）。在Aim 2中，我们将研究由尖肺泡表面氧化还原电路的扰动引起的细胞器氧化还原状态的破坏是否会增加细胞毒素诱导损伤的风险。在Aim 3中，我们将确定基底外侧表面的扰动是否会破坏1)顶端表面的氧化还原梯度，2)线粒体、细胞质、细胞核和AT1和AT2细胞的分泌途径的氧化还原梯度，以及3)氧化还原信号传导。在目标4中，我们将检查基底外侧表面的扰动会增加损伤风险的程度。最后，我们将在Aims 3和4中确定在根尖或基底外侧表面恢复这些梯度如何恢复氧化还原信号并降低对损伤的敏感性。这些研究将提供前所未有的见解，了解氧化应激和肺泡间隙GSH/GSSG或Cys/CySS比例的变化如何传递给细胞器，从而在这些不同的亚细胞区室中引起氧化应激。公共卫生相关性：肺泡衬里液和血浆之间的硫醇/二硫浓度和氧化还原状态存在显著的极性。这项研究将确定这两个细胞外池之间极性的破坏是否会改变上皮细胞亚细胞区室的氧化还原状态、氧化还原敏感蛋白的氧化还原状态或对肺损伤的易感性。这些研究的结果将提供关于肺氧化应激亚细胞区隔化的前所未有的知识。

DESCRIPTION (provided by applicant): There is a significant polarity in the concentrations and redox potentials between the epithelial lining fluid (ELF) and plasma for reduced and oxidized glutathione (GSH/GSSG) and cysteine and cystine (Cys/CySS). Further, these thiol pairs are not in equilibrium in either compartment. Using methods that we developed, we demonstrated that the thiol/disulfide redox gradients between the mitochondria, nuclei, cytoplasm and secretory pathways are not in equilibrium and perturbation within these compartments regulates the function of redox-sensitive proteins. The remarkable differences in steady-state redox states among lung compartments and the changes in these redox states associated with risk of lung injury led us to hypothesize that disruption of the extracellular redox circuitry between the alveolar space and plasma will 1) alter organelle redox states, 2) activate redox-sensitive signaling, and 3) alter the susceptibility to lung injury. Equally important, we hypothesize that restoration of extracellular redox states will normalize organelle redox states and decrease the risk of injury. The focus of this application is to demonstrate that perturbations of the redox circuitry on the apical or basolateral surface do not simply reflect oxidant stress. Rather, these extracellular changes impact the redox state of the different organelles and initiate cell signaling independent of the reactive oxygen species generated. Using alveolar type I (AT1) cell and type II (AT2) cell lines and primary AT2 cell monolayers, we will determine if oxidation of the GSH/GSSG or Cys/CySS redox gradients on the apical alveolar surface will disrupt 1) the redox gradients on the basolateral surface, 2) the redox gradients in the mitochondria, cytoplasm, nuclei and the secretory pathway of AT1 and AT2 cells, and 3) redox signaling (Aim 1). In Aim 2, we will examine if disruption of organelle redox states induced by perturbations of redox circuitry on the apical alveolar surface will increase the risk of cytotoxin-induced injury. In Aim 3, we will determine if perturbations on the basolateral surface will disrupt 1) the redox gradient on the apical surface, 2) the redox gradients in the mitochondria, cytoplasm, nuclei and the secretory pathway of AT1 and AT2 cells, and 3) redox signaling. In Aim 4 we will examine the extent by which perturbations on the basolateral surface will increase the risk of injury. Finally, we will determine in Aims 3 and 4 how restoration of these gradients on the apical or basolateral surface will restore redox signaling and decrease the sensitivity to injury. These studies will provide unprecedented insight into how oxidative stress and changes in the GSH/GSSG or Cys/CySS ratio in the alveolar space is transmitted to organelles to cause oxidative stress within these different subcellular compartments. PUBLIC HEALTH RELEVANCE: There is a significant polarity in the thiol/disulfide concentrations and redox states between the alveolar lining fluid and plasma. This study will determine if disruption of this polarity between these two extracellular pools will alter the redox state of subcellular compartments of epithelial cells, the redox state of redox-sensitive proteins or the susceptibility to lung injury. The results of these proposed studies will provide an unprecedented knowledge concerning the subcellular compartmentalization of oxidative stress in the lung.