ZINC HOMEOSTASIS AND PULMONARY ENDOTHELIAL CELL INJURY

超出所提供的空间。除了激活可溶性鸟苷酸环化酶（sGC）/cGMP依赖性途径外，NO还通过靶蛋白中半胱氨酸的翻译后修饰（例如S-亚硝化）发挥其许多生理作用。在前一个资助期，我们能够揭示一个非常重要的作用，S-亚硝化的小细胞内金属结合蛋白，金属硫蛋白（MT），在介导NO诱导的增加不稳定的锌。在培养的肺内皮细胞的观察表明，不稳定的锌有助于pronecrotic途径或相反，NO介导的锌释放降低培养的小鼠肺内皮细胞（MLEC）的敏感性促凋亡刺激。这些研究仅限于外源性NO供体在分离培养细胞中的作用。多模式成像和选择性地修改这些途径的肺内皮成分的能力的进展是当前更新的总体目标的基础，该目标是检查内源性NO对完整肺内皮中的信号通路的作用。因此，这次更新的具体目标是确定：内源性NO诱导完整肺内皮锌释放的分子基础。全光谱共聚焦和多光子激光扫描显微镜（MPLSM）将用于监测分离的灌注小鼠肺中的eNOS衍生的NO诱导的基于荧光共振能量转移的报告物的变化：a）S-亚硝化的FRET-MT;和B）cygnet 2，一种cGMP依赖性蛋白激酶A FRET嵌合体，其报告sGC的活化。二.锌依赖性蛋白激酶C（PKC）活化在完整肺内皮细胞氧化（偶氮引发的过氧）损伤中的作用。将通过MPLSM定量GFP-PKC-γ的易位来确定在隔离灌注的小鼠肺的内皮中PKC的活化（及其对锌螯合的敏感性），并且将确定PKC活化在氧化剂介导的肺水肿中的作用。II.通过蛋白质组学分析，在肺内皮中被NO S-亚硝化的靶点（除了MT）。将来源于野生型或（eNOS-1-mce作为对照）的MLEC暴露于eNOS的活化剂（钙离子载体，卡巴胆碱）。eNOS衍生的NO翻译后（例如S-亚硝化）修饰将通过比较蛋白质表达的变化来确定，所述蛋白质表达的变化通过2D电泳和质谱来进行。性能现场=

EXCEED THE SPACE PROVIDED. In addition to activation of soluble guanylyl cyclase(sGC)/cGMP dependent pathways, NO exerts many of its physiological effects by posttranslational modification (e.g. S-nitrosation) of cysteines in target proteins. In the previous funding period, we were able to reveal a singularly important role for S-nitrosation of the small intracellular metal binding protein, metallothoinein (MT), in mediating NO induced increases in labile zinc. Observations in cultured pulmonary endothelial cells suggested that labile zinc contributed to pronecrotic pathways or conversely, NO mediated Zn release reduced the sensitivity of cultured murine lung endothelial cells (MLEC) to proapoptotic stimuli. These studies were confined to the effects of exogenous NO donors in isolated cultured cells. Advances in multimode imaging and the ability to selectively modify pulmonary endothelial components of these pathways in situ underlies the overall goal of the current renewal which is to examine the role of endogenous NO on signaling pathways in the intact pulmonary endothelium. Accordingly, the specific aims of this renewal are to determine the: I. molecular basis of endogenously derived NO-induced zinc release in intact pulmonary endothelium. Full spectral confocal and multiphoton laser scanning microscopy (MPLSM) will be used to monitor eNOS derived NO induced changes in fluorescence resonance energy transfer-based reporters: a) FRET-MT for S-nitrosation; and b) cygnet2, a cGMP dependent protein kinase A FRET chimera that reports activation of sGC), in isolated perfused mouse lungs. II. role of zinc dependent activation of protein kinase C (PKC) in oxidative (azo-initiated peroxyl) injury to the intact pulmonary endothelium. Activation of PKC (and its sensitivity to zinc chelation) will be determined in endothelium of iso ated perfused mouse ung by MPLSM quantification of translocation of GFP-PKC-gamma and role of PKC activation in oxidant mediated pulmonary edema will be determined. II1.targets (in addition to MT) that are S-nitrosated by NO in pulmonary endothelium by proteomic analysis. MLEC derived from w dtype or (eNOS-l- m ce as control) will be exposed to activators (calcium ionophore, carbachol) of eNOS. eNOS derived NO posttranslational (e.g. S-nitrosation) modifications will be determined by comparison of changes in protein express on by 2d gee ectrophoresis and mass spectrometry. PERFORMANCE SITE ========================================Section End===========================================