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, are thought to injure the endothelial barrier. Although iNOS is known to be primarily transcriptionally regulated, our evidence shows that iNOS activity and NO production in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) is highly regulated via G-protein coupled receptor signaling pathways. We have identified a novel phosphorylation site in iNOS that results in the generation of prolonged (90 minutes) "super-high output" NO from endothelial cells. We are currently identifying the novel signaling pathways by which the kinin B1 receptor stimulates iNOS activity. So far, we have identified the heterotrimeric G protein, Gαi, and Src kinase as important components of a pathway that leads to activation of MAP kinases and eventual iNOS phosphorylation. Very recently, we have found that B1 receptor activation and super-high output NO result actually promotes recovery of the endothelial barrier, as measured by increases in transendothelial electrical resistance in HLMVEC.

Model of GPI-Anchored CPM on the Membrane

In another set of studies, we are seeking to understand the important roles of regulatory carboxypeptidases in physiological and pathological processes. Known functions include peptide hormone processing, regulation of peptide hormone activity, regulation of fibrinolysis, binding hepatitis B-virus and providing the precursor Arg for nitric oxide synthase. Our studies revealed that carboxypeptidase M (CPM) on the surface of stimulated endothelial cells efficiently converts bradykinin (a B2 receptor agonist) into des-Arg⁹-bradykinin, a B1 agonist, generating a significant and prolonged output of nitric oxide. In addition, we recently determined the 3 dimensional structure of human CPM and the catalytic subunit of human plasma carboxypeptidase N. These studies have revealed structural features that could affect the orientation of CPM on the cell surface or the interaction of the 50 kDa catalytic subunit of CPN with the 83 kDa non-catalytic subunit that is a member of the leucine-rich repeat family of proteins. We are investigating the structure and function of CPM on the plasma membrane by investigating its role as an important regulator of the B1 kinin system via its ability to generate the B1 receptor agonist and efficiently deliver it to the receptor on the membrane by forming a receptor-enzyme complex.

*Click on CPN, CPM, or B1 receptor to download related publications

Lab Members