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Department of Biochemistry, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONNO. AND PROSTAGLANDIN...INTERACTIONS OF PROSTAGLANDIN...PEROXYNITRITE AND PGHSREFERENCES |
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Key Words: PGHS • NOS • NO. • interferon • peroxynitrite
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONNO. AND PROSTAGLANDIN...INTERACTIONS OF PROSTAGLANDIN...PEROXYNITRITE AND PGHSREFERENCES |
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)(1
, 2)
. Once formed, PGH2 is
converted by different enzymes to produce prostaglandins and
thromboxane (again see ) (3
, 4)
. The reactions
catalyzed by PGHS occur at two biochemically and structurally distinct
sites in PGHS termed the cyclooxygenase and peroxidase active sites,
respectively. Although the active sites are separate, the heme
prosthetic group is absolutely required for both activities
(5)
. X-ray analysis indicates that it is interposed
between the two active sites (6
7
8)
.
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Recently, it has been demonstrated that there are two isoforms of PGHS,
each of which catalyzes the cyclooxygenase and peroxidase reactions
(9
, 10)
. PGHS-1 is constitutively expressed in a variety
of cells such as gastrointestinal epithelial cells and platelets. In
these cells, PGH2 is processed to prostaglandins
critical for gastric cytoprotection and to thromboxane
A2, which is a powerful stimulus for platelet
aggregation (3
, 4
, 11)
. Conversely, PGHS-2 is an inducible
enzyme expressed transiently in a variety of cells including
fibroblasts, macrophages, endothelial cells, vascular smooth
muscle cells, neurons, and astrocytes in response to pathophysiological
challenge (1
, 3
, 4
, 12)
. For example, PGHS-2 is a critical
player in the propagation of the inflammatory response.
Like prostaglandins, nitric oxide (NO.) has been
implicated in a wide variety of physiological processes including
vasodilation, neurotransmission, and inflammation
(13
14
15)
. NO. also is generated by
constitutive and inducible enzymes. As with PGHS, inducible nitric
oxide synthase (iNOS) is associated primarily with inflammation and
other immunological processes (13
, 14)
.
The inflammatory response involves the production of highly
reactive compounds including
O2.-,
NO., ONOO-,
H2O2, and HOCl. Although
these compounds and the processes responsible for their generation are
necessary for successful defense against foreign invaders, their
production also results in `collateral damage' to host tissue
components, including proteins, lipids/membranes, and DNA. Therefore,
it is not surprising to find an association between sustained or
chronic inflammation and development of autoimmune disease,
cardiovascular disease, and cancer (3
, 4
, 16
17
18
19)
.
Inhibitors of both PGHS and nitric oxide synthase (NOS) have shown
promise not only as antiinflammatory agents, but also as cancer
preventive agents (3
, 4
, 15
, 19)
.
Although both NO. and prostaglandins are involved in inflammation and related diseases, only recently have investigations suggested a direct link between NO. production and synthesis of prostaglandins by PGHS. In the long run, such an interaction may prove to be an additional point of manipulation of the inflammatory response to prevent the development of a range of debilitating illnesses.
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NO. AND PROSTAGLANDIN BIOSYNTHESIS |
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TOPABSTRACTINTRODUCTIONNO. AND PROSTAGLANDIN...INTERACTIONS OF PROSTAGLANDIN...PEROXYNITRITE AND PGHSREFERENCES |
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21
22
23
24
25
26
27
28)
. Several investigators
have also demonstrated that in cells expressing both inducible PGHS and
NOS, NO. potentiates the formation of
prostaglandins. Salvemini and co-workers (29)
have shown
that addition of the NOS inhibitors
NG-monomethyl-L-arginine (L-NMMA) or
aminoguanidine to lipopolysaccharide (LPS) -stimulated mouse
macrophages (RAW 264.7) results not only in a decrease in nitrite
formation, but also a four- to fivefold decrease in prostaglandin
E2 (PGE2) biosynthesis.
Conversely, the addition of the PGHS inhibitor indomethacin only
inhibits PGE2 synthesis, and not nitrite
formation.
Modulation of NOS substrate levels also affects prostaglandin
biosynthesis in this macrophage system. Inhibition of
PGE2 and nitrite formation by L-NMMA or
aminoguanidine is reversed by the addition of L- but not D-arginine
(29)
. Furthermore, when macrophages are grown on
L-arginine-deficient media, they do not produce nitrite, and
PGE2 synthesis is decreased fivefold upon LPS
stimulation compared with cells grown in complete medium. As above,
addition of L-arginine restores full NO.
biosynthesis and PGHS activities, but D-arginine has no effect. In a
similar manner, addition of NO. as an
NO.-saturated solution or generation of
NO. from a donor (e.g., sodium nitroprusside
[SNP]) fully restores PGE2 biosynthesis. PGHS
activity restored by L-arginine addition is sensitive to NOS
inhibitors, but these same inhibitors have no effect when activity is
restored using NO. or NO.
donor molecules (29)
.
The relationship between prostanoid biosynthesis and
NO. appears to result from a direct interaction
between NO. and the PGHS-2 protein. Salvemini and
co-workers (29)
have evaluated the possible participation
of soluble guanylate cyclase (sGC), the first known
NO. receptor, in stimulation of prostaglandin
biosynthesis. Human fetal fibroblasts incubated with arachidonate and
an NO. donor produce increasing levels of
PGE2 and cGMP with increasing donor
concentration. The formation of cGMP is inhibited by hemoglobin, which
binds and oxidizes NO.; methylene blue, an
inhibitor of sGC. PGE2 synthesis, is blocked only
by hemoglobin, not methylene blue (29)
. This suggests that
the effect of NO. on prostaglandin synthesis is
cGMP independent. Similarly, using RAW267.4 cells, Eling and others
(30)
have demonstrated that NO. from
several different sources does not increase either PGHS-2 mRNA or
protein levels (Fig. 1
), eliminating transcriptional control as a mechanism for
NO.-stimulated prostaglandin biosynthesis in
these cells.
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The ability of NO. to enhance prostaglandin
biosynthesis has been observed in other cell culture and in
vivo models. These include various rat tissues (21
, 31
32
33
34)
, bovine endothelial cells (21
, 27)
, human
airway epithelial cells (A459) (35)
, and a rabbit renal
inflammation model (36)
. Likewise, McDaniel and others
(20)
report the enhancement of prostaglandin biosynthesis
by NO. in rat islets of Langerhans and propose
that interaction between PGHS and NO. may play a
role development of autoimmune diabetes.
The administration of LPS to rats results in the expression of both
iNOS and PGHS-2, with concomitant development of cerebral hyperemia.
Consistent with the proposed direct activation of PGHS by
NO., Okamoto and others (28)
note
that NO. enhances PGHS-2 activity without
significantly changing PGHS-2 protein levels.
In a recent study, Iadecola (37)
observed similar results
in a cerebral ischemia model. It is striking that administration of
aminoguanidine inhibits PGE2 biosynthesis only in
the area of cerebral infarct, and not in unaffected areas of the brain
such as the olfactory bulb. This correlates with the recruitment of
neutrophils expressing iNOS to the area of cerebral injury.
Furthermore, ischemia-induced PGE2 biosynthesis
is significantly reduced in iNOS knockout mice compared with wild-type
animals. PGHS-2 expression induced by cerebral injury is not diminished
in the iNOS null mice (37)
, further indicating a direct
interaction between the PGHS protein and NO..
Although many studies indicate that NO. directly
interacts with PGHS to stimulate prostaglandin production, some have
suggested that this phenomenon may result (at least in part) from an
effect of NO. on transcription of the PGHS-2
gene. Tetsuka et al. (23)
and Watkins et al.
(35)
suggest that this transcriptional effect is mediated
through a cGMP-dependent mechanism in rat mesangial cells and human
lung epithelial (A459) cells, respectively. Hughes et al.
(22)
noted that NO. may regulate
PGHS-2 expression at transcriptional and post-transcriptional levels in
rat osteoblasts. These investigators show that the effect of
NO. is highly dependent on the cytokine involved
in cellular stimulation. For example, NO. has a
profound effect on PGHS-2 expression in interferon-
-stimulated
osteoblasts, whereas the same cells stimulated with IL-1ß are
relatively insensitive to NO. (22)
.
The inhibition of PGHS-2 expression by NO. has
been observed in mouse (J774.2) and rat macrophages and chondrocytes
from osteoarthritic patients. In these studies, NOS inhibitors act to
increase PGHS-2 expression and prostaglandin biosynthesis
in response to cytokine stimulation (24
, 25
, 38)
, and the
addition of NO. donors reverse this effect
(24
, 25)
. In addition to inhibition of PGHS-2 expression
by NO., Swierkosz et al. (25)
and
Stadler et al. (26)
suggest that NO.
also directly inhibits PGHS activity. Addition of the
NO. donor SNP to broken J774.2 cell preparations
inhibits prostaglandin (specifically, prostacyclin) biosynthesis as
monitored by the extent of 6-keto-prostaglandin
F1
(6-keto-PGF1)
production (25)
. Recently, NO. has
been reported to have a similar effect on prostacyclin synthesis in
human umbilical vein endothelial cells (39)
.
6-Keto-PGF1 is often used in these
investigations as an indicator of PGHS activity. This prostaglandin is
a breakdown product of prostacyclin, and therefore introduces another
point of NO. interaction. Indeed, Wade and
Fitzpatrick (40)
have noted that NO.
can both activate and inhibit the heme-containing enzyme prostacyclin
synthase, depending on NO. concentration. For
this reason, it is important to study the effect of
NO. on more than one prostanoid product to help
rule out effects on downstream enzymes.
Some suggest that there is no interaction between
NO. synthesis and prostaglandin biosynthesis.
Hamilton and Warner observed that the selective iNOS inhibitor 1400W
had no effect on prostaglandin production in a rat platelet aggregation
model (41)
. They suggest that the supposed link between
iNOS and PGHS-2 activity results from the inability of NOS inhibitors
to block iNOS specifically. However, these authors observed the same
result with a nonselective NOS inhibitor (41)
.
It is clear that a large group of investigators have observed NO.-stimulated prostaglandin biosynthesis, apparently by direct interaction with PGHS; however, the field is by no means unified regarding the relationship between NO. and prostaglandin biosynthesis. Matters are not helped by the fact that NO. does not consistently enhance or inhibit prostanoid biosynthesis across cellular or in vivo models. This is compounded by the complexity arising from NO. effects on transcription, post-transcriptional processing, translation, posttranslational processing, or PGHS catalysis. This wide variability in observations may result from the use of differing cell lines and tissues, as well as differences in methods of cell activation and NO. delivery.
Of course, more investigation will also be necessary in order to evaluate the number of possible interactions that might take place between the NO. and prostanoid biosynthetic pathways. These include, but are not limited to: 1) downstream enzymes such as PGE isomerase, prostacyclin synthase, thromboxane synthase, 2) other arachidonate metabolizing enzymes such as lipoxygenase and cytochromes P450, and 3) transcriptional regulation of these enzymes by NO..
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INTERACTIONS OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE WITH NO. |
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TOPABSTRACTINTRODUCTIONNO. AND PROSTAGLANDIN...INTERACTIONS OF PROSTAGLANDIN...PEROXYNITRITE AND PGHSREFERENCES |
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, 43)
. Others observe
either no stimulation or slight inhibition of the enzyme by
NO. (30
, 44
, 45)
. In light of the
conflicting findings, several possible mechanisms of
NO./PGHS interaction are discussed along with the
evidence for and/or against each interaction as a mechanistic
explanation for NO.-enhanced arachidonate
oxygenation by PGHS in vivo.
Heme iron coordination
It is well established that NO. binds
efficiently to many iron-containing proteins including
cis-aconitase, NADH-ubiquinone oxidoreductase, lipoxygenase,
and most heme proteins (13
, 14
, 45
46
47
48)
. Indeed, the first
established receptor for NO. in vivo is the
heme-containing protein sGC (49
50
51)
. Thus, a reasonable
hypothesis is that an interaction between the PGHS heme and
NO. results in activation of the enzyme. Kulmacz
and others have established that NO. does bind to
PGHS heme in its ferric (native) form (44)
. The spectral
shifts observed upon NO. binding (Fig. 2
A) are similar to those observed with other hemoproteins,
including metmyoglobin and horseradish peroxidase. These spectral
changes are consistent with the formation of a hexacoordinate complex
similar to that observed upon CN- binding to
most ferric heme proteins. The reaction between PGHS heme and
NO. is reversible, with a Kd
of nearly 1 mM (44)
. The low affinity of
FeIII PGHS for NO.
indicates that coordination of NO. with
FeIII PGHS under physiological conditions (i.e.,
picomolar to nanomolar NO. concentrations)
(14
, 15
, 52)
is unlikely.
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Although the interaction between FeIII PGHS and
NO. is relatively weak,
FeII PGHS reacts rapidly with
NO., resulting in Soret (435 nm 
402 nm) and
/ß (557 nm 536 nm and 567 nm) band shifts consistent with the
formation of a five-coordinate FeII–NO complex
(Fig. 2B
), indicating that the heme is displaced from its
proximal histidine ligand (44
, 48)
. In this way,
FeII PGHS behaves more like
FeII sGC than FeII
myoglobin, hemoglobin, or horseradish peroxidase (48
, 53)
.
NO. does not displace the proximal ligand in
these proteins. Instead, a six-coordinate, low-spin
FeII–NO complex with a Soret maximum near 420 nm
is observed (44
, 48
, 53)
. The formation of the
five-coordinate FeII–NO complex is indicative of
a relatively weak interaction between the heme and its proximal ligand
His388.
With such a dramatic structural change upon NO.
binding to FeII PGHS, one might propose that this
interaction results in enhanced prostaglandin biosynthesis by PGHS. An
analogous mechanism suggested for sGC upon NO.
binding results in a 400-fold activation of that protein
(54
55
56)
. However, the significant differences between
PGHS and sGC make it difficult to draw parallels between them. First,
sGC is isolated in the FeII form. This
FeII protein does not bind
O2 and only weakly interacts with CO in
comparison to other FeII heme proteins (50
, 56
, 57)
. Conversely, PGHS is isolated in the
FeIII form; once formed after reduction in
vitro, the FeII intermediate is highly
reactive with both O2 and CO (58
, 59)
. This indicates a substantial difference in the structure of
the heme binding site and the
FeIII/FeII reduction
potentials of PGHS and sGC, and suggests that in vivo sGC
and PGHS may exist in different redox states. In fact, there is no
evidence that FeII PGHS forms in vivo.
Second, although NO. binding to sGC activates the
enzyme, the metal center does not participate in the catalytic
reaction. In contrast, the ferric heme of PGHS undergoes oxidation to a
ferryl-oxo form, which then oxidizes Tyr385 to a tyrosyl radical that
oxidizes arachidonic acid (60
61
62
63
64)
. There is no apparent
role for the ferrous enzyme, and if it did form, coupling to
NO. would result in heme dissociation and the
loss of cyclooxygenase activity. Thus, it seems unlikely that the
FeII–NO complex of PGHS forms in vivo
or that its formation results in the activation of the enzyme.
As the spectroscopic and kinetic parameters of
NO. binding suggest, Kulmacz and co-workers
(44)
observed that NO. has little
effect on cyclooxygenase activity in vitro. At
concentrations of NO. exceeding 1 mM, these
authors detected no increase in PGHS activity. In fact, only slight
inhibition was observed. These data would indicate that
NO. does not enhance PGHS activity by
coordination to the heme iron or by any other mechanism.
NO. interaction with PGHS thiols
In one respect, the work of Hajjar et al. (42)
confirms the findings of Kulmacz and colleagues (44)
. They
note that NO. does not stimulate PGHS turnover
through an interaction with the heme prosthetic group. However, these
authors and others have observed the apparent stimulation of purified
PGHS-1 in vitro by NO. (42
, 43)
. In their hands, this stimulatory effect (about fourfold) is
similar to that reported by Salvemini and co-workers in RAW 264.7 cells
(29)
. Hajjar et al. suggest that reaction between
NO. (more precisely, some oxidized derivative of
NO.) and one or more of three free PGHS-1
cysteines (C313, C512, C540) results in a structural change leading to
the increased catalytic activity of the enzyme (42)
.
Consistent with this hypothesis, exposure of PGHS to
NO. results in nitrosylation of 3 mol thiol/mol
of enzyme, and the dependence of thiol nitrosylation and PGHS activity
on NO. concentration is highly similar. Addition
of relevant concentrations of NO. produces no
dramatic shift in the PGHS heme absorption spectrum; however,
substantial changes in the far-UV CD spectrum of PGHS are detected.
Indeed, a 40% increase in ß sheet structure is calculated
(42)
.
Because cysteine 512 is not conserved between PGHS-1 and PGHS-2
(6
7
8
, 65)
, it is unlikely that modification of this
residue is responsible for the PGHS activation observed in various
cellular models. However, both cysteine 313 and cysteine 540
(Fig. 3
) are conserved between all known PGHS-1 and PGHS-2 sequences
(65)
. Furthermore, it has been shown that modification of
cysteines 313 and 540 by either chemical means (maleimides)
(66)
or site-directed mutagenesis (67)
results in substantial changes in PGHS activity. It is important to
point out, however, that these modifications result in the near
complete inactivation of PGHS, even with the conservative substitution
of serine for cysteine. With this in mind, it is difficult to envision
how nitrosylation of these residues by an oxidation product of
NO. along with the apparent dramatic structural
changes could result in PGHS activation instead of inactivation.
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NO. as a tyrosyl radical trap
Another reaction commonly observed with NO.
is the one with phenoxyl radicals to form the corresponding
O-nitrosophenol (reaction 1) or nitrosocyclohexadienone (reaction 2)
products. This reaction is observed not only with small phenolic
compounds but also
 |
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69
70
71
72
73)
. Indeed, NO. reacts rapidly
with ribonucleotide reductase, silencing its EPR tyrosyl radical signal
and blocking enzymatic activity (72
, 73)
. The reaction is
reversible in that removal of NO. results in
restoration of the tyrosyl radical and enzymatic activity (72
, 73)
.
PGHS also produces a tyrosyl radical critical to its catalytic
mechanism. Unlike ribonucleotide reductase, the tyrosyl radical is not
detected with resting enzyme. Instead, this radical must be formed by
the transfer of an electron from a tyrosine in the cyclooxygenase
active site to the heme prosthetic group of the enzyme (61
, 62)
. This two-step process, called cyclooxygenase activation, is
carried out by the peroxidase activity of PGHS. First, the heme
prosthetic group is oxidized by a hydroperoxide by two electrons from
its FeIII form to a ferryloxo porphyrin 
cation radical derivative (reaction 3). The porphyrin radical is then
reduced to its fully covalent form by an electron taken from a tyrosine
residue (reaction 4). The resulting tyrosyl radical is an integral
player in the
 | (3) |
 | (4) |
). Since a hydroperoxide (PGG2) is produced as a
result of cyclooxygenase turnover, only nanomolar starting
concentrations of fatty acid hydroperoxide are required to achieve full
activation of PGHS (74)
. Contaminating peroxides in
preparations of arachidonic acid are sufficient to achieve full
activation of PGHS. Indeed, incubation of PGHS with arachidonic acid
results in the formation of a tyrosyl radical detectable by EPR
spectroscopy (61)
.
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Similar to ribonucleotide reductase, the PGHS tyrosyl radical reacts
rapidly with NO.; however, instead of forming a reversible
adduct, the initial nitrosocyclohexadiene-one product is oxidized
further. Indeed, Gunther et al. (75)
have observed an
iminoxyl radical upon incubation of PGHS-2 with its substrate,
arachidonate, and NO. (Fig. 4
A). This radical decays with time to an EPR silent
intermediate. Both Western blotting and peptide mapping studies
indicate that the final product of the reaction is nitrotyrosine
(75
, 76)
. This suggests that upon reaction between
NO. and the PGHS tyrosyl radical, the
cyclohexadienone adduct is oxidized further, first to the iminoxyl
radical intermediate and finally to nitrotyrosine (Scheme III). Oxidation of the initial nitrosocyclohexadienone adduct appears to
occur as a result of the peroxidase activity of PGHS (75)
.
Substitution of the normal FeIII protoporphyrin
IX prosthetic group of PGHS with the MnIII
derivative produces an enzyme with full cyclooxygenase activity, but
only 0.8% peroxidase activity (5
, 77
78
79)
. As Fig. 4B
indicates, this substitution results in an impeded rate
of iminoxyl radical formation and decay.
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Given the necessity for a tyrosyl radical in the mechanism of arachidonate oxygenation by PGHS, reaction between NO. and this tyrosyl radical would tend to indicate an inhibitory effect on PGHS activity, especially considering the stability and relative difficulty of oxidizing a nitrated aromatic ring. Therefore, it is unlikely that the observed activation of PGHS in vivo by NO. is the result of this particular interaction. In fact, it is surprising that NO. is not a strong inhibitor of cyclooxygenase activity.
This particular oddity may be resolved by noting that
NO. acts as a potent reducing substrate for the
peroxidase activity of PGHS (30)
, and as such should
dramatically stimulate arachidonate oxygenation (80)
. More
than one group has observed that NO. is a slight
inhibitor of the enzyme at high concentrations (30
, 44)
.
It may be that stimulatory effects expected from
NO. as a reducing substrate are counteracted by
its role as a tyrosyl radical trap. Also, like most trapping reactions,
NO. reaction with tyrosyl radical and the
subsequent conversion of the adduct to nitrotyrosine may not be
efficient, especially considering the relatively low concentrations of
NO. compared with many spin trapping protocols.
Finally, in the presence of high concentrations of arachidonate (i.e.,
100–500 x Km), NO. may not
compete as effectively for the PGHS tyrosyl radical.
Identification of PGHS tyrosyl radicals
Although nitration of PGHS tyrosines in the presence of
NO. and arachidonic acid may not explain
NO. activation of PGHS in vivo, this
unique interaction proved helpful in resolving a longstanding issue
regarding the mechanism of activation of PGHS: identification of the
tyrosine oxidized to a radical during cyclooxygenase catalysis. Smith
and co-workers demonstrated by site-directed mutagenesis that Tyr385 is
essential to cyclooxygenase activity (63
, 81
, 82)
.
Although the Tyr385Phe mutant lacks cyclooxygenase activity, reaction
of this protein with a peroxide leads to the formation of a
spectroscopically detectable tyrosyl radical (81
82
83)
.
Also puzzling is the fact that the PGHS mutant, His386Ala, fails to
produce a tyrosyl radical detectable by EPR, yet this protein retains
significant cyclooxygenase activity (1
, 65)
. Thus, it has
been difficult to conclude unequivocally that Tyr385 is in fact
oxidized to a radical intermediate during cyclooxygenase catalysis.
Likewise, it has been difficult to conclude that the tyrosyl radical
observed by EPR is essential to the cyclooxygenase mechanism.
The reaction between PGHS tyrosyl radical and NO.
combined with the stability and unique absorption spectrum of
nitrotyrosine (the final product of the reaction) provided for a new
approach to identify the tyrosine oxidized during cyclooxygenase
turnover. We investigated by peptide mapping and subsequent amino acid
sequence analysis that PGHS tyrosines are nitrated in the presence of
NO. and arachidonic acid, and observed that only
Tyr385 is nitrated (Fig. 5
) (76)
. Along with the results of Tyr385 mutagenesis
(63
, 81
, 82)
, these data provide strong support for the
proposed mechanism of PGHS cyclooxygenase activation and sustained
turnover, as well as the central role for Tyr385 in cyclooxygenase
catalysis.
|
Tsai and co-workers (64)
, using an elegant combination of
site-directed mutagenesis and EPR spectroscopy, have also shown that
Tyr385 is oxidized to a radical intermediate, and this radical is
uniquely able to oxidize arachidonate. As established previously,
tyrosyl radicals are detected with both wild-type and Tyr385Phe PGHS
(81
82
83)
; however, these investigators have shown that
only the radical detected with the wild-type protein is able to
abstract a hydrogen from arachidonic acid to initiate the
cyclooxygenase catalytic cycle. Clearly, the PGHS tyrosyl radical is
centered on Tyr385, and this radical is essential for PGHS catalysis.
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PEROXYNITRITE AND PGHS |
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TOPABSTRACTINTRODUCTIONNO. AND PROSTAGLANDIN...INTERACTIONS OF PROSTAGLANDIN...PEROXYNITRITE AND PGHSREFERENCES |
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Peroxynitrite as a peroxidase substrate
Much of the work demonstrating a link between
NO. and prostaglandins has been undertaken using
inflammation models where neutrophils or macrophages are involved. In
addition to producing NO., these cells produce
copious amounts of O2.- using
an NADPH oxidase (84
, 85)
or NOS itself (86)
.
NO. reacts rapidly (k =
1.9 x1010 M-1
s-1) with
O2.- to form the peroxide
ONOO- (87)
. Like other peroxides,
ONOO- reacts rapidly with
FeIII ((5x107
M-1 s-1) and
MnIII (1.8x106
M-1 s-1) porphyrins to
form the corresponding MIV=O complexes
(88
89
90)
. Likewise, FeIII myelo-,
lacto-, and horseradish peroxidases react with
ONOO- at comparable rate constants to form
ferryl-oxo complexes, most probably compound I (i.e.,
FeIV=O[porphyrin radical])
(91)
. Once formed, these reactive porphyrins (free and
protein bound) rapidly oxidize reductants such as guaiacol,
glutathione, trolox, and ascorbate to regenerate the
FeIII porphyrin at rates at least three orders of
magnitude higher than the direct reaction between
ONOO- and these electron donors (88
, 90
, 92
, 93)
. This constitutes typical peroxidase activity where
ONOO- acts as the peroxide source of oxidizing
equivalents. As discussed, the peroxidase activity of PGHS is proposed
to be critical to activation of the PGHS cyclooxygenase. That is,
peroxides oxidize the PGHS heme prosthetic group, which then oxidizes
Tyr385 to the tyrosyl radical intermediate that oxidizes arachidonic
acid. Therefore, it is reasonable to suggest that
ONOO- may activate cyclooxygenase catalysis as a
typical peroxide substrate.
ONOO- supports PGHS-catalyzed guaiacol
oxidation, and therefore is a substrate for PGHS peroxidase activity
(94)
. The PGHS peroxidase can use a wide variety of
peroxides to support the oxidation of small organic reducing substrates
(80
, 95)
, and the kinetic parameters for
ONOO- reaction with PGHS are generally
consistent with those observed for other hydroperoxides (Table 1
). Although the Km of PGHS for peroxynitrite is
considerably larger than that of the fatty acid hydroperoxide,
15-HPETE, it is lower than that of
H2O2. Moreover,
ONOO- supports a greater extent of guaiacol
oxidation than either 15-HPETE or
H2O2. Given the bolus
method of addition and the rapid decomposition of
ONOO- under the assay conditions used, these
kinetic values should be taken as estimates.
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Peroxynitrite, glutathione peroxidase, and PGHS activation
Peroxides are required to activate PGHS, and preparations of
arachidonic acid contain sufficient quantities of fatty acid
hydroperoxide impurities to accomplish full activation of
cyclooxygenase. To block activation by these peroxides, PGHS must be
placed in a reaction with high concentrations of
glutathione/glutathione peroxidase (GSH/GPx) (74
, 96
, 97)
. Any hydroperoxides present in the arachidonic acid are
consumed and the cyclooxygenase remains inactive. Under these
conditions, the ability of various exogenously added hydroperoxides to
accomplish activation can be evaluated.
ONOO- is able to initiate cyclooxygenase
turnover even in the presence of GSH/GPx (Fig. 6
). Although reaction of ONOO- with GSH can result
in O2 consumption independent of prostaglandin
synthesis, most of the O2 consumption observed in
Fig. 6
is the result of arachidonate oxygenation, not GSH oxidation.
This is confirmed by the fact that the majority of
O2 uptake is inhibited by the cyclooxygenase
inhibitor indomethacin and that prostaglandin products are detected
when ONOO- is added to PGHS and arachidonate in
the presence of GSH/GPx (94)
. Under identical conditions
GSH/GPx is able to block cyclooxygenase activation by addition of other
hydroperoxides such as H2O2
or 15-HPETE. Only ONOO- is capable of activating
PGHS to oxygenate arachidonic acid. This suggests that
ONOO- may be a more effective cyclooxygenase
activator in vivo than fatty acid hydroperoxides (e.g.,
PGG2, HPETEs).
|
It is possible that the bolus addition of ONOO-
overwhelms GSH/GPx, preventing effective scavenging and allowing PGHS
activation (98)
. Moreover, addition of
ONOO- by this method does not accurately reflect
the continuous production of this oxidant in the cellular environment.
The continuous production of ONOO- by the
combination of a NO. donor and xanthine/xanthine
oxidase reverses GSH/GPx inhibition of PGHS (Fig. 7
), but withholding either component prevents PGHS activation. Similarly,
decomposition of SIN-1 (to produce both NO. and
O2.-) in the presence of PGHS,
GPx, up to 2.5 mM GSH, and arachidonate results in synthesis of
prostaglandins (Fig. 8
). Inclusion of SOD in this system prevents cyclooxygenase activation.
These data confirm that ONOO- produced even at a
low steady state can evade GSH/GPx to initiate arachidonate
oxygenation by PGHS. Neither NO. nor
O2.- alone is sufficient to
reverse inhibition by GSH/GPx. Similarly, nitrate, nitrite, and
S-nitrosoglutathione are unable to activate PGHS (94)
.
|
|
Recent investigations by Sies and co-workers have revealed that
like its heme-containing counterparts, GPx also reacts with
ONOO- to form nitrite (99)
.
Concomitantly, 2 mol of thiol is oxidized, indicating that
ONOO- behaves as a typical peroxide in the GPx
catalytic cycle (99)
. It also has been demonstrated by
stopped-flow kinetic methods that the reduced form of GPx reacts with
ONOO- at rates comparable to other peroxidases
(k =2x106 M-1
s-1 per monomer) (100)
. It is
surprising that ONOO- (either by bolus addition
or in situ generation) is capable of activating PGHS in the
presence of GPx/GSH.
Two important facts should be borne in mind when considering this
apparent paradox. First, the rate constant for PGHS reaction with
ONOO- has not yet been determined. Our
preliminary stopped-flow investigations indicate that this reaction
takes place at least as rapidly as that observed for reaction of
ONOO- with myeloperoxidase. Myeloperoxidase
reduces ONOO- with a pH-independent rate
constant of 2.0 x107 M-1
s-1, nearly one order of magnitude faster than
GPx (91)
. The rate constants for
ONOO- reduction by these two peroxidases were
calculated under different temperature conditions (GPx at 25°C,
myeloperoxidase at 12°C). Considering that the rate constant for
ONOO- reduction by the
FeIII porphyrin
5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinatoiron(III) is around 5
x107 M-1
s-1 (88)
, it is reasonable to
suggest that the reaction of PGHS with ONOO- may
be considerably faster than with GPx.
Second, it is important to recognize that to achieve full activation of PGHS, only one equivalent of ONOO- or any other peroxide is necessary, because once activated, the peroxide activator is not required to sustain the cyclooxygenase catalytic cycle. It is entirely plausible that GPx/GSH is unable to scavenge ONOO- to this level before PGHS can be activated, especially considering the possibility that PGHS may react at the same or greater rates with ONOO- than GPx/GSH. Regardless, it is clear that much work remains to characterize the kinetics of ONOO- reaction with PGHS as well as GPx/GSH.
Cellular model of PGHS activation by peroxynitrite
Upon activation, mouse macrophage-like RAW267.4 cells produce
NO. and
O2.-. As many investigators have
demonstrated, prostaglandin biosynthesis is also dramatically enhanced
in these activated cells, and prevention of NO.
formation by various methods dramatically reduces formation of
prostaglandins. If ONOO- is the direct activator
of PGHS rather than NO., one would expect that
the removal of O2.- would also
retard prostaglandin biosynthesis. We have observed the dose-dependent
inhibition of prostaglandins in activated RAW267.4 cells by two SOD
mimetic agents, CuDIPS (Fig. 9
) and MnTMPyP (94)
.The extent of inhibition by these agents
(about a 4–5 fold decrease in PGD2 and
PGE2) is similar to the effect observed upon
removing NO. (29)
. Furthermore, the
parallel decrease in PGE2 and
PGD2 suggests that the effect of the SOD mimics
is not on the prostaglandin isomerases. Several control experiments
indicate that the only effect of these compounds is the removal of
O2.-: 1) neither
CuDIPS nor MnTMPyP inhibits expression of NOS or PGHS-2, 2)
they do not directly inhibit PGHS-1, PGHS-2, or NOS, and 3)
neither appears to inhibit phospholipase A2
because both CuDIPS and MnTMPyP inhibit prostaglandin synthesis whether
arachidonate is derived from endogenous or exogenous sources. These
results with SOD mimetic agents parallel the effects of Cu/Zn SOD upon
cyclooxygenase activation by peroxynitrite in vitro with
purified PGHS (see Fig. 9
).
|
Based on these results, we propose that NO.
enhances PGHS activity under inflammatory conditions by acting as a
precursor to ONOO-, a potent peroxide activator
of PGHS (Scheme IV). In response to inflammatory signals (e.g., LPS, cytokines, and other
messengers), cells express iNOS and PGHS-2. Furthermore, inflammatory
cells such as neutrophils and macrophages produce large quantities of
O2.- using an NADPH oxidase. The
coupling of NO. and
O2.- from these sources produces
ONOO-, which can then activate PGHS.
ONOO- activates PGHS as a typical peroxide
substrate, oxidizing the FeIII heme of PGHS to
the FeIV = O[porphyrin.+]
intermediate. Intramolecular electron transfer from tyrosine 385 to the
heme produces the tyrosyl radical necessary for arachidonate
oxygenation. The net effect is the enhancement of prostaglandin
biosynthesis. Thus, blocking ONOO- formation by
removal of NO. inhibits prostaglandin
biosynthesis, and replacement of NO. with a donor
compound restores PGHS activity.
|
Of course, there is much left to learn regarding the roles of
ONOO- and NO. in the
production of prostaglandins. The kinetics of PGHS heme oxidation by
ONOO- have received only cursory examination.
This is critical as ONOO- has been shown to also
react with GPx; thus, it is not entirely certain how sufficient
ONOO- `survives' to react with PGHS in the
presence of GSH/GPx. This is an issue that may be resolved kinetically;
therefore, detailed kinetic characterization of
ONOO- reactions with GPx and PGHS is required.
ONOO- has been reported to inactivate GPx
(98)
. The role this might play in the unique ability of
ONOO- to activate PGHS in the presence of high
GSH/GPx concentrations remains to be clarified. It is also important to
determine what role ONOO- might play in PGHS
inactivation.
NO. may not act exclusively as a precursor to
ONOO-. Indeed, in some cell types,
NO. appears to affect expression of the PGHS
protein. This may be highly dependent on the cell type examined and the
method by which the cells are stimulated and treated with
NO., NO. donors, and NOS
inhibitors. The effect of these variables on the observed affects of
NO. must be characterized. Finally, the
interactions of NO. and
ONOO- with other enzymes of arachidonate
metabolism have received some attention (40
, 45
46
47
, 101
102
103
104)
, but more work is needed to fully understand how
NO. and ONOO- may
specifically regulate synthesis of individual prostanoid hormones.
In conclusion, there have been numerous reports of interactions between the NO. and prostaglandin biosynthetic pathways. Investigators have suggested that NO. enhances prostaglandin formation by direct activation of PGHS. Although no clear mechanism by which NO. may interact directly with the PGHS protein to stimulate activity has been ventured, NO. has proved valuable in the resolution of key questions of PGHS catalytic mechanism. We suggest that the lack of clarity on the ability of NO. to stimulate PGHS indicates that NO. only acts as a precursor to the stimulating species. ONOO- from NO. and O2.- coupling is able to activate PGHS to synthesize prostaglandins. As such, ONOO- may represent an important player in prostaglandin biosynthesis and inflammation, and represents an important link between prostaglandin and NO. biosynthetic pathways. This provides exciting new possibilities for the control of the inflammatory response and the debilitating consequences that can result from conditions of chronic inflammation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Department of Biochemistry,
Center in Molecular Toxicology, School of Medicine, Vanderbilt
University, Nashville, TN 37232-0146, USA. E-mail:
marnett@toxicology.mc.vanderbilt.edu.
3 Abbreviations: PGHS, prostaglandin endoperoxide
synthase; sGC, soluble guanylate cyclase; GSH/GPx,
glutathione/glutathione peroxidase; SOD, superoxide dismutase; NOS,
nitric oxide synthase; iNOS, inducible NOS; PGH2,
prostaglandin H2; PGG2, prostaglandin
G2; PGD2, prostaglandin D2,
PGE2, prostaglandin E2;
6-keto-PGF1, 6-keto-prostaglandin F1;
SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetyl-penicillamine;
SIN-1, 3-morpholinosydnonimine; L-NMMA,
NG-monomethyl-L-arginine; 15-HPETE,
15-hydroperoxyeicosatetraenoic acid; IFN-, interferon ; LPS,
lipopolysaccharide; CuDIPS, Cu(II)
(3,5-diisopropylsalicylate)2; MnTMPyP, Mn(III) tetrakis
(1-methyl-4-pyridyl) porphyrin.
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