Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2.
A lysate of unstimulated human umbilical vein endothelial cells (HUVEC) exhibited phospholipase A2 (PLA2) activity, which hydrolyzed phospholipids bearing arachidonate more preferentially than those bearing linoleate at the sn-2 position. An anti-rabbit cytosolic PLA2 monoclonal antibody absorbed the activity, whereas an anti-human type II PLA2 monoclonal antibody did not. HUVEC treated with thrombin generated prostaglandin I2 (PGI2), and the PLA2 activity of the thrombin-stimulated cells was absorbed almost completely by the anti-cytosolic PLA2 antibody. HUVEC treated with tumor necrosis factor (TNF) also generated PGI2. PGI2 generation by TNF-treated cells was suppressed partially by extracellular addition of the anti-type II PLA2 antibody. PLA2 activity in a lysate of TNF-stimulated cells was increased about 2-3-fold, and about half of the increased activity was suppressed by the anti-type II PLA2 antibody. Addition of heparin together with TNF resulted in release of type II PLA2 in the medium. Thus, both cytosolic and type II PLA2s may be involved in agonist-stimulated PGI2 synthesis in HUVEC. Furthermore, exogenously added type II PLA2 was bound to the cell surface and synergistically enhanced PGI2 generation in TNF-stimulated HUVEC. This binding was blocked by either heparin or a monoclonal antibody recognizing the heparin-binding domain of type II PLA2. Taken together, type II PLA2 generated endogenously as well as added exogenously may be captured on the HUVEC surface via heparan sulfate proteoglycan and may contribute to cellular arachidonate metabolism.
Murakami
M
M
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Kudo
I
I
Inoue
K
K
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D003470:Culture Media; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000201:enzymology*; D011464:Epoprostenol / Q000096:biosynthesis*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007527:Isoenzymes; D011464:Epoprostenol; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin