Hydroperoxide-Induced Diacylglycerol Formation and Protein Kinase C Activation in Vascular Endothelial Cells

Abstract

We examined the effects of hydrogen peroxide (H2O2) and other hydroperoxides on endothelial cell protein kinase C (PKC) activity. Bovine pulmonary artery endothelial cells (BPAEC) were grown to confluent monolayers and PKC activity was determined in an in vitro assay by measuring phosphorylation of H1 histone. In control unstimulated BPAEC, PKC was primarily localized in the cytosol and treatment of BPAEC with H2O2 resulted in a concentration (10−5 M-10−3 M) and time (15 min-60 min.) dependent translocation of the enzyme from the cytosol to the membrane fraction. In addition to H2O2, linoleic acid hydroperoxide treatment of BPAEC also resulted in PKC activation while tert-butyl hydroperoxide and cumene hydroperoxide were not effective. In addition to causing an increase in membrane-associated PKC activity, H2O2 treatment also resulted in the partial loss of cytosolic PKC activity. As diacylglycerol (DAG) is a critical endogenous activator of PKC, we evaluated whether H2O2 exposure resulted in the increased production of DAG. Exposure to 1.0 mM H2O2 resulted in biphasic accumulation of DAG (two- to threefold). The first phase of DAG formation occurred within 5 min of H2O2 exposure while the second phase of accumulation began at 15 min of treatment and plateaued at about 45 min. The metal ions Cu2+ and Fe3+ augmented the H2O2-induced loss of total (cytosolic and particulate) PKC activity in BPAEC. These studies suggest that oxidants modulate PKC activity and increase DAG levels in vascular endothelial cells.