Oxidant stress may participate in the evolution of vascular damage and loss of barrier function leading to the development of atherosclerosis, adult respiratory distress syndrome and vasculitis. Mechanisms of oxidant-induced endothelial barrier dysfunction are not well understood but likely involve modulation of lipid signalling pathways. Protein kinase C (PKC) has been implicated in oxidant-mediated disruption of normal barrier function, as activation of PKC alters in vitro endothelial cell permeability. Protein kinase C is activated by an endogenous activator, diacylglycerol (DAG) , a product of phospholipase C (PLC) catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). However, PLC-catalyzed PIP2 hydrolysis cannot account for all DAG accumulation suggesting other important DAG generating mechanisms must exist. One such mechanism is phospholipase D (PLD) catalyzed hydrolysis of membrane phospholipids generating phosphatidic acid (PA) which is subsequently dephosphorylated by PA phosphatase to yield DAG. Thus, PLD-mediated generation of PA and DAG produces sustained activation of PKC which is important for the development of increased endothelial permeability leading to vascular dysfunction. The hypothesis that oxidant-induced activation of phospholipase D causes sustained activation of protein kinase C resulting in increased vascular permeability will be tested in endothelial cells from bovine pulmonary artery and human umbilical cord vein.
The specific aims are: 1) to determine if oxidants mediate activation of endothelial cell PLD; 2) to investigate the potential role of Ca 2+ in the regulation of oxidant-mediated PLD activation; 3) to determine the regulatory role of protein kinase C in oxidant-mediated PLD activation and 4) To determine which membrane phospholipids serve as substrates for oxidant-induced PLD. 5) To determine the effect of oxidants on PLD activation in cell-free system. The long-term objectives are to provide new and important knowledge about signalling mechanisms in endothelial cells, and further establish mechanisms of oxidant-induced vascular permeability. A better understanding of signal transduction pathways and second-messengers may lead to the design of therapeutic agents directed against pulmonary oxidative injury.
