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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL047671-01A2
Application #
3366909
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1993-06-10
Project End
1998-04-30
Budget Start
1993-06-10
Budget End
1994-04-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202