CRITICAL TARGETS IN HYPEROXIC MITOCHONDRIAL INJURY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 支气管肺发育不良(BPD)是一种常见的、致残的、有时甚至是致命的慢性肺部疾病。它经常伴随着早产和呼吸窘迫的治疗，通过人工通风和高浓度的吸入氧气(高氧)。BPD的生化基础还不是很清楚。在狒狒中，超早产型(125天)和早产型(140天)的BPD都需要接受高氧暴露的治疗。高氧损伤线粒体，导致乌头酸酶活性丧失，线粒体和细胞呼吸减少，ATP丧失。在此基础上，这一建议的主要假设是，适应高氧应激需要糖酵解和/或谷氨酰胺分解酶的上调。由于己糖激酶限制了肺内糖酵解的速率，故推测肺内己糖激酶(S)的表达上调。替代缺乏线粒体的抗氧化剂可以缓解早期呼吸窘迫和由此引起的BPD，并减少糖酵解酶的上调。这些假说将在这些目标上得到验证：1)确定线粒体孔蛋白复合体(HKS)、孔蛋白和腺苷核苷酸转运体(ANT)、这些相同的蛋白质以及相关的糖酵解和谷氨酰胺分解酶的mRNA编码成分的差异表达；2)明确关键抗氧化剂(谷胱甘肽、硫氧还蛋白、超氧化物歧化酶)及其前体(S-腺苷蛋氨酸)和氧化靶标(乌头酸酶)的早期状态；(3)确定早期持续输注细胞和线粒体谷胱甘肽前体S-腺苷蛋氨酸对BPD这些标志物和肺组织病理学的影响。己糖激酶与线粒体的结合或从线粒体释放，将使用免疫金电子显微镜进行定量。还将检测HK、磷酸果糖激酶以及其他关键的糖酵解和谷氨酰胺分解酶的活性。肺GSH、TRX和ADOMet以及循环中的GSH、ADOMet和硫氨基酸将在125d模型的早期进行测量。总之，这些方法将有助于确定己糖激酶活性表达的变化是否也发生在早产新生狒狒身上。已知的己糖激酶活性表达变化发生在耐氧成年大鼠的肺中。此外，他们还将指出肺组织谷氨酰胺利用酶的表达增加是否也发生在肺氧化应激过程中，以及抗氧化剂应激和抗氧化剂补充是否改变了这些适应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bronchopulmonary dysplasia (BPD) is a common, disabling and sometimes fatal chronic lung disease. It frequently accompanies premature birth and treatment of respiratory distress with artificial ventilation and high concentrations of inspired oxygen (hyperoxia). The biochemical basis of BPD is not well understood. In the baboon, exposure to hyperoxia is required treatment in both the ultra-premature (125 d) and premature (140) model of BPD. Hyperoxia damages mitochondria results in loss of aconitase activity, decreased mitochondrial and cell respiration, and loss of ATP. On that basis, this proposal's principal hypothesis is that adaptation to hyperoxic stress requires up-regulation of glycolytic, and/or glutaminolytic, enzymes. Because hexokinase rate- limits glycolysis in lung, it is hypothesized that expression of lung hexokinase(s) is up-regulated. Replacement of deficient mitochondrial anti-oxidants may alleviate early respiratory distress and resulting BPD and decrease up regulation of glycolytic enzymes. These hypotheses will be tested in these AIMS; 1) Determine differential expression of mRNA's encoding components of the mitochondrial porin complex-hexokinases (HKs), porins, and adenine nucleotide translocators (ANT), these same proteins, and relevant glycolytic and glutaminolytic enzymes; 2) Define early status of critical anti-oxidants (glutathione [GSH], thioredoxin [TRX], superoxide dismutases]), their precursors (S-adenosylmethionine), and oxidant target (aconitase) markers, and (3) Define the efficacy of early, continuous infusion of S-adenosylmethionine (AdoMet), a precursor of cellular and mitochondrial glutathione (GSH), on these markers and pulmonary histopathology of BPD. Hexokinase binding to, or release from, mitochondria will be quantitied using immunogold electron microscopy. Activities of HK, phosphofructokinase, and additional critical glycolytic and glutaminolytic enzymes also will be assayed. Lung GSH, TRX, and AdoMet, as well as circulating GSH, AdoMet, and sulfur amino acids will be measured early in the 125 d model. Together, these approaches will help define whether changes in hexokinase activity expression, known to occur in lungs of adult rats made oxygen-tolerant, also occur in the premature newborn baboon. In addition, they will indicate whether or not increased expression of pulmonary glutamine- utilizing enzymes also occurs during pulmonary oxidative stress and whether anti-oxidant stress and whether anti-oxidant supplementation modifies these adaptations.