Biol. Pharm. Bull. 30(2) 272—278 (2007)

ero-3-phosphocholine) is a phospholipid that is synthesized by a variety of different cells and tissues in response to different stimuli, and is involved in numerous biological responses. Consequently, PAF is recognized as a major mediator that plays a central role in a variety of host defense system mechanisms and inflammatory diseases. Two pathways for PAF synthesis have been identified. One route is a de novo pathway, which is thought to constitutively produce a small amount of PAF to serve some basal physiological roles. The second route is a remodeling pathway, which synthesizes PAF via two enzymatic reaction steps. In this pathway, phospholipase A2 (PLA2) hydrolyzes 1-alkyl-2arachidonyl-sn-glycero-3-phosphocholine to 1-alkyl-2-lysoglycero-3-phosphocholine (lyso-PAF), which is then acetylated by acetyl-CoA: lyso-PAF acetyltransferase to generate PAF. The remodeling pathway appears to be involved in synthesizing most of the PAF that is generated in response to agonist receptor binding. Since acetyl-CoA: lyso-PAF acetyltransferase is unstable, it is difficult to purify, and consequently, little is known about the enzyme. However, the use of rat spleens, human neutrophils, guinea pig parotid glands and mouse mast cells as sources of the enzyme for in vitro assays has shown that the enzymatic activity appears to be regulated by the intracellular calcium levels and involve a phosphorylation–dephosphorylation mechanism. The kinase responsible for the phosphorylation is unknown, but catalytic subunits of cyclic AMP-dependent protein kinase and calcium/calmodulin–dependent protein kinase are capable of activating the acetyltransferase in vitro. Furthermore, it has also been shown that p38 mitogen-activated protein kinase (MAPK) can lead to increased activation of the acetyltransferase. It has previously been reported that hydrogen peroxide (H2O2) causes cytotoxic injury to endothelial cells, 15,16) but, under some conditions, it initiates the synthesis of biologically active molecules such as PAF. The reactive oxygen species (ROS)-mediated damage to intracellular molecules is limited by cellular antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase, and catalase. Although direct evidence for the involvement of antioxidant enzymes in the production of PAF is still lacking, it has been shown that selenium deficiency, which causes a drop in GPx activity along with a corresponding rise in hydroperoxide levels, increases PAF production in cultured human umbilical vein endothelial cell (HUVEC). These observations suggest that oxidative stress could modulate PAF synthesis and/or that scavenger enzymes could regulate the activity of the enzymes involved in PAF biosynthesis by controlling intracellular redox. On the basis of these observations, we investigated whether intracellular redox could regulate PAF synthesis. To achieve this, we used HUVEC and show here that PAF synthesis in these cells was caused by H2O2 in a concentrationand time-dependent manner that was consequent to the activation of acetyl-CoA: lyso-PAF acetyltransferase. The PAF synthesis caused by H2O2 was reduced by antioxidants but intensified by the depletion of the endogenous antioxidant, glutathione. In addition, a tyrosine kinase inhibitor and a calmodulin antagonist suppressed the activity of lyso-PAF acetyltransferase. Thus, intracellular redox affects the signal transduction system, involving a tyrosine kinase and/or a calmodulin-dependent protein kinase, and thereby modulates PAF synthesis.