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J Biol Chem, Vol. 274, Issue 40, 28264-28269, October 1, 1999
,,,
From the Center for Cardiopulmonary Pharmacology,
University of Milan, Via Balzaretti 9, Milan 20133, Italy and the
§ Division of Basic Science, Department of Pediatrics,
National Jewish Medical and Research Center,
Denver, Colorado 80206
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Leukotrienes can be produced by cooperative
interactions between cells in which, for example, arachidonate derived
from one cell is oxidized to leukotriene A4
(LTA4) by another and this can then be exported for
conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by
yet another. Neutrophils do not contain LTC4 synthase but
are known to cooperate with endothelial cells or platelets (which do
have this enzyme) to generate cys-LTs. Stimulation of human neutrophils
perfusing isolated rabbit hearts resulted in production of cys-LTs,
whereas these were not seen with perfused hearts alone or isolated
neutrophils. In addition, the stimulated, neutrophil-perfused hearts
generated much greater amounts of total LTA4 products,
suggesting that the hearts were supplying arachidonate to the
neutrophils and, in addition, that this externally derived arachidonate
was preferentially used for exported LTA4 that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were
shown to generate higher proportions of nonenzymatic LTA4 products from exogenous arachidonate (deuterium-labeled) than from
endogenous (unlabeled) sources. The endogenous arachidonate, on the
other hand, was preferentially used for conversion to LTB4 by the LTA4 hydrolase. This result was not because of
saturation of the LTA4 hydrolase, because it occurred at
widely differing concentrations of exogenous arachidonate. Finally, in
the presence of platelets (which contain LTC4 synthase),
the LTA4 synthesized from exogenous deuterium-labeled
arachidonate was converted to cys-LTs to a greater degree than that
from endogenous sources. These experiments suggest that exogenous
arachidonate is preferentially converted to LTA4 for
export (not intracellular conversion) and raises the likelihood that
there are different intracellular pathways for arachidonate metabolism.
Enzymatic oxidation of arachidonic acid has long been implicated
as a critical step in the production of mediators of inflammatory events (1-3). Cyclooxygenase, in particular COX-2 (4), as well as the
leukotriene pathway (5) are known to play important roles in this
process. Human neutrophils express 5-lipoxygenase (6),
5-lipoxygenase-activating protein (7), and
LTA41 hydrolase
(8) and are, therefore, able to convert free arachidonic acid into the
potent chemotactic and chemokinetic factor, LTB4. The
importance of the leukotriene pathway in general is demonstrated by the
significantly limited inflammatory response to specific stimuli seen in
mice with a targeted disruption of the 5-lipoxygenase gene (9).
Neutrophils also express cytosolic phospholipase A2, which
specifically releases arachidonic acid from glycerophosphocholine lipids for eicosanoid generation (10), and targeted disruption of this
gene also reduces inflammation (11). However, the processes involved
have, for the most part, been studied within a uniform cell population;
in whole tissues eicosanoid biosynthesis, and specifically leukotriene
biosynthesis, is much more complex resulting from the intricate
interplay between different cells, cellular events, and enzymatic
reactions. For example, contact between cooperating cells can lead to
the multidirectional transfer of arachidonate or reactive intermediates
of this fatty acid such as LTA4.
Adhesion of neutrophils to vascular endothelial cells is a
characteristic feature of inflammation involving numerous adhesion proteins that regulate their transmigration across the vascular endothelial barrier (12). The microenvironment at the interface between
adherent neutrophils and endothelial cells is suggested to represent a
strategic site for exchange, transcellular synthesis, and metabolism of
lipid mediators. Neutrophils by themselves do not synthesize cys-LT
but, in cooperation with endothelial cells (or platelets), lead to the
production of these mediators by the exchange of newly synthesized
LTA4 to the endothelial cell or platelet that contains
constitutively active leukotriene synthase (13, 14). This cooperative
formation of cys-LT has been shown to cause coronary vasoconstriction
and severe inflammatory changes in the rabbit heart (15, 16).
Furthermore, pharmacological manipulations aimed at altering the
neutrophil-endothelial cell adhesion process, such as the application
of nitric oxide and prostacyclin, have also been shown to reduce not
only the cys-LT production but also, secondarily, to alter coronary
vascular reactivity and cardiac function (17, 18).
Previous studies from our group have suggested that the transcellular
biosynthesis process is even more complicated and may often involve an
additional exogenous supply of arachidonate from the platelet or
endothelial cell, which is taken up by the neutrophil for conversion to
the LTA4, that is then handed back to the donor cell for
final conversion to cys-LT (19). To gain further insight into the
biochemistry of this neutrophil-endothelial cell interaction for
generation of bioactive leukotrienes within the context of a
functioning organ system, we investigated the metabolic profile of
LTA4-derived metabolites in isolated neutrophils as well as when perfused through a spontaneously beating, isolated rabbit heart.
The study demonstrates that the metabolic fate of arachidonate released
from the endogenous pool of neutrophil phospholipids is different from
that of arachidonate derived from exogenous sources.
Isolated Cell Preparation--
Human neutrophils were obtained
from blood (40 ml) withdrawn from healthy donors that had not taken
medication for at least 1 week and were purified by dextran
sedimentation followed by centrifugation over a discontinuous
Ficoll-Paque density gradient as described previously (20).
Alternatively, human neutrophils were prepared from buffy coat obtained
at a local blood bank through direct centrifugation over a
discontinuous Percoll gradient (d = 1.077 and 1.098).
Cells collected at the interface between 1.077 and 1.098 were washed,
and red blood cells were eliminated by hypotonic lysis. Washed human
platelets were prepared from platelet-rich plasma according to
Patscheke (21). Washed platelets were resuspended in a
phosphate-buffered saline solution without Ca2+ and
Mg2+ (Dubelco, Weston, IL). Priming of neutrophils was
carried on using granulocyte macrophage colony-stimulating factor
(GM-CSF, 1 nM) (Amersham Pharmacia Biotech), for 30 min in
phosphate-buffered saline. Neutrophils (107
ml Isolated Perfused Heart Preparation--
Albino rabbits
(Hartley) weighing between 2.5 and 3.0 kg were utilized. Hearts were
isolated and perfused in a retrograde direction at 37 °C through the
aorta as described previously (22). The rate of perfusion was
maintained with a roller pump (Gilson Minipulse 2, Biolabo, Milan,
Italy). All hearts were equilibrated for 30 min at a flow rate of 20 ml
min Leukotriene Analysis by RP-HPLC--
The heart perfusate (~45
ml) was thawed and centrifuged at 3500 × g for 15 min.
After the addition of 5 ml of 1 M phosphate buffer, pH 7.4, the sample was extracted using a solid phase cartridge (Mega Bond-Elut
C8, Varian, Harbor City, CA). The cartridge was washed with 5 ml of
hexane and eluted first with 4 ml of ethyl acetate:methanol (99:1) and
then using 4 ml of methanol:water (90:10). The ethyl acetate fractions,
containing PGB2 as well as monohydroxy- and
dihydroxy-arachidonic acid derivatives, were dried, reconstituted in
0.6 ml of mobile phase A, and injected into an HPLC gradient pump
system (Beckman model 126) connected to a diode array UV detector
(Beckman model 168). UV absorbance was monitored at 280 nm, and full UV
spectra (210-340 nm) were acquired at a rate of 0.5 Hz. A multilinear
gradient from solvent A (methanol/acetonitrile/water/acetic acid,
10:10:80:0:02, v/v/v/v, pH 5.5, with ammonium hydroxide) to solvent B
(50% methanol, 50% acetonitrile) was used to elute a 3 × 125-mm
column, at the flow rate of 0.5 ml min
The same number of neutrophils that were used in the isolated heart
perfusion were suspended at the concentration of 107
ml
Enzymatic-LTA4 metabolites were used as a collective name
for LTC4, LTB4, 20-hydroxy-LTB4 and
20-carboxy-LTB4; nonenzymatic-LTA4 metabolite
was used as a collective name for other products (namely Leukotriene Analysis by Electrospray Ionization-Mass
Spectrometry--
LTA4 metabolites obtained from
experiments carried out using deuterium-labeled arachidonate (5 µM) were extracted as described above by a solid phase
extractor. The organic solvent eluates were taken to dryness then
dissolved in 0.3 ml of methanol/water (7/3, v/v). An aliquot of
extracted samples (50 µl) was injected into a RP-HPLC column
(Columbus 3 µm, 1 × 125 mm, Phenomenex, Torrance, CA) and
directly interfaced into the electrospray source of a triple quadrupole
mass spectrometer (Sciex API III+, Perkin-Elmer). The column
was eluted at a flow rate of 50 µl min
The analysis of each leukotriene was carried out by liquid
chromatography/mass spectrometry measuring the ion abundance for the
following collision induced transformation at the corresponding retention times: LTB4 and
6-trans-LTB4 isomers (m/z
335 Data Analysis--
Comparison of LTA4 metabolites
and nonenzymatic/enzymatic ratios in different groups was carried out
by analysis of variance (ANOVA) and post hoc analysis
performed with the Tukey-Kramer test. Values were expressed as
mean ± S.E. of 3-5 different neutrophil preparations. A value of
p < 0.05 was considered to be of statistical significance.
Initial experiments evaluated the profile of 5-lipoxygenase
products formed after the challenge of neutrophils alone,
i.e. utilizing endogenous sources of arachidonate. Human
neutrophils (107 ml
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) were supplied with Ca2+ (2 mM) and Mg2+ (0.5 mM) and allowed
to equilibrate for 5 min at 37 °C prior to perfusion and/or
challenge. Alternatively, to mimic the condition observed during
isolated heart perfusion, neutrophils were allowed to adhere to plastic
in 6-well cell dishes (Costar, Cambridge, MA) at a density of 2 × 105 cm2 (37 °C, 5 min). Arachidonic acid
(arachidonate, 1-10 µM) or deuterium-labeled arachidonate (d8-arachidonate, 5 µM, Cayman Chemical, Ann Arbor, MI) was added together
with fMLP when used.
1 to allow extensive rinsing of the vascular bed; the
hearts were then perfused in a recirculating system with a total volume
of 50 ml. Human neutrophils (1 × 107 cells) were
diluted 3-fold to 3 × 106/ml in phosphate-buffered
saline, and to avoid mechanical obstruction of coronary vasculature,
they were slowly infused into the recirculating medium of isolated
rabbit hearts. The system was challenged with fMLP (0.3 µM, Sigma) 10 min after the addition of GM-CSF-primed cells to the recirculating reservoir. The entire volume of
recirculating buffer was collected 60 min after the fMLP challenge,
spiked with 50,000 dpm [3H]LTD4 as well as 25 ng of PGB2, and kept at 
80 °C until analysis.
1. Solvent B was
increased to 35% over 6 min, to 65% over 25 min, and to 100% over 3 min. This method permitted separation of LTB4 from
(5S,12S)-dihydroxyeicosatetraenoic acid as well
as from nonenzymatic LTA4 metabolites. The dried methanolic
extracts, containing cysteinyl leukotrienes only, were reconstituted in
HPLC solvent A (0.6 ml) containing 25 ng of PGB2, and to
assess recovery of cysteinyl leukotrienes (50-80%), an aliquot (50 µl) was used to measure radioactivity by scintillation counting
(Packard 4000). The remaining sample was injected into the same HPLC
system used for LTB4. The use of
[3H]LTD4 to monitor recovery was necessary
because of the double extraction protocol used. Positive identification
of cysteinyl leukotriene was obtained through UV spectral analysis of
chromatographic peaks eluting at appropriate retention times with the
characteristic UV absorption chromophore with a maximum at 280 nm.
Quantitation was performed on positively identified peaks only using
standard curves of synthetic compounds (Cayman Chemical), and values of cys-LT were corrected for the recovery of radioactive tracer.
1 and challenged with fMLP (0.3 µM).
Stimulation was terminated after 60 min by the addition of 2 ml of
ice-cold methanol containing 25 ng of PGB2, and the samples
were stored at 20 °C overnight. Incubates were then centrifuged
for 15 min at 3500 × g; the supernatant were diluted
to 15 ml with H2O and extracted using a solid phase cartridge (Oasis, Waters, Milford, MA). Ninety percent aqueous methanol
eluates were taken to dryness using a SpeedVac evaporating centrifuge
(Savant Instruments, Farmingdale, NY), reconstituted, and analyzed by
RP-HPLC as described above.
6-trans-LTB4 isomers,
5,6-dihydroxyeicosatetraenoic acids and
12-O-methyl-6-trans-LTB4
isomers) arising from water or methanol-catalyzed, nonenzymatic
hydrolysis of LTA4. Total LTA4 metabolites were
defined as enzymatic LTA4 metabolites + nonenzymatic
LTA4 metabolites.
1 using a linear
gradient from 40 to 100% solvent B (A, water, 0.05% acetic acid, pH
5.7, with NH4OH; B, acetonitrile/methanol, 65/35) over 30 min, and the ratio between d8-labeled and
unlabeled LTA4 metabolites was evaluated by a selected
reaction monitoring technique.
195), 20-hydroxy-LTB4 (m/z
351 195), 20-carboxy-LTB4 (m/z
365 195), LTC4 (m/z 624 272),
and 5-hydroxyeicosatetraenoic acid (m/z 319 203). Deuterium-labeled leukotrienes were measured by the following
transitions: d8-LTB4 and
6-trans-LTB4 isomers (m/z
343 197), d8-20-hydroxy-LTB4
(m/z 359 197),
d8-20-carboxy-LTB4 (m/z 373 197), and
d8-LTC4 (m/z
632 272). A separate aliquot was analyzed for arachidonate and
d8-arachidonate by single ion recordings at
m/z 303 and 311, because the concentration of
free arachidonate in the extracts of stimulated cells was considerably greater than the leukotriene products. The ion abundance ratios were
calculated for each specific eicosanoid to directly determine isotope
enrichment (specific activity) for each exogenous
d8-arachidonate experiment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 in suspension) were
primed with GM-CSF and stimulated with fMLP. Significant amounts of
LTA4 metabolites were synthesized with maximal production
at concentrations higher than 0.1 µM (Fig. 1). The most abundant metabolite observed
was 20-COOH-LTB4 with lower amounts of
20-OH-LTB4 and LTB4. Nonenzymatic
LTA4 metabolites were not seen, and in the absence of
endothelial cells or platelets, no LTC4 was produced
(Fig. 2A).
View larger version (13K):
[in a new window]
Fig. 1.
Effect of fMLP on LTA4 metabolite
formation by GM-CSF-primed human neutrophils. Production of
LTA4 metabolites by isolated human neutrophils upon priming
with GM-CSF (1 nM, 30 min, room temperature) and a
challenge at 37 °C with different concentrations (0.1 nM-10 µM) of fMLP. Total LTA4
metabolites were measured by RP-HPLC as described under "Materials
and Methods." Results are expressed as mean ± S.E.
(n = 3).
View larger version (26K):
[in a new window]
Fig. 2.
RP-HPLC analysis of LTA4
metabolites produced by isolated rabbit hearts perfused with
GM-CSF-primed human neutrophils. Representative chromatograms are
shown from: (A) isolated human neutrophils (107
cells) challenged with fMLP (0.3 µM) after priming with
GM-CSF (1 nM, 30 min); (B) the entire
recirculating perfusate obtained 60 min after the fMLP (0.3 µM) challenge of isolated rabbit heart with no
neutrophils; and (C) the entire recirculating perfusate of
isolated rabbit heart in the presence of GM-CSF-primed human
neutrophils (107 cells) 60 min after the challenge with
fMLP (0.3 µM). PGB2 (25 ng) was used as an
internal standard. UV absorbance spectra of peaks a-f eluting at the characteristic retention time of
20-COOH-LTB4, 20-OH-LTB4, LTB4
LTC4, LTD4 and LTE4, respectively,
are shown in insets. Chromatographic peaks labeled with
x represent uncharacterized background UV-absorbing material
resulting from the extraction of the large volumes of recirculating
buffer in the heart perfusion experiments.
In agreement with previous results (16), an fMLP challenge of the same
number of neutrophils in the reperfused, spontaneously beating,
isolated rabbit heart resulted in the production of large amounts of
cys-LT (Fig. 2C), presumably as a result of
neutrophil-endothelial cell cooperative synthesis of cys-LT (see Ref.
23). Whereas no LTA4 products were seen in the perfusate of
fMLP-challenged isolated rabbit hearts alone, quantitative analysis
revealed that in the neutrophil-perfused hearts the amount of
LTA4 metabolites detected was greatly increased and,
importantly, was much more that that seen when the same number of
neutrophils were stimulated in vitro (Fig.
3). The data suggested that neutrophil
interaction with the coronary vasculature of the heart not only led to
the new synthesis of cys-LTs (transcellular biosynthesis) but also to a
4-fold increase in total LTA4 metabolites, likely because of additional sources of externally derived (nonneutrophil)
arachidonate.
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Under the working hypothesis that arachidonate released from
endothelial cells (therefore "exogenous" arachidonate) might represent a privileged precursor responsible for both the increase in
LTA4 metabolites observed and also the preferential
conversion of LTA4 into products of transcellular
metabolism, the metabolic fate of exogenous arachidonic acid supplied
to human neutrophils was investigated. To minimize the potential
homotypic transcellular metabolism occurring during neutrophil
activation in suspension (20), the number of neutrophils was limited to
2 × 106, and cells were allowed to adhere to a
plastic surface at the density of 2 × 105
cm2 for 5 min prior to the challenge. Simultaneous
evaluation of the metabolic fate of endogenous and exogenous
arachidonate was carried out using GM-CSF-primed neutrophils challenged
with fMLP in the presence of d8-arachidonate (5 µM). The results shown in Fig.
4 indicate a significantly increased
abundance of deuterium label (increased
d8/d0 ratio) in
nonenzymatic LTA4 hydrolysis products (e.g.
6-trans-LTB4 isomers and
5,6-dihydroxyeicosatetraenoic acids) when compared with
LTA4 hydrolase metabolites (e.g.
LTB4, 20-OH-LTB4 and 20-COOH-LTB4).
The LTA4 derived from exogenous arachidonate therefore
seemed to be preferentially excluded from the enzymatic conversion to
LTB4 and its subsequent metabolites. Relatively low levels
of 20-OH-LTB4 and 20-COOH-LTB4 were seen in
this experiment compared with Fig. 1 (data not shown) probably
reflecting the absence of cell-cell contact and the transcellular
metabolism needed for 
-oxidation of LTB4 (20, 24).
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One possible explanation for the observed shift with exogenous
arachidonate toward a preferential release of LTA4, rather than conversion into LTB4, was that the LTA4
hydrolase might have been saturated with substrate. Accordingly, the
relative ratio of enzymatic to nonenzymatic metabolites of
LTA4 resulting from the addition of increasing amounts of
arachidonate in unprimed neutrophils was investigated. As shown in Fig.
5, this ratio remained unchanged even
when the concentration of exogenous arachidonate was varied (1-10
µM). A comparison of LTA4 product level
showed that administration of 2-5 µM exogenous
arachidonate together with fMLP in unprimed neutrophils led to amounts
of metabolites not different from that observed in the GM-CSF-primed,
fMLP-stimulated cells (Fig. 6). In
keeping with the results observed with deuterium-labeled arachidonate,
when unprimed neutrophils were challenged with fMLP and exogenous
arachidonate (2-5 µM) more nonenzymatic LTA4
metabolites were produced than seen with GM-CSF-primed cells stimulated
with fMLP in the absence of added arachidonate (Fig.
7 A), and the ratio of
nonenzymatic to enzymatic products showed a 2-fold increase (Fig.
7B). To determine if the increased production of
nonenzymatic metabolites reflected an increased availability of intact
LTA4 for transcellular metabolism, GM-CSF-primed
neutrophils and platelets were co-incubated at a ratio of 1:40
(neutrophil:platelet) and challenged with fMLP (which does not activate
the platelets) in the presence of deuterium-labeled arachidonate (5 µM). This resulted in the preferential formation of
deuterium-labeled LTC4 (ratio of
d8-LTC4/LTC4 is 2.3)
when compared with enzymatic metabolites of LTA4-hydrolase
(ratio of
d8-20-OH-LTB4/20-OH-LTB4
is 1.2) (Fig. 8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Studies with isolated neutrophil preparations have illustrated the mechanisms underlying 5-lipoxygenase activation and the production of biologically relevant amounts of leukotrienes. In addition, however, transcellular metabolic events involving cooperation between different cell species in the synthesis of eicosanoids critically determine the overall quantity, as well as profile, of arachidonate metabolites generated by cell activation at the organ level. We and others (25, 26) have previously reported transcellular synthesis of cys-LT in mixed cell systems and more recently, the functional consequences of this process, in a model of a spontaneously beating, neutrophil-perfused rabbit heart (15, 20).
In the present study we addressed the qualitative and quantitative
changes in the profile of arachidonate metabolites produced by the
activation of human neutrophils during perfusion of the isolated rabbit
heart. In this environment, fMLP stimulation of GM-CSF-primed human
neutrophils resulted in the production of very large amounts of cys-LT.
In the absence of neutrophils the isolated perfused hearts did not
generate detectable LTA4 products. In sharp contrast, the
same number of GM-CSF-primed neutrophils stimulated with fMLP in
vitro produced significantly lower amounts of overall
LTA4 metabolites and only LTA4
hydrolase-derived products, namely LTB4 and its
-oxidized derivatives, whereas LTC4 as well as
nonenzymatic LTA4 metabolites could not be observed. This
observation led to the hypothesis that additional arachidonate was
provided to the neutrophils by rabbit heart cells, likely endothelial
cells, thereby significantly enhancing the production of leukotrienes. The alternative suggestion that the leukotrienes came from the rabbit
cells has been previously ruled out, showing that their formation is
completely dependent on neutrophil 5-lipoxygenase (15). Furthermore it
appeared likely that this additional arachidonate supplied to
neutrophils was undergoing a preferential metabolism into a pool of
LTA4 that was readily exported outside of the neutrophil and therefore available for transcellular metabolism into
LTC4 by adjacent endothelial cells.
The suggestion that exogenous arachidonate plays a critical role in this process was tested in GM-CSF-primed isolated neutrophils challenged in the presence of exogenous, deuterium-labeled arachidonate. This allowed a distinction between metabolites derived from endogenous or exogenous arachidonate. The relative amount of labeled and unlabeled enzymatic (LTA4 hydrolase) metabolites of LTA4 was compared with that of nonenzymatic metabolites as an index of exported LTA4. The results obtained showed that significantly larger amounts of the exogenous arachidonate underwent preferential metabolism to an LTA4 pool that was not apparently available to cytosolic LTA4 hydrolase, thus resulting in export and nonenzymatic hydrolysis. As predicted, in the presence of a limited number of platelets, the LTA4 arising from metabolism of exogenous arachidonate was preferentially converted into LTC4 by the platelets (rather than into LTB4) as a result of transcellular cooperation between neutrophils and platelets, as described previously.
One possible explanation for the different pathway exhibited for exogenous versus endogenous arachidonate was that the LTA4 hydrolase was readily saturated and/or suicide inactivated so that excess arachidonate supplied from outside the neutrophil was oxidized to LTA4 but was unable to be enzymatically hydrated. However, the arachidonate dose-response experiments suggested that this was not the case in that external arachidonate supplied to fMLP-stimulated, unprimed neutrophils at a concentration that yielded the same amount of total LTA4 metabolites as that seen from only endogenous sources (fMLP with GM-CSF-primed neutrophils with no added arachidonate) still resulted in preferential production of nonenzymatic products.
The data led to the suggestion that exogenous arachidonate is
metabolized to leukotrienes by a pathway that is physically separate in
the cell involved in conversion of arachidonate from endogenous
sources. The simplest version of this concept is that a portion of the
5-lipoxygenase is located at (or translocated to) the plasma membrane
and there preferentially metabolizes arachidonate reaching the cell
from the outside. LTA4 produced at this site would be
available for export and may have some difficulty gaining access to the
cytosolic LTA4 hydrolase. More complex pathways may exist
but again would require maintenance of separate pools of arachidonate
and protection of the exogenous arachidonate-derived LTA4
from the hydrolase. These possibilities are currently under investigation. Whatever the mechanism, the observations suggest that
this unique pathway of arachidonate metabolism and eicosanoid generation occurs with physiologic cell stimuli not just with deliberately added arachidonate but also with arachidonate derived from
adjacent cells and can also be extended to an intact organ, the
isolated perfused heart. In this system, the complex transcellular metabolism event(s), namely arachidonate from endothelial cells to
neutrophils and LTA4 back to the endothelial cells, seems
to result in preferential production of cysteinyl leukotrienes and, as
we have shown previously, significant physiologic consequences to the
organ as a whole (16).
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ACKNOWLEDGEMENTS |
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We thank Lindsay Guthrie for neutrophil isolation, Charis Johnson for outstanding support in the mass spectrometric analyses, and Giuseppe Rossoni for the isolated heart preparations.
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FOOTNOTES |
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* This work was supported in part by Grant HL34303 from the National Institutes of Health and support from the International Program of the National Heart, Lung, and Blood Institute (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-388-4461; Fax: 303-398-1381; E-mail: hensonp@njc.org.
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ABBREVIATIONS |
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The abbreviations used are: LTA4, leukotriene A4; cys-LT, cysteinyl leukotriene; GM-CSF, granulocyte macrophage colony-stimulating factor; fMLP, formylmethionylleucylphenylalanine; RP-HPLC, reverse-phase high pressure liquid chromatography; PGB2, prostaglandin B2.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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