Hydrogen Peroxide Activation of Ca 2+ -Independent Phospholipase A 2 in Uterine Stromal Cells He ´le `ne Birbes, Emmanuel Gothie ´, Jean-Franc ¸ois Pageaux, Michel Lagarde, and Christian Laugier 1 Biochimie & Pharmacologie, INSERM U352, INSA-Lyon, Ba ˆ t. 406, 69621 Villeurbanne Cedex, France Received August 1, 2000 In rat uterine stromal cells (U III cells), an oxidative stress induced by H 2 O 2 caused a dose-dependent re- lease of arachidonic acid (AA) that was independent of intracellular Ca 2 concentration and was not inhibited by Ca 2 -dependent phospholipase A 2 (cPLA 2 ) inhibi- tors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H 2 O 2 treatment did not impair AA esterification but significantly increased Ca 2 - independent PLA 2 (iPLA 2 ) activity. Since iPLA 2 spe- cific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H 2 O 2 , we con- clude that iPLA 2 activity represents the major mech- anism by which H 2 O 2 increases the availability of non- esterified AA in U III cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H 2 O 2 , suggesting a regula- tory mechanism of iPLA 2 by PKC that remains to be clarified. © 2000 Academic Press Key Words: oxidative stress; arachidonic acid; Ca 2 - independent PLA 2 ; PKC inhibitors; uterine stromal cells. In uterine stromal cells, AA acting either directly or after conversion to oxygenated products, mediates sev- eral physiological events such as proliferation and dif- ferentiation of stromal to decidual cells (1–3). The cel- lular level of non-esterified AA depends on the rate of deacylation by phospholipases A 2 partly counterbal- anced by reacylation of lysophospholipids by non- esterified AA into phospholipids (4, 5). A major path- way for the release of AA from phospholipids involves cPLA 2 activation (6). However, the involvement of an iPLA 2 in agonist-induced AA release has also been suggested in different cell lines (7, 8). Several studies have shown that reactive oxygen species enhance AA release and metabolism in differ- ent cell systems. In vascular smooth muscle cells and striatal neurons, H 2 O 2 was shown to trigger AA release through cPLA 2 activation (9 –11). In other cell systems, the mechanism of oxidant-induced AA release appears to be independent of Ca 2+ , resulting either from the activation of an iPLA 2 (12–14) or the inhibition of AA esterification into phospholipids (15, 16). Since rat uterine stromal cell in culture (U III cells) possess dif- ferent PLA 2 including secretory PLA 2 , iPLA 2 , and cPLA 2 (17–19), it was of interest to investigate the ability of H 2 O 2 to stimulate AA release from these cells and to identify the target. We found that H 2 O 2 -induced AA release was independent of intracellular Ca 2+ con- centration and was not inhibited by cPLA 2 inhibitors, nor by PKC inhibitors or by PKC down-regulation. H 2 O 2 treatment did not impair fatty acid esterification but significantly increased iPLA 2 activity. Since the iPLA 2 specific inhibitor bromoenollactone (BEL, 20) markedly suppressed H 2 O 2 -induced AA release, we conclude that increased iPLA 2 -mediated fatty acid re- lease represents the major mechanism by which H 2 O 2 triggers accumulation of free AA in U III cells. MATERIAL AND METHODS Chemicals. Tissue culture medium, L-glutamine, penicillin, strep- tomycin, fetal calf serum (FCS) were obtained from Life Technologies (Cergy-Pontoise, France). [5,6,8,9,11,12,14,15- 3 H]arachidonic acid (210 Ci/mmol), [4,5- 3 H]docosahexaenoic acid (DHA) (58 Ci/mmol), L-- dipalmitoyl-[2-palmitoyl-1- 14 C]phosphatidylcholine (55.5 mCi/mmol) and L--1-palmitoyl-[2-arachidonoyl-1- 14 C]-phosphatidylcholine (55 mCi/mmol) were obtained from New England Nuclear (Boston, MA). Fatty acid-free bovine serum albumin (BSA), shingosine and calphostin C were from Sigma (St. Louis, MO). Calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) were from Calbiochem (La Jolla, CA). (E)-16-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H- pyran-2-one (bromoenollactone, BEL) was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were of analytical grade. Cell culture. For stock culture, U III cells were grown in medium 199 supplemented with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin and 100 g/ml streptomycin. They were incubated in 75 Abbreviations used: PLA 2 , phospholipase A 2 ; AA, arachidonic acid; DHA, docosahexaenoic acid; PKC, protein kinase C; BEL, bromoenol- lactone; PMA, phorbol myristate acetate; EGTA [ethylene-bis (oxy- ethylenenitrilo)] tetraacetic acid; BAPTA, [1,2-bis(o-aminophenoxy)- ethane-N,N,N',N'-tetraacetic acid, sodium]. 1 To whom correspondence should be addressed at INSERM U352, Biochimie & Pharmacologie, INSA-Lyon, 20 Ave. A. Einstein, Ba ˆt. 406, 69621 Villeurbanne Cedex. Fax: 33(0)4-72-43-85-11. E-mail: Christian.Laugier@insa-lyon.fr. Biochemical and Biophysical Research Communications 276, 613– 618 (2000) doi:10.1006/bbrc.2000.3479, available online at http://www.idealibrary.com on 613 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.