REDOX ACTIVITY OF THE PULMONARY ENDOTHELIAL SURFACE

肺内皮细胞跨质膜电子传递(TPMET)系统通过细胞内供体向细胞外电子受体的运输来影响细胞外电子受体的减少。肺组织TPMET的生理作用和机制尚不清楚。激励这项研究的一个普遍假设是，肺内皮细胞TPMET影响全身动脉血液成分的氧化还原状态。根据通过混合静脉血到达的电子受体的性质，这种功能可能具有抗氧化剂或促氧化剂的后果。在其抗氧化作用中，肺内皮细胞TPMET再生血浆脂蛋白抗氧化剂有助于抗氧化防御体循环。这在一定程度上是巨大的肺内皮细胞表面积和肺在循环系统中的位置的功能结果。当TPMET系统中存在氧化还原活性毒素或自由过渡金属电子受体时，肺TPMET促进其保护作用的特性也有可能引发氧化损伤。拟议研究的目的是检验这些概念，并进一步阐明肺内皮细胞TPMET的细胞机制。本研究的具体目的如下：1.确定肺内皮细胞TPMET对生理或毒理氧化还原活性物质胞外氧化还原状态的影响。具体的假设是，肺内皮细胞TPMET系统(1)将辅酶Q0和Trolox C的氧化形式还原为其抗氧化对苯二酚形式，(2)再生还原形式的血浆脂蛋白抗氧化辅酶Q10，作为保护血浆脂蛋白免受氧化的内皮保护的机制，以及(3)将肺毒素百草枯还原为其氧化前单定位形式。一般的方法是证明当完整的细胞暴露在氧化形式下时，这些电子受体的还原产物出现在细胞外介质中，并确定细胞表面是否有能够介导完整细胞进行还原的蛋白质。II.阐明TPMET的细胞机制。具体的假设是，肺内皮细胞TPMET系统之一的活性(1)取决于细胞内NAD(P)H/NAD(P)+比率所反映的细胞内氧化还原状态，以及(2)涉及TPMET黄素蛋白，可能还涉及其他氧化还原中心。一般的方法是将TPMET活性与完整细胞中的吡啶核苷酸氧化还原平衡联系起来，并确定氧化还原修复基团抑制剂对分离的质膜氧化还原组分的还原酶活性的一些影响。

Transplasma membrane electron transport (TPMET) systems in pulmonary endothelial cells effect reduction of extracellular electron acceptors via transport of intracellular donors to the extracellular acceptors. The physiological role and mechanisms of pulmonary TPMET are not well understood. A general hypothesis motivating the proposed research is that pulmonary endothelial TPMET influences the redox status of systemic arterial blood constituents. This function can have either antioxidant or prooxidant consequences depending on the nature of the electron acceptors arriving via the mixed venous blood. In its antioxidant role, pulmonary endothelial TPMET regeneration of plasma lipoprotein antioxidants contributes to antioxidant defense of the systemic circulation. This is, in part, a functional outcome of the large pulmomary endothelial surface area and the location of the lung in the circulatory system. The very properties of lung TPMET that promote its protective effects also have the potential to initiate oxidant injury when TPMET systems are presented with redox active toxins or free transition metal electron acceptors. The goal of the proposed studies is to examine these concepts and to further elucidate cellular mechanisms involved in pulmonary endothelial TPMET. The specific aims of the proposed research are as follows: I. Determine the influence of pulmonary endothelial TPMET on the extracellular redox state of physiological or toxicological redox active compounds. The specific hypotheses are that pulmonary endothelial TPMET systems (1) reduce the oxidized form of coenzyme Q0 and Trolox C quinone to their antioxidant hydroquinone forms, (2) regenerate the reduced forms of the plasma lipoprotein antioxidant coenzyme Q10 as a mechanism underlying endothelial protection of plasma lipoprotein from oxidation, and 3) reduce the pulmonary toxin paraquat to its prooxidant monocation form. The general approach will be to demonstrate that reduction products of these electron acceptors appear in the extracellular medium when the intact cells are exposed to the oxidized forms, and to determine whether there are proteins on the cell surface that are capable of mediating the reduction carried out by the intact cells. II. Elucidate cellular mechanisms involved in TPMET. The specific hypotheses are that the activity of one of the pulmonary endothelial TPMET systems (1) depends on intracellular redox status as reflected in the intracellular NAD(P)H/NAD(P)+ ratios, and (2) involves a TPMET flavoprotein, and possibly other, redox centers. The general approach will be to correlate TPMET activity and pyridine nucleotide redox poise in intact cells and to determine some of the effects of redox prosthetic group inhibitors on the reductase activity of isolated plasma membrane redox components.