Identification of the phospholipase A(2) isoforms that contribute to arachidonic acid release in hypoxic endothelial cells: limits of phospholipase A(2) inhibitors.
Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A(2) (PLA(2)) activity in human umbilical vein endothelial cells. Both basal PLA(2) activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)), while OPC, an inhibitor of soluble PLA(2) (sPLA(2)) only inhibited the hypoxia-induced AA release and PGF(2alpha) synthesis. Hypoxia does not alter expression of iPLA(2), sPLA(2) and cPLA(2) and cycloheximide did not inhibit PLA(2) activation, indicating that hypoxia-induced increase in PLA(2) activity is due to activation rather than induction. However, mRNA levels for sPLA(2) displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA(2) activity showed an increase in sPLA(2) activity but not in cPLA(2) activity after 2hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA(2) activity is mostly due to the activation of sPLA(2).
Michiels
Carine
C
Laboratoire de Biochimie et Biologie Cellulaire, Facultes Universitaires Notre Dame de la Paix, 61 rue de Bruxelles, 5000, Namur, Belgium. carine.michiels@fundp.ac.be
Renard
Patricia
P
Bouaziz
Najat
N
Heck
Nathalie
N
Eliaers
Francois
F
Ninane
Noelle
N
Quarck
Rozenn
R
Holvoet
Paul
P
Raes
Martine
M
D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D015237:Dinoprost / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009281:Naphthalenes; D011753:Pyrones; C081565:arachidonyltrifluoromethane; D010767:Phosphorylcholine; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; C068170:oleyloxyethylphosphorylcholine; D015237:Dinoprost; D010741:Phospholipases A