Post-Translational Regulation of High Output NO and Endothelial Barrier Dysfuncti

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Sepsis, a leading cause of acute lung injury, causes pulmonary inflammation and increased capillary endothelial permeability and is a potent stimulus for inducible nitric oxide synthase (iNOS) expression. Nitric oxide (NO) plays an important role in regulating lung vascular permeability, and high levels produced during inflammation, or combined with superoxide to form peroxynitrite, can injure the endothelial barrier. Although iNOS is thought to be primarily transcriptionally regulated, our evidence shows that iNOS activity and NO production in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) are under more finely-tuned post-translational regulation. In Project 2, we will address new and important signaling pathways by which high output NO is regulated in cytokine-stimulated HLMVECs. In Specific Aim #1, we will elucidate the mechanisms by which NO production via iNOS is efficiently induced by the release of Arg from Arg-containing peptides by the membrane carboxypeptidases (CP) M and CPD. Our hypothesis is that CPM and/or CPD form a macromolecular complex with the Arg transporter and iNOS resulting in tight coupling of the transfer of Arg released from peptide substrates to iNOS. In Specific Aim #2, we will identify the signaling pathways by which the kinin B1 receptor stimulates iNOS activity and high output NO production in cytokine-stimulated HLMVECs and determine its consequences on lung endothelial barrier function. Our hypothesis is that B1 receptor stimulation in cytokine-treated HLMVECs activates iNOS by coupling through the heterotrimeric G protein, Galpha-i, and Src kinase leading to a change in phosphorylation and/or subcellular localization of iNOS to up-regulate NO production. In Specific Aim #3, we will determine the pathway of the bradykinin B2 receptor stimulation of the prolonged, high output NO in cytokine-acfivated HLMVECs and the role of CPM and CPD in amplifying NO output by converting kinin B2 agonists to the des-Arg-kinin B1 agonists, and the resultant consequences on endothelial permeability. Our hypothesis is that the B2 receptor couples through Galpha-i in cytokine-stimulated HLMVECs to activate Src kinase and Akt, resulting in phosphorylation and the prolonged activation of eNOS. This, coupled with carboxypeptidase-mediated generation or B1 agonists and B1 receptor activation of iNOS, results in the further amplification of NO production and disruption of the endothelial barrier. These studies will identify novel mechanisms by which lung microvascular endothelial cells under inflammatory conditions can generate high-output NO as autocrine and paracrine signals to increase endothelial permeability, and thus will allow identification of potential targets for therapeutic intervention to improve endothelial barrier function.