Types of purinoceptors and phospholipase A 2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells

Abstract

Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431–17437, 1997). To understand the mechanism(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A 2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A 2 (cPLA 2) were also performed to evaluate whether downstream activation of cPLA 2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P 2u/P2Y 2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATPγS > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADPβS and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATPγS in stimulating the release of arachidonate. These results suggest that both P 2u/P2Y 2 and P 2y/P2Y 1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA 2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [ 3H]acetate incorporation into radyl[ 3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA 2 supplies the lysophospholipids as substrates for the transacetylation reaction.