Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y(1) and P2Y(2) purinoceptors that activate cytosolic phospholipase A(2) in endothelial cells

The aim of this study was to investigate the stimulating effects on arachid onic acid release of P2Y(1) and P2Y(2) receptor-selective agonists, 2-methy lthio-ATP (2MeSATP) and UTP, respectively, in bovine pulmonary artery endot helial cells. Exposure of cells to 2MeSATP and UTP led to the release of ar achidonic acid, a response which was abolished by the removal of extracellu lar Ca2+ and methyl arachirionyl fluorophosphonate. Phorbol 12-myristate 13 -acetate (PMA) itself not only stimulated arachidonic acid release but also played a permissive role in the response to UTP. However, PMA failed to en hance the arachidonic acid response induced by 2MeSATP, probably due to gre ater attenuation of the [Ca2+](i) increase caused by 2MeSATP than UTP. Inhi bition of protein kinase C with Ro 31-8220 (1-[3-(amidinothio) propyl-1H-in doyl-3-yl]-3-(1-methy-1H-indoyl-3-yl)-maleimide-methane sulphate) and staur osporine, but not with Go 6976 (12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13- methyl-5-oxo-indolo(1,3-a)pyrrolo(3,4-c)carbazole), reduced the arachidonic acid response of 2MeSATP, UTP and PMA. PMA-induced potentiation of the UTP response reached a maximum after a I-h preincubation, then declined and ev entually lost its effect when the preincubation lasted up to 8 h. Among the protein kinase C isoforms present in endothelial cells, beta I and epsilon could be down-regulated by treatment with PMA for 4-24 h. PD 098059 (2-(2- Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) inhibited extracellular signa l-regulated protein kinase activation, cytosolic phospholipase A(2) phospho rylation and arachidonic acid release caused by 2MeSATP, UTP and PMA. Taken together, our results demonstrate that P2Y(1) and P2Y(2) purinoceptors med iate arachidonic acid release by activating cytosolic phospholipase A(2) th rough an elevation of [Ca2+](i) and protein kinase C epsilon-, extracellula r signal-regulated protein kinase-dependent phosphorylation. (C) 1999 Elsev ier Science B.V. All rights reserved.