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-

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

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

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

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

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

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

[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

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.

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

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

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

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
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,

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-

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

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

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

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

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

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