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INFLAMMATION AND IMMUNOPHARMACOLOGY
Departments of Medicine (D.M.A., J.A.O.), Pharmacology (O.B., J.A.O.), and Biochemistry (L.J.M.), Vanderbilt University, Nashville, Tennessee
Received August 5, 2002; accepted October 4, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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A number of mechanisms have been hypothesized to account for the
pharmacological profile of salicylate. Because it is a weak or negligible
inhibitor of both PGHS isoenzymes (Vane,
1971
; Mitchell et al.,
1994; Riendeau et al.,
1997), actions other than direct PGHS inhibition have been
explored, including the suppression of activators of gene transcription,
inhibition of regulatory protein kinase activity, suppression of inflammatory
cytokine and adhesion molecule expression, and stimulation of the production
of adenosine by leukocytes (for reviews, see
Aronoff and Neilson, 2001;
Tegeder et al., 2001).
Salicylate has been found to inhibit the expression of PGHS-2 in human
umbilical vein endothelial cells and foreskin fibroblasts
(Xu et al., 1999), but not in
other cell types (Mitchell et al.,
1997; Giuliano et al.,
2001). Salicylate has been reported to block PGHS-2 transcription
in endotoxin-stimulated murine peritoneal macrophages
(Xu et al., 1999), but not in
RAW 264.7 cells (Hinz et al.,
2000), whereas PGHS-2 expression was increased by salicylate in
endotoxin-treated human monocytes (Amann et
al., 2001). The salicylate metabolite gentisic acid inhibits
PGE2 production in RAW 264.7 cells
(Hinz et al., 2000).
Although numerous investigations have demonstrated only weak inhibitory
effects of salicylate on the purified PGH-synthases
(Humes et al., 1981; Mitchell
et al., 1994,
1997;
Riendeau et al., 1997;
Amann et al., 2001;
Giuliano et al., 2001),
Mitchell et al. (1997)
demonstrated that salicylate inhibited PGE2 production by A459
cells with an IC50 value of approximately 30 µM when exogenous
arachidonic acid was absent or present in low concentrations; this occurred
without changes in PGHS-2 expression. However, increasing the concentration of
exogenous arachidonic acid abrogated the inhibition by salicylate, an effect
considered to be consistent with salicylate acting as a competitive inhibitor
of PGHS-2 in these cells. This inverse relationship between substrate
concentration and the inhibitory potency of salicylate was confirmed in vivo
(Giuliano et al., 2001).
An explanation of the unique human pharmacology of salicylate, however,
cannot be readily derived from the above-mentioned findings. Of interest is
the analogy between the pharmacological action of salicylate and that of
acetaminophen, which also exerts antipyretic and analgesic effects at
doses/concentrations having little or no antiplatelet or antiinflammatory
action. We recently demonstrated that the cellular selectivity of
acetaminophen as an inhibitor of PGH-synthases is dependent on hydroperoxide
concentrations within the cell (Boutaud et
al., 2002).
The effect of peroxide concentration on the action of acetaminophen is
consistent with the concept of PGHS isoforms as bifunctional enzymes operating
in a branched chain mechanism in which a tyrosyl radical in the cyclooxygenase
site is required for the dioxygenation of arachidonic acid to yield
PGG2, which is subsequently reduced to PGH2 by the
hemecontaining peroxidase site (Dietz et
al., 1988; Karthein et al.,
1988) (see Discussion). Acetaminophen, acting as a
cosubstrate of PGHS-peroxidase, reduces the higher oxidative state of the heme
prosthetic group, inhibiting the regeneration of the catalytic tyrosyl radical
within the cyclooxygenase site, thereby blocking arachidonic acid metabolism
(Boutaud et al., 2002).
Elevated levels of peroxide antagonize the reductant function of acetaminophen
and prevent it from inhibiting the enzyme. Thus, cells with high levels of
peroxide, such as the activated platelet, are resistant to the action of
acetaminophen, whereas the drug blocks PG formation in endothelial cells at
low micromolar concentrations (Boutaud et
al., 2002). Because PGG2 is a substrate for the
PGHS-peroxidase, elevating the concentration of PGG2 by increasing
the concentration of arachidonic acid will antagonize the inhibitory action of
acetaminophen (Boutaud et al.,
2002).
Given the evidence that increasing the concentration of substrate similarly abrogates the inhibitory effect of salicylate, we considered whether salicylate inhibition could be suppressed by peroxides. These investigations address the hypothesis that inhibition of PGH-synthases by salicylate is contingent on a low concentration of the lipid peroxides that drive the enzyme to its higher oxidative state.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Incubation of PGHS-1 and PGHS-2 with [14C]Arachidonic Acid
and o-Anisic Acid, Benzoic Acid, or Salicylate. PGHS-1 was
purified from ram seminal vesicles as described previously
(Marnett et al., 1984).
Wild-type murine PGHS-2 was expressed in Sf9 cells (Novagen, Madison, WI) and
purified as described previously
(Rowlinson et al., 1999).
Ovine PGHS-1 (specific activity 202 mol of arachidonic acid/min/mol of enzyme) or wild-type murine PGHS-2 (specific activity 109 mol of arachidonic acid/min/mol of enzyme) was preincubated on ice for 20 min with 2 M equivalents of hematin in Tris-HCl buffer, pH 8.0, 500 µM phenol. This solution was then warmed for 5 min at 37°C in the presence or absence of sodium salicylate (final concentrations 0.3, 0.5, 1, and 2 mM), o-anisic acid, or benzoic acid (final concentrations 1 and 2 mM). [14C]Arachidonic acid (20 µl) (4.8 nCi, 0.5 µM final concentration) in Tris-HCl buffer, pH 8.0, was preincubated at 37°C for 2 min. The reaction was initiated by adding equivalent activities of PGHS-1 (5.4 nM final concentration) or PGHS-2 (10 nM final concentration) to a total volume of 200 µl. The reaction was terminated after 8 s by the addition of 200 µl of ice-cold diethyl ether/methanol/4 M citric acid (30:4:1) containing 8 µg of butylated hydroxyanisole as antioxidant and 8 µg of unlabeled arachidonic acid as a carrier.
The organic layer was loaded on a silica plate and eluted with the organic phase of ethyl acetate/isooctane/water/glacial acetic acid (45:25:50:1). Thin layer chromatography plates were analyzed for radioactivity by a Bioscan AR-2000 imaging scanner (Bioscan, Washington, DC). Graphical analysis was performed using Win-Scan software (Bioscan). PGHS-1 and PGHS-2 activity was expressed as activity relative to control (no sodium salicylate added). Activity was determined as picomoles of arachidonic acid converted into product per minute per picomole of enzyme. Salicylate experiments were performed in duplicate on three separate occasions. o-Anisic acid and benzoic acid experiments were performed in duplicate on two separate occasions.
Incubation of PGHS-2 with Salicylate and Variable Substrate Concentrations. Murine PGHS-2 was prepared and preincubated as described above and then warmed for 5 min at 37°Cinthe presence or absence of sodium salicylate (final concentrations 0.5, 1, and 2 mM). [14C]Arachidonic acid (20 µl) (0.5, 1, 2, and 10 µM final concentration) in Tris-HCl buffer, pH 8.0, was preincubated at 37°C for 2 min. The reaction was initiated by adding PGHS-2 (10 nM final concentration) to a total volume of 200 µl and was terminated and products analyzed by thin layer chromatography as described above. Experiments were performed in duplicate on three separate occasions.
Incubation of Variable Concentrations of PGHS-2 with Salicylate. Experimental protocol was similar to that described above with the exception that the final [14C]arachidonic acid concentration was 10 µM and the reaction was initiated by adding PGHS-2 to achieve final concentrations of 10, 20, 30, 50, and 100 nM. Reactions were terminated after 8 s and products analyzed as noted above. The 10 nM PGHS-2 reaction was terminated after 15 s to allow for sufficient substrate utilization. Experiments were performed in duplicate on three separate occasions.
Incubation of Mn Protoporphyrin IX-Reconstituted PGHS-1 with
Salicylate. Before reconstitution with hematin, ovine PGHS-1 purified as
described above demonstrated 36% holoenzyme activity compared with the
activity in the presence of hematin (data not shown). Further removal of heme
was accomplished as published previously
(Odenwaller et al., 1990) and
resulted in a preparation of PGHS-1 with no detectable holoenzyme activity
(data not shown). We used this apoenzyme preparation for reconstitution with
Mn protoporphyrin IX.
Apo-PGHS-1 was preincubated on ice for 20 min with 2 M equivalents of Mn
protoporphyrin IX in Tris-HCl buffer, pH 8.0, 500 µM phenol. This solution
was then warmed for 5 min at 37°C in the presence or absence of sodium
salicylate (final concentrations 0.3, 0.5, 1, and 2 mM).
[14C]Arachidonic acid (20 µl) (4.8 nCi, 0.5 µM final
concentration) in Tris-HCl buffer, pH 8.0, was preincubated at 37°C for 2
min. The reaction was initiated by adding Mn-PGHS-1 (5.4 nM final
concentration) to a total volume of 200 µl. Mn-PGHS-1 demonstrates a lag
period before full cyclooxygenase activity begins
(Odenwaller et al., 1992).
Thus, the reaction was terminated after 120 s as described above, permitting a
comparable amount of substrate utilization (in the absence of inhibitor) as
seen within 8 s using hematin-reconstituted PGHS-1 (Fe-PGHS-1; data not
shown). Specimens were then processed as described for Fe-PGHS-1. Experiments
were performed in duplicate on three separate occasions.
Incubation of PGHS-1 with 12-HPETE or 12-HETE. When needed, 10 µl of 12-HPETE or 12-HETE in Tris-HCl buffer, pH 8.0 (final concentration 0.3 µM for both), was added to the 20-µl solution containing [14C]arachidonic acid immediately before the initiation of the reaction with Fe-PGHS-1 or Mn-PGHS-1. Tris-HCl buffer, pH 8.0, was added to control samples. All experiments were performed in duplicate on three separate occasions.
Incubation of PGHS-2 with 12-HPETE. Where indicated, 10 µl of 12-HPETE in Tris-HCl buffer, pH 8.0 (final concentration 0.2 µM), was added to the 20-µl solution containing [14C]arachidonic acid immediately before the initiation of the reaction with PGHS-2. Tris-HCl buffer, pH 8.0, was added to control samples. All experiments were performed in duplicate on three separate occasions.
Assay of PGHS-Peroxidase Activity with PPHP and Inhibitors.
Peroxidase activity was determined by the reduction of PPHP to
5-phenyl-4-pentenyl alcohol (PPA) with a modification of a previously
described method (Markey et al.,
1987). PGHS-1 was preincubated on ice for 20 min with 2 M
equivalents of hematin in Tris-HCl buffer, pH 8.0, 500 µM phenol. This
solution was then warmed for 5 min at 37°C in the presence or absence of
sodium salicylate (final concentrations 0.3, 0.5, 1, and 2 mM) or
acetaminophen (final concentrations 0.3, 0.5, and 1 mM). PPHP (20 µl) (10
µM final concentration) in Tris-HCl buffer, pH 8.0, was preincubated at
37°C for 2 min. The reaction was initiated by adding PGHS-1 (5.4 nM final
concentration) to a total volume of 200 µl. The reaction was terminated
after 8 s by the addition of 200 µl of ice-cold diethyl ether/methanol/4 M
citric acid (30:4:1). The organic layer was then evaporated under nitrogen to
near-dryness and reconstituted with 65% (v/v) methanol/water (final volume 500
µl) for HPLC analysis. Experiments were performed in duplicate on three
separate occasions.
Isocratic HPLC Analysis. The HPLC system consisted of an L-6200A Intelligent pump (Hitachi, Tokyo, Japan) and a spectro-Monitor 3000 UV detector (LDC/Milton Roy, Riviera Beach, FL). Samples were chromatographed isocratically on a 5-µm Discovery C18 reverse-phase HPLC column (Supelco, Bellefonte, PA) with 65% (v/v) methanol/water as the mobile phase at a flow rate of 1 ml/min. Ultraviolet peaks for PPHP and PPA were detected at 250 nm and integrated using Laura Software version 1.2 (Lab Logic, Sheffield, England) and PGHS-1 peroxidase activity was expressed as activity relative to control (no inhibitor added). Activity was determined as picomoles of PPHP converted to PPA per minute per picomole of enzyme.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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12-HPETE Opposes Salicylate Inhibition of PGHS-1 and PGHS-2. Studies
have reported that the inhibitory potency of many phenolic PGHS inhibitors is
inversely related to ambient peroxide concentrations
(Hemler and Lands, 1980;
Hanel and Lands, 1982;
Ouellet and Percival, 2001;
Boutaud et al., 2002). We
therefore added the platelet-derived lipid hydroperoxide (and PGHS-peroxidase
substrate) 12-HPETE to the PGHS assay. As illustrated in
Fig. 2A, a concentration of
12-HPETE (0.3 µM) below that of substrate fully reversed PGHS-1 inhibition
by salicylate. Only a slight decrease in inhibitory potency for salicylate was
seen with the reduced form of 12-HPETE, 12-HETE. A similar effect was observed
when 12-HPETE was added to PGHS-2 (Fig.
2B). We limited the concentration of 12-HPETE to 0.2 µM because
we have shown that it inactivates PGHS-2 at higher concentrations
(Boutaud et al., 2002). At this
concentration of hydroperoxide, the potency of salicylate as an inhibitor of
PGHS-2 was substantially and significantly reduced, even though there was not
the complete reversal of salicylate effect that was produced by addition of
0.3 µM 12-HPETE to PGHS-1.
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Increasing PGG2 Production Reverses Salicylate Inhibition of PGHS-2. The primary substrate of PGHS-peroxidase in investigations of the purified enzyme in vitro is PGG2, the hydroperoxide product of arachidonic acid oxygenation by PGHS-cyclooxygenase. Because increased peroxidase activity is capable of reversing salicylate inhibition, it follows that enhancing PGG2 production should diminish the inhibitory effect of salicylate. We therefore modified our in vitro assay to assess the effect of increasing either arachidonic acid or PGHS-2 concentration on salicylate inhibition. For these studies the salicylate level was kept constant at 2 mM, because this amount showed greater than 75% inhibition of PGHS-2 under usual conditions (Fig. 1A). Increasing either substrate (Fig. 3A) or enzyme concentrations (Fig. 3B) largely reversed the inhibition of 2 mM salicylate.
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Salicylate Is Not a PGHS-Peroxidase Cosubstrate and Does Not Inhibit
Peroxidase Activity. Although many phenolic PGHS inhibitors serve as
reducing cosubstrates for the PGHS-peroxidase active site, it has been
reported that salicylate does not (Markey
et al., 1987). We sought to determine whether salicylate had
important redox interactions with PGHS-peroxidase cycling under our in vitro
conditions. Reduction of the PGHS-peroxidase substrate PPHP to the alcohol PPA
can be followed chromatographically
(Markey et al., 1987). Under
the same reaction conditions used for testing salicylate against ovine PGHS-1,
we substituted PPHP as the substrate and confirmed that salicylate fails to
serve as a reducing cosubstrate for the peroxidase activity
(Fig. 4). As illustrated, this
contrasts with acetaminophen, an effective reducing cosubstrate for the
peroxidase (Markey et al.,
1987). Importantly, salicylate does not inhibit PPHP reduction,
implicating the PGHS-cyclooxygenase site as the primary target for the
drug.
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Salicylate Inhibition of Mn-PGHS-1 Is Not Reversed by 12-HPETE. To
assess whether the effect of 12-HPETE on salicylate inhibition observed with
Fe-PGHS-1 is mediated through an electron transfer between the peroxidase
ferriprotoporphyrin radical and the catalytic tyrosine of the cyclo-oxygenase
site (Tyr385 in PGHS-1), we repeated the 12-HPETE experiments using Mn-PGHS-1,
which has little peroxidase activity. Reconstituting apo-PGHS-1 with Mn
protoporphyrin instead of iron protoporphyrin (hematin) generates an enzyme
retaining full cyclooxygenase function but <1% of normal peroxidase
activity (Odenwaller et al.,
1990,
1992), permitting a study of
cyclooxygenase inhibition with minimal contribution from the peroxidase site.
We found that inhibition of Mn-PGHS-1 by salicylate was slightly greater than
that observed with Fe-PGHS-1 (IC50 value of 437 ± 34 µM
for Mn-PGHS-1; mean ± S.D.; IC50 value 648 ± 130
µM for Fe-PGHS-1). Notably, in the presence of 0.3 µM 12-HPETE,
salicylate does not inhibit the Fe-PGHS-1, whereas salicylate is able to
inhibit Mn-PGHS-1 in the presence of 12-HPETE
(Fig. 5).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) that inhibition
of PGE2 synthesis by salicylate in A549 cells is reversed by
elevating the concentration of exogenous substrate. The influence of PGHS-peroxidase activity on the action of salicylate is further substantiated by the observation that 12-HPETE does not prevent salicylate from inhibiting the Mn-PGHS-1, which cannot reduce the hydroperoxide effectively, whereas this hydroperoxide completely abrogates the effect of salicylate on the Fe-PGHS-1. Taken together, these results indicate that the molecular activity of the PGHS-peroxidase determines the inhibition of PGHS-cyclooxygenase by salicylate. Thus, it can be predicted that salicylate will act as an inhibitor of PGH synthases only in cellular environments in which the levels of lipid hydroperoxide are low.
Inhibition of the PGHS-cyclooxygenase by both salicylate and acetaminophen is reversed by high concentrations of hydroperoxide. This similarity of salicylate and acetaminophen, as well as fundamental differences between their mechanism of action, are best considered in the context of the current understanding of the molecular linkage between PGHS-peroxidase activity and catalysis by the PGHS-cyclo-oxygenase site.
Oxygenation of arachidonic acid by the PGHS-cyclooxygenase requires the
generation of a tyrosyl radical within this active site. Activation of this
cyclooxygenase tyrosine to the radical state is initiated within the
PGHS-peroxidase site, as a hydroperoxide oxidizes the ferriprotoporphyrin
(heme) prosthetic group by two electrons to a ferryloxoprotoporphyrin cation
radical (known as compound I) (eq. 1). Intramolecular electron transfer yields
the tyrosyl radical (Y·), reducing the heme to its fully
covalent state (compound II) (eq. 2). A one-electron reduction of compound II
regenerates the resting Fe(III) state of the enzyme (eq. 3)
(Landino and Marnett, 1996;
Marnett, 2000).
 | (1) |
 | (2) |
 | (3) |
The tyrosyl radical abstracts the pro-S-hydrogen from carbon-13 of
arachidonic acid to initiate its oxygenation, and thereby is reduced to
tyrosine (eq. 4). Regeneration of the tyrosyl radical occurs, however, in the
final step of the cyclo-oxygenase catalytic cycle when tyrosine is oxidized by
the peroxyl radical precursor to PGG2 (eqs. 5 and 6)
(Marnett, 2000).
PGG2 itself then serves as a substrate for the peroxidase site
(Hemler et al., 1978) where it
is reduced to the alcohol product of the PGHS, PGH2 (eq. 1).
 | (4) |
 | (5) |
 | (6) |
Some "leakage" of the PGG2 peroxyl radical from the
cyclooxygenase active site can occur, thereby interrupting the cyclooxygenase
cycle that regenerates the necessary tyrosyl radical; this necessitates
reactivation of the cyclooxygenase activity by heme-dependent hydroperoxide
reduction (eq. 2) (Marnett,
2000).
Our findings have confirmed that acetaminophen serves as a PGHS-peroxidase
cosubstrate, supplying the electrons that return the ferryloxoprotoporphyrin
radical to the ferric or resting state necessary for another cycle of peroxide
reduction (Markey et al.,
1987). Thus, acetaminophen effectively competes with the
cyclooxygenase active site tyrosine for reduction of the heme radical and
thereby inhibits the catalytic function of the PGHS-cyclooxygenase; this
inhibition is evident at low concentrations of peroxide, but increasing
peroxide concentration drives the enzyme to the protoporphyrin radical state
and antagonizes the inhibitory action of acetaminophen
(Boutaud et al., 2002).
Salicylate, however, is not a cosubstrate for the peroxidase, as
demonstrated by Markey et al.
(1987) and confirmed under the
conditions used in the present study. Rather, the evidence indicates that the
action of salicylate is linked to occupancy of the PGHS-cyclooxygenase active
site. Examination of the crystal structure of the PGH synthases has localized
salicylic acid within the cyclooxygenase pocket just above the narrowest part
of the channel (Loll et al.,
1995). Localization of salicylate in the active site is consistent
with the evidence that it antagonizes the acetylation of PGHS by aspirin
(Vargaftig, 1978;
Merino et al., 1980;
Dahl et al., 1983;
Rizk and Abdel-Rahman, 1994)
and blocks the inhibition of the enzyme by indomethacin
(Humes et al., 1981;
Dahl et al., 1983).
The mechanism whereby the oxidative state of the PGHS synthases determines
the action of salicylate has not been elucidated. The hypotheses include
reduction of the cyclooxygenase tyrosyl radical by salicylate, an effect that
would be overcome by high concentrations of peroxides that drive the
regeneration of the tyrosyl radical. Although salicylate is not a cosubstrate
for the PGHS-peroxidase, the ability for the drug to act as a reductant has
been observed with other hemoproteins with peroxidase activity. Formation of a
phenoxyl radical of salicylate by methemoglobin and horseradish peroxidase has
been demonstrated by electron paramagnetic resonance under peroxidative
conditions (Shiga and Imaizumi,
1973,
1975), and salicylate has been
shown to serve as a reducing cosubstrate for myeloperoxidase
(Hermann et al., 1999).
Alternatively, the binding affinity of salicylate in the
PGHS-cyclooxygenase site could be determined by hydrogen bonding with the
reduced catalytic tyrosine, in which case peroxide-driven tyrosyl radical
formation would prohibit hydrogen bonding. By analogy, hydrogen bonding of
aspirin with Tyr-385 of PGHS-1 has been proposed as a requisite step in the
acetylation of the active site serine by aspirin
(Hochgesang et al., 2000). Our
finding that the function of salicylate as a PGHS inhibitor is dependent on
its hydroxyl group would be consistent with either an action as a phenolic
reductant of the tyrosyl radical or with a binding affinity that is determined
by hydrogen bonding of the hydroxyl to tyrosine.
In conclusion, the finding that inhibition of the catalytic activity of the PGH synthases by salicylate is antagonized by lipid hydroperoxides provides a basis for considering that cellular levels of hydroperoxides determine the selective action of the drug in vivo.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PGHS, prostaglandin H2 synthase; PG, prostaglandin; 12-HPETE, 12-hydroperoxyeicosatetraenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; PPHP, 5-phenyl-4-pentenyl hydroperoxide; PPA, 5-phenyl-4-pentenyl alcohol; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Olivier Boutaud, Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602. E-mail: olivier.boutaud{at}vanderbilt.edu
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) was added, immediately followed by [14C]arachidonic acid
(0.5 µM final concentration). The reaction was stopped after 8 s. Each data
point represents the mean of six values. The significance of differences with
the control experiment done in absence of 12-HPETE was determined (*,
p < 0.05; **, p < 0.005).
