Tài liệu Báo cáo Y học: Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils pot

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 A
2
inhibitor,
arachidonyl trifluoromethyl ketone (AACOCF
3
), on
arachidonic acid (AA) release and lipid mediator biosyn-
thesis by ionophore-stimulated human neutrophils. Initial
studies indicated that AACOCF
3
at concentrations 0–10 l
M
did not affect AA release from neutrophils. In contrast,
AACOCF
3
potently inhibited leukotriene B
4
formation by
ionophore-stimulated neutrophils (IC
50
 2.5 l
M
). Like-

wise, AACOCF
3
significantly inhibited the biosynthesis of
platelet activating factor. In cell-free assay systems, 10 l
M
AACOCF
3
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 B
4
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
AACOCF
3
is also an inhibitor of CoA-independent trans-
acylase and 5-lipoxygenase. Thus, caution should be exer-
cisedinusingAACOCF
3
as an inhibitor of cytosolic
phospholipase A
2

in whole cell assays because of the com-
plexity of AA metabolism.
Keywords: arachidonic acid; lipid mediators; neutrophils;
phospholipase A
2
; inhibitor.
Phospholipases A
2
(PLA
2
) are enzymes that hydrolyse
acyl bonds at the sn-2 position of phospholipids generat-
ing free fatty acids and lysophospholipid moieties [1].
Several mammalian PLA
2
isotypes have been cloned and
sequenced [2–10]. The most characterized of these
enzymes are a hormonally regulated, cytosolic high
molecular mass enzyme (cPLA
2
) [11], a calcium-indepen-
dent PLA
2
(iPLA
2
) [12] and various secretory low
molecular mass isotypes (sPLA
2
)[6].ThesemajorPLA
2

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 cPLA
2
with lipid mediator formation [20].
Because of the importance of PLA
2
, various approaches
have been designed to influence PLA
2
levels within cells
[21–29]. Of various inhibitors that have been used,
analogues of AA such as arachidonoyl trifluoromethyl
ketone (AACOCF
3
) have been purported to be specific in
inhibiting cPLA
2
.Thus,AACOCF
3
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 AACOCF
3
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 PLA
2
, 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:
Abbreviations: AA, arachidonic acid; AA-CP, arachidonic acid
in cell pellets; AA-SF, arachidonic acid in supernatant fluids;
cPLA
2
, cytosolic phospholipase A
2
; GPC, sn-glycerol-3-PCho;
GPE, sn-glycerol-3-PEtn; GPI, sn-glycero-3-PIns; iPLA

2
,
calcium-independent phospholipase A
2
; 5-LO, 5-lipoxygenase;
NICI-GC/MS, negative ion-chemical ionization gas chromatography/
mass spectrometry; PAF, platelet activating factor; PLA
2
,
phospholipase A
2
; sPLA
2
, secretory phospholipase A
2
;SA,
specific activity.
(Received 14 February 2002, revised 16 May 2002,
accepted 24 June 2002)
Eur. J. Biochem. 269, 3760–3770 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03070.x
[37–39,50–53]. Evidence using cell-free preparations sug-
gests that the selective transfer of arachidonate from
1-alkyl-2-arachidonoyl-GPC (sn-glycero-3-PCho) to lyso-
phospholipid acceptors leads to the formation of 1-alkyl-
2-lyso-GPC. This intermediate can be converted to platelet
activating factor (PAF) by acetyl transferase [54–56].
Rapid remodeling involving many enzyme activities and
phospholipid substrates makes it difficult to study these
molecular events [37,57,58]. Earlier studies demonstrated
that inflammatory cells released relatively large quantities

of AA from several phospholipids (PtdEtn > Ptd-
Cho > PtdIns) [58,59]. However, when cells are pulse-
labeled, AA release is accompanied by changes in the
specific activities (SA) of all major phospholipid subclasses
[58,59]. These changes in SA are due to rapid acylation,
deacylation and remodeling reactions. Thus, while PLA
2
isotypes may provide most of the AA that is utilised
for eicosanoid biosynthesis, reacylation and remodeling
between phospholipid subclasses may also be a crucial
factor involved in the regulation of free AA levels and the
generation of potent lipid mediators.
The objective of these studies was to determine the
effects of a putative cPLA
2
inhibitor (AACOCF
3
)onAA
release and lipid mediator biosynthesis. Our data suggest
that AACOCF
3
decreases lipid mediator formation by
neutrophils without affecting free AA levels by inhibiting
CoA-independent transacylase (CoA-IT) and 5-lipoxygen-
ase (5-LO) activities. SA measurements show that lipid
mediators and free AA are derived from different phospho-
lipid pools. Together, these data suggest that there is
segregation of AA pools within neutrophils and caution
should always be exercised in the use of AA analogues
such as AACOCF

3
as specific inhibitors in whole cell
assays because of the complexity of AA metabolising
pathways and because these compounds influence many
enzyme activities.
MATERIALS AND METHODS
Materials
[
3
H]Acetic acid (3.3 CiÆmmol
)1
) and [5,6,8,9,11,12,14,
15–
3
H]AA (76 CiÆmmol
)1
) were purchased from New
England Nuclear (Boston, MA, USA). AACOCF
3
was
purchased from Biomol (Plymouth Meeting, PA, USA),
lipid standards from Avanti Polar Lipids (Birmingham, AL,
USA), and Hanks Balanced Salt Solution (HBSS) and
NaCl/P
i
purchased from Gibco Laboratories (Grand
Island, NY, USA). Silica gel G plates were purchased from
Analtech (Newark, DE, USA), with silica gel columns
purchased from J. T. Baker Inc. (Phillipsburg, NJ, USA).
Ionophore A23187 was purchased from Calbiochem (La

Jolla, CA, USA), Ficoll-Paque from Pharmacia (Piscata-
way, NJ, USA) and Dextran 70 from Abbot Laboratories
(North Chicago, IL, USA). Arachidonic acid, leukotriene
B
4
(LTB
4
) and 20-hydroxy-leukotriene B
4
were purchased
from Cayman (Ann Arbor, MI, USA). Essentially fatty-
acid-free human serum albumin was purchased from Sigma
(St Louis, MO, USA). Pentafluorobenzyl bromide and
diisopropylethylamine were purchased from Alltech/
Applied Science Associate (Deerfield, IL, USA) and HPLC
grade solvents purchased from Fisher Scientific (Norcross,
GA, USA).
Neutrophil isolation and stimulation
Neutrophils were obtained from venous blood of healthy
human donors as described previously [60]. Neutrophils
(5 · 10
6
mL
)1
) in HBSS were incubated at 37 °C(5min)
prior to stimulation. Different concentrations of freshly
made AACOCF
3
in dimethylsulfoxide were added to the
cells 5 min before stimulation with 2.5 l

M
ionophore
A23187, for periods of time indicated in the figure legends.
The incubations were terminated by the addition of
methanol/chloroform (2 : 1, v/v). Lipids were extracted
from the reaction mixture by the method of Bligh & Dyer
[61]. In experiments where the quantities of leukotrienes
were determined, neutrophils were removed from the
supernatant fluid by centrifugation (400 g,5min).Pro-
staglandin B
2
(PGB
2
, 250 ng) was added as an internal
standard and eicosanoids were extracted twice from the
supernatant fluids using 3 mL ethyl acetate.
Determination of AA release from
stimulated neutrophils
After the addition of 100 ng [
2
H
8
]AA as an internal
standard, solvents were removed from ethanol extracts of
supernatant fluids using a stream of nitrogen. Fatty acids
were then converted to pentafluorobenzyl esters and the
molar quantities of free fatty acids were determined by
combined negative ion-chemical ionization gas chromatog-
raphy/mass spectroscopy (NICI-GC/MS), using a Hewlett
Packard model 5989 instrument. Carboxylate anions (m/z)

were monitored at 303 and 311 nm for AA and [
2
H
8
]AA,
respectively, in the single ion-monitoring mode. In experi-
ments where cellular AA was determined, lipids were
extracted from cellular pellets. A fatty acid enriched fraction
was obtained using Bakerbond silica gel disposable columns
[62]. After solvent removal using a stream of nitrogen, molar
quantities of AA were determined as described above.
Determination of molar quantities of leukotrienes
PGB
2
(250 ng) was added to supernatant fluids as an
internal standard prior to sample concentration using a
stream of nitrogen. Leukotrienes were suspended in 30%
methanol in water and injected onto an Ultrasphere ODS
column (2.0 · 250 mm, Rainin Instrument Co, Woburn,
MA, USA) that had been conditioned in a solvent
that consisted of methanol/water/phosphoric acid
(550 : 450 : 0.2, v/v/v, pH 5.7). The solvent was delivered
at a flow rate of 0.3 mLÆmin
)1
and products were monitored
(270 and 206 nm) using a Hewlett Packard diode array
detection system. After 5 min, eicosanoids were eluted
from the column by increasing the amount of methanol to
100% over 50 min. Leukotrienes and free AA were
collected and the radioactivity in these fractions determined

by liquid scintillation counting. Molar quantities of leuko-
trienes were determined by UV spectroscopy.
PAF biosynthesis
The incorporation of [
3
H]acetateintoPAFwasusedto
quantitate PAF biosynthesis in human neutrophils. Briefly,
neutrophils in HBSS (5 · 10
6
mL
)1
) were incubated with
4 lCiÆmL
)1
[
3
H]acetate for 15 min at 37 °C. The cells were
Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur. J. Biochem. 269) 3761
stimulated in the presence and absence of 10 l
M
AACOCF
3
and reactions terminated by the addition of methanol/
chloroform (2 : 1, v/v). After phospholipid extraction,
PAF was isolated by TLC on silica gel G developed
in chloroform/methanol/acetic acid/water (50 : 25 : 8 : 4,
v/v/v/v). Radioactivity on TLC plates was detected using a
Bioscan radioactivity imaging system (Washington, DC,
USA) and the amount of radioactivity migrating with PAF
was determined by zonal scraping, followed by liquid

scintillation counting.
Determination of 5-LO activity
Neutrophils (5 · 10
6
mL
)1
) were stimulated as described
above. Cells were removed from supernatant fluids by
centrifugation (400 g, 5 min) and cell pellets were suspended
in 1mL of 50m
M
phosphate buffer containing 1 m
M
dithiothreitol, 1.6 m
M
EDTA, 1 lgÆmL
)1
leupeptin,
1 lgÆmL
)1
pepstatin and 0.5 m
M
phenylmethanesulfonyl
fluoride. Cells were then broken by sonication (10 s, three
times) using a model W-220 sonicator (Heat System
Ultrasonic Inc., Farmingdale, NY, USA) set at a power
scale of two and 10% output. Unbroken cells were removed
from sonicates by centrifugation (10 000 g,10min).Cyto-
solic and pellet fractions were obtained after ultracentrifu-
gation of sonicates (100 000 g, 60 min). 5-LO activity was

determined in cytosolic fractions in a final volume of 1 mL
of 200 m
M
Tris/HCl (pH 7.5) containing 1.8 m
M
ATP,
1.6 m
M
EDTA and 10 m
M
CaCl
2
. 5-LO activity was
initiated by the addition of [
3
H]AA (100 nCiÆnmol
)1
)and
2.6 nmol hydroxy-9-cis-11-trans-octadecadienioc acid as a
hydroxyperoxide activator. 5-LO products [5-hydroxyeico-
satetraenoic acid (HETE), LTB
4
and 20-OH-LTB
4
]andAA
were isolated by reverse phase HPLC as described previ-
ously [16] and radioactivity determined by liquid scintilla-
tion counting.
Determination of CoA-IT activity
Neutrophils were suspended in CoA-IT sonication buffer

[50 m
M
Hepes buffer, pH 7.4, containing 1 m
M
EDTA and
20% sucrose (w/v)]. Cells were broken using a probe
sonicator as described above, and cytosolic and membrane
fractions obtained after ultracentrifugation (100 000 g,
60 min, 4 °C). The membrane fraction was diluted in
NaCl/P
i
containing 1 m
M
EGTA with 10 lg total protein
utilised for determining CoA-IT activity. The reaction
was initiated by the addition of [
3
H]1-alkyl-2-lyso-GPC
(0.1 lCi) and 1 nmol 1-O-hexadecyl-2-lyso-GPC in a final
volume of 100 lL. After 10 min at 37 °C, the reaction was
stopped and lipids were extracted [61]. Phospholipids were
separated by TLC on silica gel G developed in chloroform/
methanol/acetic acid/water (50 : 25 : 8 : 4, v/v/v/v). The
product ([
3
H]1-alkyl-2-acyl-GPC) was visualized by radio-
scaning (Bioscan), scrapped and quantified by liquid
scintillation spectroscopy.
[
3

H]AA labeling of glycerolipids and determination
of specific activities (SA)
Human neutrophils were labeled by adding [
3
H]AA (1 lCi
per 2 · 10
7
cells) complexed to fatty-acid-free human serum
albumin (0.25 mgÆmL
)1
) in HBSS for 0.5 h. Unincorporated
[
3
H]AA was removed by washing cells three times with
HBSS containing 0.25 mgÆmL
)1
human serum albumin.
Cells were incubated in a water bath (37 °C, 15 min) to
allow complete reacylation of cellular free AA. After
stimulation with ionophore, cells were removed from
supernatant fluids by centrifugation (400 g,5min).Super-
natant fluids were added to 4 vol. of ethanol and molar
quantities of leukotriene and AA determined by HPLC and
NICI-GC/MS, respectively.
Lipids in the cell pellets were extracted and glycerolipid
classes were isolated using normal phase HPLC as
described previously [63]. Radioactivity was determined
in a portion of the isolated fractions by liquid scintilla-
tion counting using a Beckman liquid scintillation
counting system (Fullerton, CA, USA). Portions of

PtdCho and PtdEtn fractions were hydrolysed using 10 U
of Grade 1 Bacillus cereus phospholipase C (Boehringer
Mannheim) for 2.5 h. Diradylglycerols obtained from
phospholipase C hydrolysis were converted to acetate
derivatives [63]. 1-Acyl-, 1-alkyl-, and 1-alk-1¢-enyl-
subclasses were separated by TLC on silica gel G
developed in benzene/hexane/ether (50 : 25 : 4, v/v/v).
Molar quantities of AA in phospholipid classes and
subclasses were determined after base hydrolysis by
NICI-GC/MS as described above. SA in phospholipid
classes and subclasses were calculated and expressed as
radioactivity (nCi)Ænmol
)1
arachidonate.
Determination of SA of AA and leukotrienes
The neutral lipid fraction obtained from normal phase
HPLC was separated into classes by TLC using silica gel G
developed in hexane/ether/formic acid (90 : 60 : 6, v/v/v).
Radioactivity in products was determined using a radio-
chromatogram imaging system (Bioscan). The region cor-
responding to free fatty acids was scraped into vials while an
equal amount was used to determine molar quantities of
arachidonate by NICI-GC/MS. SA of cellular AA was
calculated and expressed as radioactivity (nCi)Ænmol
)1
arachidonate.
Leukotrienes and free AA were isolated by reverse phase
HPLC, as described above. Fractions corresponding to
leukotrienes and free AA were collected and the amount of
radioactivity in each determined by scintillation counting.

Free AA was also converted to pentafluorobenzylesters and
the molar quantity of AA determine by NICI-GC/MS.
SA of leukotrienes and AA from supernatant fluids
(AA-SF) were calculated and expressed as radioactivity
(nCi)Ænmol
)1
.
Statistical analysis
All data are expressed as the means ± SEM of separate
experiments. Statistics (P-values) were obtained from Stu-
dent’s t-test for paired samples. Notations used on figures
and legends are for P < 0.05.
RESULTS
Effect of AACOCF
3
on AA release from neutrophils
During activation of neutrophils, AA is released from
phospholipid pools by cPLA
2
. Our studies and those of
3762 A. N. Fonteh (Eur. J. Biochem. 269) Ó FEBS 2002
Wykle and colleagues have shown that AA levels and lipid
mediator formation are also modulated by changes in CoA-
IT activity [53,64–69]. As cPLA
2
and CoA-IT are selective
for AA, we hypothesized that AACOCF
3
may influence AA
levels in activated neutrophils by modulating these activities.

Therefore, we examined AA levels within cells or in
supernatant fluids of neutrophils that had been stimulated
in the presence of different concentrations of AACOCF
3
.
As shown in Fig. 1, AACOCF
3
(0–10 l
M
) did not inhibit
AA release from stimulated neutrophils. Paradoxically,
there was an increase in AA levels within neutrophils and in
supernatant fluids as the concentration of AACOCF
3
was
increased. These data suggested that AACOCF
3
at these
concentrations did not affect PLA
2
activity. However,
at higher concentrations ( 20 l
M
), AACOCF
3
reduced
AA release from A23187-stimulated neutrophils (78.7 ±
54.9 pmol per 10 million neutrophils, n ¼ 4, for AA in
supernatant fluids and 178 ± 23.8 pmol per 10 million
neutrophils, n ¼ 4, for cellular AA). To make sure that

AACOCF
3
was presented for sufficient time to penetrate
the cells and inhibit cellular enzymes, neutrophils were
incubated with 10 l
M
AACOCF
3
for different periods of
time. As shown in Fig. 2A, AA released into supernatant
fluids was not inhibited even after neutrophils were
incubated with AACOCF
3
for 60 min. Similarly, cellular
free AA levels were not inhibited under similar conditions
(Fig. 2B). Together, these data suggest that there are two
types of AA pools in neutrophils, one that is not inhibited
by low concentrations of AACOCF
3
and another that is
inhibited by high concentrations of AACOCF
3
.The
increase in AA at low concentrations of AACOCF
3
could
also suggest that an AACOCF
3
-sensitive pool was
destined for product formation, and the inhibition of this

product formation may have accounted for the build up of
free AA.
Influence of AACOCF
3
on lipid mediator biosynthesis
To determine whether the increase in AA levels was due
to a decrease in AA-derived mediators, we examined
leukotriene biosynthesis. AACOCF
3
inhibited the biosyn-
thesis of LTB
4
and 20-OH-LTB
4
at a concentration
(10 l
M
) that did not affect AA release (Fig. 3). These
data suggest that free AA and AA destined for
leukotriene formation are derived from different phospho-
lipid pools or are regulated by different signaling
pathways.
As LTB
4
and PAF share a common precursor [13,70], we
next determined whether AACOCF
3
also influenced PAF
synthesis. Ionophore A23187-induced [
3

H]acetate incorpo-
ration into PAF (Fig. 4). Pre-incubation with 10 l
M
Fig. 1. Dose-dependent effects of AACOCF
3
on AA release. Human
neutrophils incubated with different concentrations of AACOCF
3
for
5 min were stimulated for 5 min with 2.5 l
M
ionophore A23187.
Stimulation was stopped by centrifugation and molar quantities of AA
in the supernatant fluid (AA-SF) and AA associated with cell pellet
(AA-CP) were determined by NICI-GC/MS as described in Materials
and methods. These data are the mean ± SEM of six separate
experiments (*P <0.05comparedto0l
M
AACOCF
3
).
Fig. 2. Time-dependent effects of AACOCF
3
on AA release. Human
neutrophils incubated with 10 l
M
AACOCF
3
for 5 min were stimu-
lated for different periods of time with 2.5 l

M
ionophore A23187.
Stimulation was stopped by centrifugation and molar quantities of
AA-SF (A) for cells stimulated with or without AACOCF
3
were
determined by NICI-GC/MS as described in Materials and methods.
Similarly, AA-CP (B) with or without AACOCF
3
was determined.
These data are the mean ± SEM of six separate experiments.
Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur. J. Biochem. 269) 3763
AACOCF
3
resulted in significant inhibition of radiolabeled
PAF formation. Similar to LTB
4
, these data suggest that the
formation of the PAF precursor, lyso PAF, was indepen-
dent of free AA release.
Influence of AACOCF
3
on enzyme activities
An explanation for the inhibition of LTB
4
and PAF
biosynthesis independent of AA inhibition is that
AACOCF
3
may inhibit other enzymes that are directly

linked to the formation of these lipid mediators while higher
concentrations of AACOCF
3
are required to inhibit the
PLA
2
activity responsible for AA release in whole cells. We
next examined the effects of AACOCF
3
on 5-LO and CoA-
IT activities.
5-LO activity. In unstimulated cells, the bulk of radioac-
tivity resided in the AA peak. After ionophore stimulation,
there was an increase in radiolabel coeluting with 5-LO
products, LTB
4
and 5-HETE (Fig. 5A). In the presence of
10 l
M
AACOCF
3
, there was a significant decrease
(89.8 ± 5.6%, n ¼ 3) in 5-LO product formation
(Fig. 5B). These studies suggest that AACOCF
3
at a
concentration that does not affect AA release in whole
neutrophils effectively inhibits 5-LO activity.
Fig. 5. Influence of AACOCF
3

on 5-lipoxygenase activity. Human
neutrophils incubated without or with 10 l
M
AACOCF
3
for 5 min
were stimulated for 5 min with 2.5 l
M
ionophore A23187. Cytosol
and membrane fractions were prepared by ultracentrifugation. 5-LO
activity in membranes from unstimulated neutrophils (Control) or
ionophore A23187-stimulated (A23187) neutrophils (A) and A23187-
stimulated neutrophils in the presence of 10 AACOCF
3
(B) was
determined as described in Materials and methods. Radioactivity
(DPM) coeluting with 5-LO products (LTB
4
, 5-HETE) or with free
AA is indicated by arrows. These data are representative of three
separate experiments.
Fig. 3. Influence of AACOCF
3
on leukotrienes biosynthesis. Human
neutrophils incubated without or with 10 l
M
AACOCF
3
for 5 min
were stimulated for 5 min with 2.5 l

M
ionophore A23187. Cell stim-
ulation was stopped by centrifugation and molar quantities of LTB
4
and 20-OH-LTB
4
in supernatant fluids were determined by reverse
phase HPLC as described in Materials and methods. These data are
the mean ± SEM of five separate experiments (*P < 0.05 compared
to 0 l
M
AACOCF
3
).
Fig. 4. Effects of AACOCF
3
on PAF formation. Human neutrophils
incubated without or with 10 l
M
AACOCF
3
for 5 min were stimu-
lated for 5 min with 2.5 l
M
ionophore A23187. PAF biosynthesis
assessed by [
3
H]acetate incorporation was determined as described in
Materials and methods. These data are the mean ± SEM of five
separate experiments (*P < 0.05 compared to 0 l

M
AACOCF
3
).
3764 A. N. Fonteh (Eur. J. Biochem. 269) Ó FEBS 2002
CoA-IT activity. Inhibition of CoA-IT by AACOCF
3
could potentially account for the decrease in PAF formation.
We tested this hypothesis by measuring microsomal CoA-IT
activity. As shown in Fig. 6, microsomes prepared from
neutrophils contained CoA-IT activity (4.06 ± 0.15 nmolÆ
mg
)1
Æmin
)1
, n ¼ 4). AACOCF
3
dose-dependently inhibited
CoA-IT activity (IC
50
 7.5 l
M
). These data suggest that
inhibition of CoA-IT activity by AACOCF
3
might account
for the decrease in PAF formation.
Relationship between SA of phospholipids, arachidonic
acid and leukotrienes
To obtain further evidence for different AA pools,

neutrophils were labeled with [
3
H]AA for 30 min to
achieve nonisotopic equilibrium. This strategy differen-
tially labels the major phospholipid pools within neu-
trophils and thus allows comparisons to be made between
the SA of phospholipids and products. Five minutes
after stimulation, molar quantities of arachidonate in
phospholipid subclasses are reduced except in 1-alk-
1-enyl-2-AA-GPC (Table 1). 1-Alk-1-enyl-2-AA-GPE
and 1-alkyl-2-AA-GPC account for most of the AA
released from PtdEtn and PtdCho, respectively. In
PtdCho and PtdEtn subclasses, the rank order of SA is
1-acyl-2-AA-GPC (51.7 ± 12.5 nCiÆnmol
)1
, n ¼ 3) >
1-alkyl-2-AA-GPC (7.2 ± 0.8 nCiÆnmol
)1
) > 1-acyl-2-AA-
GPE (3.9 ± 0.8 nCiÆnmol
)1
) > 1-alk-1-enyl-2-AA-GPE
(0.6 ± 0.2 nCiÆnmol
)1
). During cell activation, there is a
decrease in the SA in 1-acyl-linked phospho-
lipids (38.1 ± 9.33% decrease in 1-acyl-2-AA-GPC;
22.7 ± 2.2% decrease in 1-acyl-2-AA-GPI and 26.9 ±
10.8% decrease in 1-acyl-2-AA-GPE). Concomitantly,
there is an increase in the SA in 1-ether-linked phosp-

holipid subclasses (19.0 ± 6.0% increase in 1-alkyl-2-AA-
GPC; 48.3 ± 10.3% increase in 1-alk-1-enyl-2-AA-GPE).
There is a twofold increase in SA of PtdEtn (Fig. 7).
These data suggest that a remodeling process involving
the release of low SA AA from ether-linked phospho-
lipid subclasses accompanied the incorporation of this AA
into 1-acyl-linked phospholipid subclasses (mainly in
PtdCho/PtdIns). Conversely, the increase in the SA of
ether-linked PtdEtn and PtdCho subclasses suggested that
high SA AA from 1-acyl-linked phospholipids (mainly
PtdCho and PtdIns) was being remodeled into the ether-
linked subclasses (1-alkyl-2-AA-GPC and 1-alk-1-enyl-2-
AA-GPE).
Examination of SA of products indicated that PtdCho/
PtdIns were the likely sources of AA that was utilized
for leukotriene biosynthesis (Fig. 7). In contrast, PtdEtn
that accounts for the bulk of free AA released from
neutrophils had a SA that was significantly lower than the
SA of leukotrienes and thus did not contribute AA for
leukotriene biosynthesis. The SA of AA associated with cell
pellet (AA-SP) closely resembled that of PtdCho/PtdIns and
leukotrienes and was different from AA that was released
into the supernatant fluid (AA-SF). Likewise, the SA of
AA-SF mimicked that of PtdEtn and was different from
that of PtdCho/PtdIns. Together, these data suggest that
there is segregation of AA pools within neutrophils.
Fig. 6. Influence of AACOCF
3
on CoA-IT activity. Membrane and
cytosolic fractions were prepared from human neutrophils by ultra-

centrifugation. Total membrane protein (10 lg) was incubated with
different concentrations of AACOCF
3
for 5 min. Subsequently, CoA-
IT activity was initiated by adding [
3
H]1-alkyl-2-lyso-GPC. Phospho-
lipids were isolated by TLC and radiolabel in PtdCho determined by
liquid scintillation counting. These data are the mean ± SEM of four
separate experiments (*P < 0.05 compared to 0 l
M
AACOCF
3
).
Table 1. Changes in arachidonate content of phospholipids after A23187 stimulation. Human neutrophils were incubated without (control) or with
ionophore A23187 for 5 min. Glycerolipids were extracted and phospholipids (PtdEtn, PtdIns, PtdCho) isolated by normal phase HPLC. PtdEtn
and PtdCho subclasses (1-acyl-, 1-alkyl-, 1-alk-1-enyl-) were separated as described under Materials and methods. Molar quantities of arachidonate
were determined after base hydrolysis by NICI-GC/MS. Net release of AA was then determined. These data are the mean ± SEM of three separate
experiments (*P < 0.05 compared to control).
Arachidonate content (nmolÆ10
)7
cells)
Phospholipids Control A23187 Net Release
1-Acyl-2-AA-GPC 0.495 ± 0.066 0.237 ± 0.070* )0.258
1-Alkyl-2-AA-GPC 1.655 ± 0.224 0.550 ± 0.058* )1.105
1-Alk-1-enyl-2-AA-GPC 0.102 ± 0.013 0.191 ± 0.141 +0.089
1-Acyl-2-AA-GPI 1.829 ± 0.041 0.991 ± 0.131* )0.838
1-Acyl-2-AA-GPE 0.858 ± 0.235 0.720 ± 0.285 )0.138
1-Alkyl-2-AA-GPE 1.279 ± 0.470 1.142 ± 0.472 )0.137
1-Alk-1-enyl-2-AA-GPE 5.491 ± 0.857 2.661 ± 0.458* )2.830

Ó FEBS 2002 Effects of AACOCF3 on lipid mediator biosynthesis (Eur. J. Biochem. 269) 3765
DISCUSSION
An important finding of the present study is that there are at
least two distinct arachidonate pools in human neutrophils.
One AA pool (from PtdCho/PtdIns) is linked to lipid
mediator formation while another AA pool that is not
linked with product formation is closely associated with
PtdEtn. The following key pieces of data support these
observations: (a) concentrations of AACOCF
3
<10l
M
do not inhibit AA release from ionophore-stimulated
neutrophils. Paradoxically, there is a slight increase in AA
levels at these concentrations. It requires > 10 l
M
AACOCF
3
for AA release to be effectively inhibited.
(b) At concentrations of AACOCF
3
that do not inhibit AA
release, there is > 85% inhibition of LTB
4
and PAF
biosynthesis suggesting that AA release may not be linked
to lipid mediator biosynthesis. (c) AACOCF
3
inhibits 5-LO
activity at concentrations that are not effective in decreasing

AA release. (d) CoA-IT activity that generates lyso PAF,
an intermediate of PAF biosynthesis, is inhibited by
AACOCF
3
at concentrations that do not affect AA release.
A decrease in LTB
4
biosynthesis concomitant with an
increase in free AA suggest that AA destined for LTB
4
biosynthesis is regulated differently from AA that remains
free. (e) Further evidence for the segregation of AA pools
within neutrophils is provided by studies showing that AA
destined for leukotriene biosynthesis has a higher SA than
AA that is released from the biggest arachidonate pool in
PtdEtn.
A schematic representing AA metabolism in human
neutrophils is shown in Fig. 8 (major enzymes are labeled
alphabetically, a–g). AA is constantly released and remod-
eled in cells. This process is slow and well controlled in
unstimulated cells and is accelerated when cells are stimu-
lated. Once presented to cells, AA is incorporated into
cellular glycerophospholipids by a distinct pathway. Free
AA is initially converted to AA-CoA by AA-CoA synthe-
tase (a) [46]. Inhibitors of AA-CoA synthetase prevent AA
incorporation into cellular lipids leading to a build-up of
free cellular AA within cells [71]. Without inhibitors, AA-
CoA is acylated to 1-acyl-linked phospholipid subclasses by
CoA-dependent acyl transferase (b). Once in 1-acyl-linked
subclasses, AA is transferred to the larger pools found in

ether-linked subclasses by CoA-IT (c) [35]. Similar to
Fig. 8. Proposed mechanism for incorporation, remodeling, release and
lipid mediator biosynthesis in human neutrophils. Free AA is converted
to AA-CoA and incorporated into 1-acyl-linked phospholipid sub-
classes by AA-CoA synthetase (a) and AA-CoA-dependent acyl
transferases (b), respectively. Under resting conditions, AA is
remodeled from 1-acyl-linked phospholipids to 1-ether-linked
phospholipids by CoA-IT activity (c). During cell activation, the
remodeling process is accelerated due to the formation of 1-alk-
1-enyl-2-lyso-GPE by PLA
2
(d). Enhanced remodeling is accompa-
nied by an increase in the formation of 1-alkyl-2-lyso-PAF (e), which
is converted, to PAF by acetyl transferase (f). AA from PtdCho/
PtdIns is simultaneously utilised by 5-LO (g) to form LTB
4
,whichis
further metabolised to 20-OH-LTB
4
. Low concentrations of
AACOCF
3
prevent PAF and LTB
4
formation by inhibiting CoA-IT
and/or 5-LO. Higher concentrations of AACOCF
3
prevent AA
release from PtdEtn resulting in a decrease in AA and lipid mediators
(§, enzyme activities inhibited by low concentrations of AACOCF

3
;
/, enzyme activity inhibited by higher concentrations of AACOCF
3
;
Y, low SA AA released into the supernatant fluid and re-incorporated
into PtdCho/PtdIns).
Fig. 7. Relationship between specific activity of phospholipids and
products. Human neutrophil phospholipids were differentially labeled
with [
3
H]-AA. After stimulation with 2.5 l
M
ionophore A23187 for
different periods of time, cell pellets and supernatant fluids were
obtained by centrifugation. Phospholipids (PtdCho, PtdIns and
PtdEtn) were isolated by normal phase HPLC, while products in the
supernatant fluid (AA-SF, LTB
4
and 20-OH-LTB
4
)wereisolatedby
reverse phase HPLC. The radioactivity recovered in phospholipids,
AA-SF, AA-CP and leukotrienes was determined by liquid scintilla-
tion counting and the SA (radioactivity) determined as described in
Materials and methods. These data are the mean ± SEM of three
separate experiments (*P < 0.05 compared to AA-CP, LTB
4
and
20-OH LTB

4
;**P < 0.05 compared to AA-SF).
3766 A. N. Fonteh (Eur. J. Biochem. 269) Ó FEBS 2002
AA-CoA synthetase, inhibition of CoA-IT results in an
increase in free AA and a corresponding build-up of AA in
triacylglycerols that can be prevented by co-incubation of
cells with CoA-synthetase inhibitors [72,73]. Whereas inhi-
bition of CoA-IT results in a decrease in lipid mediator
formation, changes in AA-CoA synthetase may or may not
accompany a decrease in lipid mediator formation
[53,71,74]. These data suggest that the initial incorporation
of AA into phospholipids may not always be critical in
mediator generation by stimulated cells, while the capacity
of cells to remodel AA via CoA-IT is closely linked to
mediator formation.
Specific activity measurements have shown that the
major lipid mediators produced by human neutrophils,
PAF and LTB
4
, share the same common precursor,
1-alkyl-2-AA-GPC [70]. The present data using AACOCF
3
are in agreement with these earlier studies by demon-
strating that inhibition of LTB
4
and PAF biosynthesis by
AACOCF
3
occurs without concomitant inhibition of AA
release. Generation of the common precursor pool by

CoA-IT occurs by the transfer of AA from 1-acyl-linked
phospholipid to 1-ether linked phospholipid classes.
During cell activation, PLA
2
(Fig. 8, d) releases AA from
mainly 1-alk-1-enyl-2-AA-GPE resulting in the formation
of 1-alk-1-enyl-2-lyso-GPE. Wykle and colleagues have
shown that 1-alk-1-enyl-2-lyso-GPE enhances PAF for-
mation by a CoA-IT-dependent generation of lyso PAF
[55].Thus,whilePLA
2
isotypes may not be directly
involved in generating lyso PAF, the activity of PLA
2
on
PtdEtn may indirectly drive the remodeling process that
generates the PAF precursor by providing AA acceptor
molecules. As AA release is not affected, these data
suggest that some activities responsible for AA metabo-
lism in whole cells are more sensitive to AACOCF
3
than
PLA
2
.
While CoA-IT may explain how PAF is formed, the
release of AA destined for LTB
4
cannot easily be explained
by a decrease in AA remodeling alone. There are several

mechanisms that may account for a decrease in LTB
4
formation. First, CoA-IT may have an intrinsic lipase
activity that is inhibited by AACOCF
3
and this process
prevents AA release from 1-alkyl-2-AA-GPC with the
corresponding decrease in LTB
4
formation. Secondly,
CoA-ITmaybelinkedtoAACOCF
3
-sensitive PLA
2
isotypes (Fig. 8, e) whose activities are also increased
during cell activation. Putative candidates include iPLA
2
(group VI PLA
2
), which is very sensitive to AACOCF
3
,or
isoforms of cPLA
2
that may be more sensitive to
AACOCF
3
. Presently, four isoforms of cPLA
2
have been

sequenced and cloned [3,7,8]. Determining which cPLA
2
isoform(s) is(are) expressed in neutrophils and its sensitivity
to AACOCF
3
will be critical in elucidating the interplay
between remodeling and AA release. Thirdly, the selective
transfer of AA to 1-alkyl-2-AA-GPC under resting condi-
tions by CoA-IT may be disrupted when cells are activated.
Evidence for this possibility comes from studies showing
that AA constitutes the bulk of the fatty acid at the sn-2
position of 1-alkyl-2-acyl-GPC under resting conditions
[70]. Upon cell activation, most of this AA is replaced by
other fatty acids. Finally, because cell activation is
accompanied by an increase in PAF biosynthesis via
acetylation of 1-alkyl-2-lyso-GPC by acetyl transferase
(Fig. 8, f) activity, competition for 1-alkyl-2-lyso-GPC
intermediate by various transferases may prevent reacyla-
tion of AA. As CoA-IT has not been cloned and
characterized, our knowledge of its interaction with
PLA
2
isotypes and other transferases and its role in
AA release, LTB
4
and PAF biosynthesis will remain
rudimentary.
While the major focus of our study has been on CoA-
IT because of its role in AA remodeling and regulating
lipid mediator biosynthesis, it is important to note that

AACOCF
3
may also inhibit other AA metabolizing
enzymes that control lipid mediator formation. For
example, our studies show that AACOCF
3
effectively
inhibits 5-LO (Fig. 8, g) activity and this inhibition could
account for the decrease in leukotriene biosynthesis.
AACOCF
3
also inhibited the incorporation of exogenous
AA into neutrophils, possibly via AA-CoA synthetase or
CoA-dependent-acyl transferase (data not shown). Inhibi-
tion of these enzyme activities that are linked to the
control of AA levels within cells could lead to the
depletion of cellular AA that would have been utilized for
leukotriene biosynthesis. Further studies that favor AA-
CoA synthetase and CoA-acyl transferase activities are
required to fully identify the role of these enzymes in lipid
mediator formation.
Overall, these data highlight the role of two main
activities (5-LO, CoA-IT) in lipid mediator biosynthesis
and the complex nature of AA metabolism. Whereas 5-LO
and CoA-IT are directly linked to LTB
4
and PAF
formation respectively, PLA
2
isotypes hydrolyse AA from

a phospholipid pool that is not linked to mediator
formation by neutrophils. Because of rapid remodeling of
AA by various AA-specific enzymes, caution should be
exercised when using AA analogues as inhibitors of AA
metabolism in whole cell studies.
ACKNOWLEDGEMENTS
This work was supported in part by AI 24985 SI from the National
Institutes of Health to A. N. F. I am grateful for expert technical
assistance from Steve Brooks, Javid Heravi and Dennis Swan. I thank
Dr A. Trimboli for helpful suggestions.
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