Types of purinoceptors and phospholipase A2 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 mechanisms(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 A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 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 cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.