From the Meakins-Christie Laboratories, Department of
Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec
H2X 2P2, Canada and the § Claude Pepper Institute and
Department of Chemistry, Florida Institute of Technology,
Melbourne, Florida 32901-6988
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ABSTRACT |
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We previously showed that 6-trans isomers of
leukotriene B4 but not leukotriene B4
itself are converted to dihydro metabolites by human neutrophils. The
first step in the formation of these metabolites is oxidation of the
5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of
the present investigation was to characterize the second step in the
formation of the dihydro metabolites, reduction of an olefinic double
bond. We found that the olefin reductase reduces the 6,7-double bond of
5-oxoeicosanoids, is localized in the cytosolic fraction of
neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol
converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro
products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro
metabolites is inhibited by EGTA and a calmodulin antagonist and
stimulated by the addition of calcium and calmodulin, whereas the
reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is
slightly inhibited by calcium. Furthermore, eicosanoid
6- and
13-reductases could be separated
by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic
acid (5-oxo-ETE) is converted by the
6-reductase to
6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE
in mobilizing calcium in neutrophils. We conclude that neutrophils
contain both 5-oxoeicosanoid
6-reductase and
prostaglandin
13-reductase. Metabolism of 5-oxo-ETE by
the
6-reductase results in loss of its biological
activity.
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INTRODUCTION |
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A major pathway in the metabolism of many eicosanoids is initiated
by oxidation of one of the hydroxyl groups by an NAD+- or
NADP+-dependent dehydrogenase. This is usually
followed by reduction of an adjacent double bond by an olefin reductase
in the presence of NADH or NADPH. A number of distinct cytosolic
15-hydroxyprostaglandin dehydrogenases oxo-dize various
prostaglandins (PGs)1 to
their biologically inactive 15-oxo metabolites (1-3). These products
can then be reduced by cytosolic PG 13-reductases to
biologically inactive 13,14-dihydro-15-oxo-PGs (1;4), which in turn can
be further reduced to dihydro-PGs by ketoreductases (5).
Lipoxygenase products can be metabolized by analogous pathways. We have
shown that leukotriene B4 (LTB4) is converted
to 12-oxo-LTB4 by an NAD+-dependent
12-hydroxyeicosanoid dehydrogenase in neutrophils (6). This is followed
by reduction of the 10,11-double bond by a cytosolic NADH-dependent 10-reductase to give
10,11-dihydro-12-oxo-LTB4, which is then reduced to the
corresponding dihydro compound by a ketoreductase (6). Metabolism of
the potent neutrophil agonist (7), LTB4, by this pathway
results in considerable loss of biological activity (8-10). 12(S)-Hydroxy-5,8,10,14-eicosate-traenoic acid is
metabolized in a similar manner by neutrophils (11). However, in this
case, the 10,11-dihydro metabolite of
12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid has been
reported to be a potent proinflammatory agent (12). LTB4 is
metabolized by a similar pathway in monocytes (9, 13) and kidney (14).
However, in the latter case, the 12-hydroxy dehydrogenase is clearly
distinct from the neutrophil enzyme (14).
We previously showed that neutrophils convert 6-trans isomers of LTB4, which are formed nonenzymatically from LTA4, to dihydro metabolites (15, 16). This reaction proceeds by a sequence analogous to that described above for LTB4, the initial step being oxidation of the 5-hydroxyl group, followed by reduction of one of the double bonds and the oxo group (16). We initially speculated that the dihydro products of these reactions might have been 6,11-dihydro metabolites, due to migration of the two remaining double bonds. However, mass spectral evidence subsequently suggested that the products were 6,7-dihydro metabolites (17). The initial step in the formation of these substances is oxidation of the 5-hydroxyl group by a microsomal NADP+-dependent dehydrogenase that is highly specific for eicosanoids containing a (5S)-hydroxyl group followed by a 6-trans double bond (18). LTB4, which has a 6-cis double bond, is not metabolized by this pathway. The best substrate for 5-hydroxyeicosanoid dehydrogenase is (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), which is converted to 5-oxo-ETE (18), a potent activator of neutrophils (19, 20) and eosinophils (21-23).
Relatively little is known about the olefin reductase that converts 5-oxoeicosanoids to their dihydro metabolites. The objectives of this study were to investigate the regulation of this enzyme, its substrate specificity, and its subcellular localization. We also wanted to determine whether 5-oxo-ETE could be converted to a dihydro metabolite by this pathway and, if so, how this would affect its biological activity.
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EXPERIMENTAL PROCEDURES |
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Materials--
Calmodulin was purchased from Biomol (Plymouth
Meeting, PA), whereas the calmodulin inhibitor calmidazolium chloride
(R24571) was obtained from Calbiochem. PGB2 was obtained
from Sigma. 6-trans-LTB4, 12-epi-6-trans-LTB4,
15-oxo-PGF2, and
13,14-dihydro-15-oxo-PGF2
were purchased from Cayman
Chemical (Ann Arbor, MI).
Preparation of Eicosanoids-- A number of the eicosanoids used in this study were prepared by total chemical synthesis. 5-Oxo-ETE, 8-trans-5-oxo-ETE, and 6,7-dihydro-5-oxo-ETE were prepared as described previously (24). 5-Oxo-[11,12,14,15-3H]ETE and 8-trans-5-oxo-[11,12,14,15-3H]ETE were prepared by reduction of an 11,14-diyne precursor with tritium gas (performed by American Radiolabeled Chemicals, St. Louis, MO) (25).
Various eicosanoids were prepared biochemically. 5-Oxo-6-trans-LTB4 and 5-oxo-12-epi-6-trans-LTB4 were synthesized by incubation of 6-trans-LTB4 (2 µM) and 12-epi-6-trans-LTB4 (2 µM) (Cayman Chemical Co.), respectively, with a microsomal fraction from human neutrophils for 90 min in the presence of NADP+ (1 mM) (18). 5-Oxo-15-hydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5-oxo-15-HETE) and 5-oxo-15-hydroxy-6E,8E,11Z,13E-eicosatetraenoic acid (8-trans-5-oxo-15-HETE) were prepared by incubation of 5-oxo-ETE and 8-trans-5-oxo-ETE, respectively, with soybean lipoxygenase (18). 5-HETE was synthesized by incubation of arachidonic acid (NuChek Prep, Inc., Elysian, MN) with potato 5-lipoxygenase (26). 5,15-Dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5,15-diHETE) was prepared by incubation of 5-HETE with soybean lipoxygenase (27). 5-Hydroxy-15-oxo-6E,8Z,11Z,13E-eicosatetraenoic acid (15-oxo-5-HETE) was prepared by incubating 5,15-diHETE (2 µM) with the cytosolic fraction obtained from pregnant rabbit lungs in the presence of NAD+ (28, 29). Similarly, 15-oxo-[5,6,8,9,11,12,14,15-3H]PGF2Preparation of Subcellular Fractions from Neutrophils-- Human neutrophils were purified by treatment of blood with Dextran T-500, centrifugation over Ficoll-Paque, and hypotonic lysis of the remaining red cells (30). The cells (25 × 106/ml) were suspended in 20 mM phosphate buffer, pH 7.4, containing 0.3 M sucrose, phenylmethylsulfonyl fluoride (1 mM), leupeptin (2 µg/ml), and aprotinin (2 µg/ml). The neutrophils were then disrupted by sonication (model 4710 Ultrasonic Homogenizer; Sonics & Materials, Danbury, CT) in an ice bath for 2 × 5 s at a power setting of 1 and for a further 5 s at a power setting of 2. The sonicate was centrifuged successively at 1500 × g for 10 min, 10,000 × g for 10 min, and 200,000 × g for 60 min. The 10,000 × g and 200,000 × g pellets were suspended in phosphate-buffered saline (1.25 times the original volume) containing calcium and magnesium.
Separation of 6- and
13-Reductases
by Ion Exchange Chromatography--
The 200,000 × g
supernatant fraction (10 ml) was incubated with 1 ml of DEAE-Sepharose
for 30 min at 4 °C with constant mixing. The gel suspension was
packed into a column that was eluted with 1) 20 mM
phosphate buffer, pH 7.4 (10 ml); 2) 50 mM NaCl in 20 mM phosphate (10 ml); and 3) 250 mM NaCl in 20 mM phosphate (10 ml). The initial unretained fraction
contained the
13-reductase, which was monitored using
15-oxo-5-HETE as substrate, whereas the 250 mM NaCl
fraction contained the
6-reductase, which was monitored
using 8-trans-5-oxo-[3H]ETE as substrate.
Analysis of Metabolites by Precolumn Extraction/Reversed-phase
High Pressure Liquid Chromatography (RP-HPLC)--
Fractions obtained
as described above were incubated with various substrates, and the
reactions were terminated by the addition of methanol (0.6 ml) and
stored at 80 °C until analysis by RP-HPLC. After the samples were
thawed, water was added to give a final volume of 4 ml (i.e.
15% methanol). Eicosanoids were analyzed by automated precolumn
extraction/RP-HPLC as described previously (31). Products were detected
and UV spectra recorded using a Waters model M991 diode array detector.
Dihydro products were quantitated on the basis of UV absorbance or
(when 5-oxo-[3H]ETE,
8-trans-5-oxo-[3H]ETE, or
15-oxo-[3H]PGF2
were the substrates)
measurement of radioactivity. Different conditions were used for the
analysis of metabolites of oxoeicosanoids (i.e. 5-oxo-ETE
and 8-trans-5-oxo-ETE), hydroxyoxoeicosanoids, and
15-oxo-PGF2
a. All mobile phases contained 0.02% acetic acid. Conditions for oxoeicosanoids were as follows: Spherisorb ODS-2
column (3.2 × 250 mm; 5-µm particle size; Phenomenex); 60% acetonitrile in water, isocratic for 40 min at 0.5 ml/min. Conditions for hydroxyoxoeicosanoids were as follows: Novapak C18
column (3.9 × 150 mm; Waters); linear gradient between 37 and
45% acetonitrile over 30 min at 1 ml/min. Conditions for
15-Oxo-PGF2
a were as follows: Novapak C18
column; 31% acetonitrile, isocratic at 1 ml/min. PGB2 (250 ng/sample) was used as an internal standard.
Localization of the Positions of the Double Bonds in Dihydro-5-oxo-12-epi-6-trans-LTB4-- 12-epi-6-trans-LTB4 (2 µM) was incubated with the 1500 × g supernatant fraction from human neutrophils for 90 min at 37 °C in the presence of NADP+ (1 mM). The reaction was terminated by the addition of methanol (0.5 volumes). Water was added to give a final concentration of methanol of 15%, and the mixture was centrifuged at 1000 × g for 10 min. The supernatant was extracted without acidification on a C18 Sep-Pak (Waters-Millipore) as described previously (32). The methyl formate fraction was evaporated to dryness under a stream of nitrogen, and the residue (containing 5-oxo-12-epi-6-trans-LTB4) was incubated with the 200,000 × g supernatant fraction from human neutrophils for 90 min at 37 °C in the presence of calcium (1 mM) and NADPH (1 mM). The products were extracted using octadecylsilyl silica as described above. RP-HPLC analysis of an aliquot of the methyl formate fraction after the first extraction (material from the incubation with the 1500 × g supernatant) confirmed that the major product was 5-oxo-12-epi-6-trans-LTB4 (18). Dihydro-5-oxo-12-epi-6-trans-LTB4, the major product of the incubation with the 200,000 × g supernatant fraction, was purified by RP-HPLC as described above and incubated at a concentration of 2 µM with the 200,000 × g pellet obtained from porcine neutrophils, prepared as described previously (6), in the presence of NAD+ (1 mM). The products of the reaction were analyzed by precolumn extraction/RP-HPLC as described above.
Protein Determination-- Protein concentrations were determined as described by Bradford (33).
Measurement of Cytosolic Calcium Levels-- Calcium levels were measured in indo-1-loaded neutrophils as described previously (34), using a Photon Technology International (PTI) Deltascan 4000 spectrofluorometer with a temperature-controlled cuvette holder equipped with a magnetic stirrer.
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RESULTS |
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Subcellular Localization of Olefin Reductase Activity in Human Neutrophils-- We had previously shown that 12-epi-6-trans-LTB4 is converted to dihydro and dihydro-5-oxo metabolites by a 1500 × g supernatant fraction from human neutrophils. To investigate the subcellular localization of the olefin reductase required for the formation of these products, 5-oxo-12-epi-6-trans-LTB4 was incubated with subcellular fractions from neutrophils in the presence of different cofactors. When 5-oxo-12-epi-6-trans-LTB4 was incubated with a microsomal fraction from neutrophils in the presence of NADPH, the major metabolite was the ketoreductase product, 12-epi-6-trans-LTB4 (12e-6t-B4) (Fig. 1A). Only a small amount of a dihydro product (dh-12e-B4) was formed under these conditions. In contrast, the major product formed when 5-oxo-12-epi-6-trans-LTB4 was incubated with the cytosolic fraction from neutrophils in the presence of NADPH was its dihydro metabolite (dh-5o-12e-B4) (Fig. 1B).
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Position of Double Bond Reduced by the Olefin Reductase--
We
had previously suggested that
12-epi-6-trans-LTB4 was converted to
a 6,11-dihydro metabolite by intact neutrophils (16). However,
identification of the positions of the double bonds in this product was
not very conclusive, because the diagnostic fragment ions in its mass
spectrum were not very intense and could possibly have arisen as a
result of rearrangements. A recent study employing mass spectral
analysis of fragments formed by oxidative ozonolysis provided evidence
that it is the 6,7-double bond of 6-trans isomers of LTB4
that is reduced by these cells (17). We used a different approach to
investigate the position of the reduced double bond of
12-epi-6-trans-LTB4. As shown in Fig.
2,
5-oxo-12-epi-6-trans-LTB4 could
potentially be reduced to three products by an olefin reductase, resulting in 6,7-dihydro, 6,11-dihydro, or 10,11-dihydro metabolites. Reduction of the 9,10-double bond in the middle of the triene chromophore is unlikely, whereas reduction of the 14,15-double bond is
theoretically possible but would not result in a change in the UV
spectrum of the product. Reduction of the 10,11-double bond of
12-epi-6-trans-LTB4 (Fig. 2) can also
be excluded, since the resulting product would have a
max around 280 nm, which is not observed.
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Time Course for the Formation of Dihydro Metabolites of
5-oxo-15-HETE--
Other 5-oxoeicosanoids were also metabolized by
cytosolic fractions from neutrophils in a manner analogous to that
shown for 12-epi-6-trans-LTB4 in Fig.
1. 5-Oxo-15-HETE was converted to 5,15-diHETE and two dihydro products,
presumably 6,7-dihydro-5-oxo-15-HETE and 6,7-dihydro-5,15-diHETE. The
time course for the formation of these three metabolites is shown in
Fig. 4. The initial
6-reductase and ketoreductase products
(dihydro-5-oxo-15-HETE and 5,15-diHETE) were formed fairly rapidly and
reached maximal levels by about 90 min, after which time the amounts
declined. The product formed by a combination of the two pathways
(dihydro-5,15-diHETE) was formed much more slowly and did not appear to
have reached maximal levels by 120 min.
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Substrate Specificity for the Formation of Dihydroeicosanoids by
Neutrophil Cytosol--
To investigate the substrate specificity of
the neutrophil cytosolic olefin reductase, a variety of oxoeicosanoids
were prepared either chemically or biochemically and incubated with
neutrophil cytosol in the presence of NADPH. The products were analyzed
by RP-HPLC, and the amounts of dihydro products (dihydro plus
dihydro-oxo) were determined. The cytosolic olefin reductase converted
a variety of 5-oxoeicosanoids to dihydro metabolites (Table
II). Of these, the best substrates were
5-oxo-6-trans-LTB4 and
8-trans-5-oxo-15-HETE. 5-Oxo-ETE and its 8-trans isomer
appeared to be metabolized more slowly, but this may have been due at
least in part to the conversion of these substances by a competitive
pathway to 15-hydroxy products due to the presence of 15-lipoxygenase
in the cytosol. The 6-reductase appears to prefer
substrates with an 8-trans double bond, since both
8-trans-5-oxo-ETE and 8-trans-5-oxo-15-HETE were metabolized more rapidly than the corresponding 8-cis compounds. However, of all of the products tested, 15-oxo-5-HETE was by far the
best substrate, being metabolized at a rate at least 3 times that of
the 5-oxoeicosanoids tested. This raised the possibility that the
cytosolic reductase was actually a
13-reductase that was
also capable of reducing the 6,7-double bond of 5-oxoeicosanoids.
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Effects of Calcium on the Formation of Dihydroeicosanoids by Neutrophil Cytosol-- All of the experiments described above were performed in the presence of calcium (1 mM). To determine whether the conversion of oxoeicosanoids to dihydro products was affected by calcium, neutrophil cytosol was incubated with various substrates in calcium-free medium in the presence of EGTA (1 mM). Removal of calcium inhibited the conversion to dihydro metabolites of the three 5-oxoeicosanoids tested (5-oxo-6-trans-LTB4, 5-oxo-15-HETE, and 8-trans-5-oxo-15-HETE) by between 70 and 80% (p < 0.01) (Fig. 5). In contrast, conversion of 15-oxo-5-HETE to its dihydro metabolite was stimulated by about 25% (p < 0.05) in the presence of EGTA. This experiment thus provides strong evidence that neutrophil cytosol contains at least two distinct olefin reductases and that the activity of one of these is enhanced by calcium.
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Effects of Calmodulin on the Metabolism of
5-Oxo-6-trans-LTB4 by Neutrophil Cytosol--
To
investigate the possibility that the calcium dependence of the
6-reductase is mediated by calmodulin, the effect of the
calmodulin inhibitor calmidazolium chloride on the conversion of
5-oxo-6-trans-LTB4 to dihydro metabolites was
determined. Neutrophil cytosol fractions were incubated with
5-oxo-6-trans-LTB4 in the presence of calcium (1 mM), NADPH (1 mM), and various concentrations
of calmidazolium chloride. Calmidazolium chloride (EC50 = 0.3 µM) strongly inhibited the formation of dihydro
metabolites from this substrate by about 85% at the highest
concentration tested (Fig. 6). The
effects of the addition of calmodulin to neutrophil cytosol fractions on
6-reductase activity was also investigated.
Calmodulin (EC50 = 0.2 µM) stimulated the
formation of dihydro metabolites from
5-oxo-6-trans-LTB4 by 80% above control at the
highest concentration tested (Fig. 6, inset).
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Separation of Eicosanoid 6- and
13-Reductases by Ion Exchange Chromatography--
The
experiments described above suggested that neutrophil cytosol contains
both
6- and
13-reductases. To attempt to
separate these two activities, the cytosol was applied to a column of
DEAE-Sepharose, which was washed with 20 mM phosphate
buffer, pH 7.4, and eluted with increasing concentrations of NaCl in
the same buffer (Fig. 7). The
6-reductase activity of each of the column fractions was
estimated by incubation with 5-oxo-6-trans-LTB4
in the presence of Ca2+ (1 mM) and NADPH (1 mM).
13-Reductase activity was determined by
incubating column fractions with 15-oxo-5-HETE in the presence of EGTA
and NADPH. The
13-reductase activity was not retained by
the DEAE-Sepharose and appeared in the flow-through fraction (Fig. 7).
On the other hand, the
6-reductase was strongly retained
by the column and was eluted with 250 mM NaCl. This
fraction did not contain significant 15-lipoxygenase activity, which
resulted in the metabolism of 5-oxo-ETE and
8-trans-5-oxo-ETE to 15-hydroxy products when they were
incubated with neutrophil cytosolic fractions (data not shown).
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Properties of the Partially Purified
6-Reductase--
The requirements of the
DEAE-Sepharose-purified
6-reductase for cofactors and
calcium were investigated using
5-oxo-6-trans-LTB4 as a substrate. Removal of
calcium by chelation with EGTA inhibited
6-reductase
activity in the 250 mM NaCl column fraction by about 40%,
whereas the addition of calmodulin resulted in an increase in enzyme
activity of about 73% (Table III). The
effect of calmodulin was nearly completely inhibited by the addition of
EGTA. The reductase reaction was dependent on NADPH. No products could
be detected in the absence of cofactors, whereas activity was
substantially reduced when NADH was substituted for NADPH.
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Formation of 6,7-Dihydro Metabolites of 5-Oxo-ETE and
8-trans-5-Oxo-ETE--
As shown in Table II, both 5-oxo-ETE and
8-trans-5-oxo-ETE are metabolized to dihydro products by the
neutrophil 6-reductase. To confirm the identities of
these products, 5-oxo-[11,12,14,15-3H]ETE and
8-trans-5-oxo-[11,12,14,15-3H]ETE, both in the
absence of unlabeled substrates, were incubated with the 250 mM NaCl DEAE-Sepharose fraction in the presence of NADPH
and Ca2+. After termination of the reactions, authentic
chemically synthesized 6,7-dihydro-5-oxo-ETE was added, and the
products were analyzed by RP-HPLC. As shown in Fig.
8A, the major metabolite of
5-oxo-[3H]ETE cochromatographed with
6,7-dihydro-5-oxo-ETE, which was detected at 200 nm, whereas a smaller
amount of 5-HETE was formed. 8-trans-5-Oxo-[11,12,14,15-3H]ETE was also
converted principally to a dihydro metabolite that had a longer
retention time than 6,7-dihydro-5-oxo-ETE, presumably because of the
different configuration of the 8,9-double bond (Fig. 8B).
Although only a small amount of 8-trans-5-HETE was detected,
a product with a slightly longer retention time, presumably identical
to 6,7-dihydro-8-trans-5-HETE, was present.
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Biological Activity of 6,7-Dihydro-5-oxo-ETE-- Because 5-oxo-ETE is a potent stimulator of calcium mobilization in neutrophils, it was important to determine whether reduction to 6,7-dihydro-5-oxo-ETE affected biological activity. Authentic 6,7-dihydro-5-oxo-ETE was capable of inducing calcium mobilization in neutrophils, but only at very high concentrations, and its potency was about 1000 times lower than that of 5-oxo-ETE (Fig. 10). The effect of 6,7-dihydro-5-oxo-ETE on calcium levels would appear to be mediated by a 5-oxo-ETE receptor, since pretreatment of neutrophils with a high concentration (10 µM) of the former compound desensitized these cells to subsequent addition of 5-oxo-ETE (Fig. 10, inset).
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Properties of the Partially Purified
13-Reductase--
The effects of cofactors and calcium
on
13-reductase activity in the DEAE-Sepharose
flow-through fraction were examined using 15-oxo-5-HETE as a substrate
(Table III). As observed for the
6-reductase, this
reaction was dependent upon NADPH, with much lower activity being
observed in the presence of NADH. In agreement with the results with
cytosol (Fig. 5), calcium inhibited enzyme activity by about 40%.
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DISCUSSION |
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We previously showed that human neutrophils convert 6-trans
isomers of LTB4 to dihydro metabolites (15, 16). The enzyme responsible for this reaction does not act directly on these substrates but rather requires prior oxidation of the 5-hydroxyl group (16, 18).
The position of the double bond that was reduced was not clear from our
initial studies (16). However, a recent study based on mass spectral
analysis of fragments formed by oxidative ozonolysis of the dihydro
metabolite of 6-trans-LTB4 provided evidence
that the reduced double bond is in the 6,7-position (17). The present
study indicates that the two remaining double bonds in the dihydro
metabolite of 12-epi-6-trans-LTB4 are
in the 8,9- and 10,11-positions, since they are conjugated with the
12-oxo group formed upon oxidation of the 12-hydroxyl group of
6,7-dihydro-5-oxo-12-epi-LTB4 by
12-hydroxyeicosanoid dehydrogenase (see Fig. 2). We also obtained evidence suggesting that a small amount of a 6,11-dihydro product may
have been formed, raising the possibility that the
6-reductase may not be completely specific. However, we
cannot exclude the possibility that another enzyme is responsible for the formation of the putative 6,11-dihydro product.
Monocytes have been reported to convert lipoxins to dihydro and
dihydro-oxo products. Lipoxin A4 is converted to 15-oxo,
13,14-dihydro-15-oxo, and 13,14-dihydro metabolites by these cells
(35), whereas lipoxin B4 is converted to dihydro products
analogous to those formed from 6-trans isomers of LTB4
(36). It would seem likely that the enzyme responsible for the
formation of 13,14-dihydro metabolites of lipoxin A4 is PG
13-reductase (35). Although the nature of the enzyme
that converts lipoxin B4 to dihydro products was not
investigated further in the above study, it would seem probable that it
is identical to the eicosanoid
6-reductase that we have
identified in neutrophils.
In the present study, we investigated the specificity of the olefin
reductase in neutrophils by synthesizing a series of substrates that
could be converted to dihydro products, which could be detected either
by UV absorbance or by radioactivity. Initial studies on the metabolism
of these substrates by cytosolic fractions from neutrophils suggested
that the olefin reductase that converts 5-oxo-12-epi-6-trans-LTB4 to its
dihydro metabolite may not be specific, since 15-oxo-5-HETE was found
to be a better substrate than any of the 5-oxoeicosanoids tested. This
raised the possibility that the enzyme responsible for this reaction
could be a PG 13-reductase. However, the markedly
different calcium requirements for reduction of 5-oxo- and
15-oxoeicosanoids suggested that this was not the case. This was
confirmed when we were able to separate the two activities on a column
of DEAE-Sepharose, which retained the
6-reductase but
not the
13-reductase. Once the two enzymes were
separated, it was apparent that the
6-reductase did not
display any
13-reductase activity and vice
versa.
The eicosanoid 6-reductase has a fairly low
Km (~130 nM) but also a relatively low
Vmax (~3 pmol/min/mg of protein). Thus, this
enzyme can efficiently metabolize low, but not high, concentrations of
substrate. This is in agreement with our earlier finding that intact
neutrophils have only a limited capacity to metabolize 6-trans isomers
of LTB4 to their 6,7-dihydro metabolites (16). The
13-reductase has a somewhat higher Km
(~220 nM) than the
6-reductase but also
has a higher Vmax (~12 pmol/min/mg of
protein). The difference in the Vmax values for
the two enzymes may be somewhat greater than this, because the
6-reductase was partially purified for this experiment
to remove enzymes that competed for the substrate, whereas
13-reductase activity was measured using the
unfractionated cytosolic fraction. Thus, the
13-reductase has a considerably higher capacity in
neutrophils than the
6-reductase. The
Km of the neutrophil
13-reductase is
similar to that of a cytosolic NADPH-dependent
prostaglandin
13-reductase in rat liver, which was
reported to be about 280 nM (4).
The dependence of the 6-reductase on calcium and
calmodulin is intriguing and suggests that the activity of this enzyme
may be tightly regulated. Other olefin reductases involved in the metabolism of eicosanoids, including PG
13-reductase and
the
10-reductase responsible for the formation of
10,11-dihydro metabolites of LTB4, are not known to be
affected by calmodulin. Similarly, there is little evidence for the
regulation of steroid olefin reductases by calmodulin, with the
possible exception of a sterol
24-reductase present in
hepatoma cells and human skin fibroblasts (37). It is not clear whether
calmodulin acts directly on the
6-reductase or whether
its actions are mediated by another protein such as a
calmodulin-dependent kinase or phosphatase. The relatively low specific activity of the enzyme after chromatography on
DEAE-Sepharose suggests that factors other than calmodulin may also be
involved. Despite removal of a substantial amount of protein by the
chromatographic procedure, the
6-reductase activity in
the presence of calmodulin after DEAE-Sepharose (13.5 ± 0.8 pmol/mg/min; Table III) is no higher than that in the cytosol in the
presence of calmodulin (15.20 ± 1.48 pmol/mg of protein/min; Fig.
6, inset).
The 5-hydroxyeicosanoid dehydrogenase/6-reductase
pathway was first discovered in studies on the metabolism of 6-trans
isomers of LTB4, which are formed nonenzymatically from
LTA4 and have little biological activity (15, 16). The
biological significance of this pathway was unclear until we found that
the preferred substrate for the first step, catalyzed by the
dehydrogenase, is 5-HETE, which is converted into a biologically active
product, 5-oxo-ETE (18). A major objective of the present study was to determine whether 5-oxo-ETE is a substrate for the
6-reductase and, if so, whether the product,
6,7-dihydro-5-oxo-ETE, is more or less potent than its precursor. To
accomplish this, we prepared tritium-labeled 5-oxo-ETE (25), which
would allow us to monitor the formation of its 6,7-dihydro metabolites,
which, unlike other lipoxygenase products, do not absorb significantly in the UV. Furthermore, to enable us to identify the putative 6,7-dihydro metabolite and to test its biological activity, we prepared
this compound by chemical synthesis (24).
Our results clearly show that both 5-oxo-ETE and its 8-trans isomer are
metabolized by the 6-reductase to dihydro metabolites.
Metabolism of 5-oxo-ETE by this enzyme results in a dramatic loss in
biological activity, as 6,7-dihydro-5-oxo-ETE is about 1000 times less
potent in stimulating calcium mobilization in neutrophils. This further
supports the argument that neutrophils possess a highly specific
recognition mechanism for 5-oxo-ETE, since a variety of minor
structural modifications cause substantial losses in biological
activity (19, 34, 38). It is becoming apparent that metabolism of
5-oxo-ETE by a variety of pathways results in dramatic reductions in
biological potency, including metabolism by 20-hydroxylase (100-fold)
(34), 12-lipoxygenase (>10,000),2 and
5-ketoreductase (100-fold) (19) enzymes. Metabolism of 5-oxo-ETE by the
6-reductase would result in a permanent loss in
biological activity, since this reaction is presumably irreversible, in
contrast to reduction of 5-oxo-ETE to 5-HETE by a 5-ketoreductase.
It is interesting that the 8-trans isomer of 5-oxo-ETE appears to be a
better substrate for the 6-reductase than 5-oxo-ETE
itself. Indeed, this enzyme shows a preference for substrates with a
5-oxo group followed by two trans double bonds. Metabolism of
8-trans-5-oxo-ETE could be of some significance, since this
substance does possess some biological activity, with a potency about
one-fifth that of 5-oxo-ETE (34, 38). Moreover, we have detected
8-trans-5-oxo-ETE after stimulation of neutrophils (34),
although it is not yet clear whether this compound can be formed
enzymatically. However, the results of our specificity studies should
be interpreted with some caution, since we do not yet understand
completely how this enzyme is regulated, and the conditions employed
may not be optimal due to possible requirements for additional
factors.
In conclusion, human neutrophils possess two distinct olefin reductases
that metabolize eicosanoids, a 6-reductase that converts
5-oxo-ETE and other 5-oxoeicosanoids to 6,7-dihydro metabolites
and a
13-reductase that converts 15-oxo-PGs and other
15-oxoeicosanoids to 13,14-dihydro metabolites. The
6-reductase is highly regulated by calmodulin and
possibly other factors, whereas the
13-reductase is, if
anything, slightly inhibited by calcium. Metabolism of 5-oxo-ETE by the
6-reductase results in a dramatic 1000-fold loss in
biological potency.
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada Grant MT-6254, the J. T. Costello Memorial Research Fund, National Institutes of Health Grant DK44730 (to J. R.), and National Science Foundation Grant CHE-90-13145 (to J. R.; for an AMX-360 NMR instrument).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 all correspondence should be addressed: Meakins-Christie Laboratories, 3626 St. Urbain St-Montreal, Quebec H2X 2P2, Canada. Tel.: 514-398-3864 (ext. 094071); Fax: 514-398-7483; E-mail: Bill{at}Meakins.LAN.McGill.ca.
The abbreviations used are: PG, prostaglandin; LTB4, leukotriene B45-HETE, (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid5-oxo-15-HETE, 5-oxo-(15S)-hydroxy-6,8,11,13-eicosatetraenoic acid15-oxo-5-HETE, (5S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid5, 15-diHETE, (5S,15S)-dihydroxy-6,8,11,13-eicosatetraenoic acidRP-HPLC, reversed-phase high pressure liquid chromatography.
2 W. S. Powell, S. Gravel, S. P. Khanapure, and J. Rokach, manuscript in preparation.
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REFERENCES |
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