CRITICAL TARGETS IN HYPEROXIC MITOCHONDRIAL INJURY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 支气管肺发育不良（BPD）是一种常见的，致残的，有时是致命的慢性肺部疾病。它经常伴随早产和呼吸窘迫的人工通气和高浓度吸入氧（高氧）治疗。BPD的生物化学基础还不清楚。在狒狒中，在超早产（125天）和早产（140天）BPD模型中均需要暴露于高氧治疗。高氧损伤线粒体导致乌头酸酶活性丧失、线粒体和细胞呼吸降低以及ATP丧失。在此基础上，该提议的主要假设是，适应高氧应激需要上调糖酵解和/或氨基葡萄糖分解酶。由于己糖激酶速率限制肺中的糖酵解，因此假设肺己糖激酶的表达上调。补充线粒体抗氧化剂可以减轻早期呼吸窘迫和BPD，减少糖酵解酶的上调。1）确定线粒体孔蛋白复合物-己糖激酶（HK）、孔蛋白和腺嘌呤核苷酸移位器（ANT）、这些相同的蛋白质以及相关的糖酵解和精氨酸分解酶的mRNA编码组分的差异表达; 2）确定关键抗氧化剂的早期状态（谷胱甘肽[GSH]，硫氧还蛋白[TRX]，超氧化物歧化酶]），其前体（S-腺苷甲硫氨酸）和氧化剂靶标（顺乌头酸酶）标志物，和（3）确定早期连续输注S-腺苷甲硫氨酸（S-adenosylmethionine，S-Met）（细胞和线粒体谷胱甘肽（GSH）的前体）的疗效，这些标志物和BPD的肺组织病理学。将使用免疫金电子显微镜定量己糖激酶与线粒体的结合或从线粒体释放。还将测定HK、磷酸果糖激酶和其他关键糖酵解酶和精氨酸分解酶的活性。将在125天模型的早期测量肺GSH、TRX和蛋氨酸，以及循环GSH、蛋氨酸和含硫氨基酸。总之，这些方法将有助于确定是否在己糖激酶活性表达的变化，已知发生在肺的成年大鼠制成耐氧，也发生在早产新生狒狒。此外，它们将表明肺谷氨酰胺利用酶的表达增加是否也发生在肺氧化应激期间，以及抗氧化应激和抗氧化剂补充剂是否改变这些适应。

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.