Tài liệu Báo cáo Y học: Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic

Differential effects of arachidonoyl trifluoromethyl ketone

on arachidonic acid release and lipid mediator biosynthesis

by human neutrophils

Evidence for different arachidonate pools

Alfred N. Fonteh

Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine,

Medical Center Boulevard, Winston-Salem, NC 27157, USA.

The goal of this study was to determine the effects of a

putative specific cytosolic phospholipase A2 inhibitor,

arachidonyl trifluoromethyl ketone (AACOCF3), on

arachidonic acid (AA) release and lipid mediator biosyn￾thesis by ionophore-stimulated human neutrophils. Initial

studies indicated that AACOCF3 at concentrations 0–10 lM

did not affect AA release from neutrophils. In contrast,

AACOCF3 potently inhibited leukotriene B4 formation by

ionophore-stimulated neutrophils (IC50
2.5 lM). Like￾wise, AACOCF3 significantly inhibited the biosynthesis of

platelet activating factor. In cell-free assay systems, 10 lM

AACOCF3 inhibited 5-lipoxygenase and CoA-independent

transacylase activities. [3

H]AA labeling studies indicated

that the specific activities of cell-associated AA mimicked

that of leukotriene B4 and PtdCho/PtdIns, while the specific

activities of AA released into the supernatant fluid closely

mimicked that of PtdEtn. Taken together, these data argue

for the existence of segregated pools of arachidonate in

human neutrophils. One pool of AA is linked to lipid

mediator biosynthesis while another pool provides free AA

that is released from cells. Additionally, the data suggest that

AACOCF3 is also an inhibitor of CoA-independent trans￾acylase and 5-lipoxygenase. Thus, caution should be exer￾cised in using AACO CF3 as an inhibitor of cytosolic

phospholipase A2 in whole cell assays because of the com￾plexity of AA metabolism.

Keywords: arachidonic acid; lipid mediators; neutrophils;

phospholipase A2; inhibitor.

Phospholipases A2 (PLA2) are enzymes that hydrolyse

acyl bonds at the sn-2 position of phospholipids generat￾ing free fatty acids and lysophospholipid moieties [1].

Several mammalian PLA2 isotypes have been cloned and

sequenced [2–10]. The most characterized of these

enzymes are a hormonally regulated, cytosolic high

molecular mass enzyme (cPLA2) [11], a calcium-indepen￾dent PLA2 (iPLA2) [12] and various secretory low

molecular mass isotypes (sPLA2) [6]. These major PLA2

isotypes have all been implicated in arachidonic acid (AA)

mobilization and eicosanoid biosynthesis by inflammatory

cells [13–19]. Specifically, knockout studies have conclu￾sively linked cPLA2 with lipid mediator formation [20].

Because of the importance of PLA2, various approaches

have been designed to influence PLA2 levels within cells

[21–29]. Of various inhibitors that have been used,

analogues of AA such as arachidonoyl trifluoromethyl

ketone (AACOCF3) have been purported to be specific in

inhibiting cPLA2. Thus, AACO CF3 has been used

extensively in several cell systems to examine AA release

[23,26,30–32]. However, no comprehensive study has been

undertaken to examine the effects of AACOCF3 on other

AA-specific pathways in whole cells, even though this

compound has been shown to inhibit other enzyme

activities [33,34].

In addition to PLA2, other enzymes have been shown to

affect arachidonate content and its release from inflamma￾tory cells [35]. Incorporation and release of AA is

accompanied by remodeling between various phospholipid

subclasses [35–39]. CoA-dependent and CoA-independent

enzymes are responsible for regulating cellular arachi￾donate levels [40–42]. Different forms of an activity

(arachidonoyl CoA synthetase) that converts free AA to

arachidonoyl-CoA (AA-CoA) at the expense of ATP have

been described previously [43–46]. Once synthesized,

AA-CoA is incorporated into lysophospholipids by CoA￾dependent acyl transferases [45,47–49]. In addition to these

CoA-dependent mechanisms, arachidonate is rapidly

shuttled from 1-acyl-linked phospholipids to 1-ether￾linked phospholipids by CoA-independent transacylase

Correspondence to A. N. Fonteh, Molecular Neurology

Program, Huntington Medical Research Institutes, 99,

North El Molino Avenue, Pasadena, CA 91101–1830, USA.

Fax: + 1 626 795 5774, Tel.: + 1 626 795 4343,

E-mail: [email protected]

Abbreviations: AA, arachidonic acid; AA-CP, arachidonic acid

in cell pellets; AA-SF, arachidonic acid in supernatant fluids;

cPLA2, cytosolic phospholipase A2; GPC, sn-glycerol-3-PCho;

GPE, sn-glycerol-3-PEtn; GPI, sn-glycero-3-PIns; iPLA2,

calcium-independent phospholipase A2; 5-LO, 5-lipoxygenase;

NICI-GC/MS, negative ion-chemical ionization gas chromatography/

mass spectrometry; PAF, platelet activating factor; PLA2,

phospholipase A2; sPLA2, secretory phospholipase A2; SA,

specific activity.

(Received 14 February 2002, revised 16 May 2002,

accepted 24 June 2002)

Eur. J. Biochem. 269, 3760–3770 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03070.x