ZINC HOMEOSTASIS AND PULMONARY ENDOTHELIAL CELL INJURY

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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===========================================