Characterization of the Cytoprotective Action of Peroxynitrite
Decomposition Catalysts*
Thomas P.
Misko
§,
Maureen K.
Highkin
,
Amy W.
Veenhuizen
,
Pamela T.
Manning
,
Michael K.
Stern¶,
Mark G.
Currie
, and
Daniela
Salvemini
From the
Discovery Pharmacology, G. D. Searle,
and ¶ Monsanto Corporate Research, St. Louis, Missouri 63167
 |
ABSTRACT |
The formation of the powerful oxidant
peroxynitrite (PN) from the reaction of superoxide anion with nitric
oxide has been shown to be a kinetically favored reaction
contributing to cellular injury and death at sites of tissue
inflammation. The PN molecule is highly reactive causing lipid
peroxidation as well as nitration of both free and protein-bound
tyrosine. We present evidence for the pharmacological manipulation of
PN with decomposition catalysts capable of converting it to
nitrate. In target cells challenged with exogenously added synthetic
PN, a series of metalloporphyrin catalysts
(5,10,15,20-tetrakis(2,4,6-trimethyl-3,3-disulfonatophenyl)porphyrinato iron (III) (FeTMPS);
5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III)
(FeTPPS); 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III) (FeTMPyP)) provided protection against PN-mediated injury
with EC50 values for each compound 30-50-fold below
the final concentration of PN added. Cytoprotection was correlated with
a reduction in the level of measurable nitrotyrosine. In addition, we
found our catalysts to be cytoprotective against endogenously generated
PN in endotoxin-stimulated RAW 264.7 cells as well as in dissociated
cultures of hippocampal neurons and glia that had been exposed to
cytokines. Our studies thus provide compelling evidence for the
involvement of peroxynitrite in cytokine-mediated cellular injury and
suggest the therapeutic potential of PN decomposition catalysts in
reducing cellular damage at sites of inflammation.
 |
INTRODUCTION |
The excess production of nitric oxide, generated primarily by the
inducible nitric-oxide synthase
(iNOS),1 has been implicated
as a mediator of cellular injury at sites of inflammation (1, 2).
However, recent evidence supports a role for peroxynitrite (PN) in the
cellular damage and death (3, 4) once attributed entirely to nitric
oxide (NO). The formation of this powerful oxidant from nitric oxide
and superoxide anion, free radical species frequently generated by
activated infiltrating leukocytes, is essentially diffusion-limited.
Peroxynitrite has been shown to cause lipid peroxidation (5), chemical
cleavage of DNA (6, 7), inactivation of key metabolic enzymes such as
aconitase (8, 9), ribonucleotide reductase, succinate dehydrogenase,
and cytochrome oxidase of the mitochondrial electron transport chain
(10, 11), and reduction in cellular antioxidant defenses by oxidation
of thiol pools (12). PN can also nitrate protein tyrosine residues,
possibly leading to inactivation of tyrosine kinase activity (13) or to
the disruption of key cytoskeletal components that may contribute to
the pathogenesis of diseases such as amyotrophic lateral sclerosis (14)
or ALS. At physiologic or acidic pH, the protonated form of PN is a
short lived molecule. As a result of this instability, the detection of
nitrotyrosine has become a reliable biochemical marker for the presence
of peroxynitrite in pathophysiological processes. Nitrotyrosine has
been detected in tissues from Alzheimer's (15), multiple
sclerosis2 (16), ALS (14,
17), and rheumatoid arthritis (18) patients, suggesting a role for
peroxynitrite in the pathogenesis of these diseases. It should be
noted, however, that evidence for PN-independent routes of
nitrotyrosine formation have been recently demonstrated (19),
indicating that careful consideration should be taken when assigning
the source of oxidative damage in a disease process.
There is accumulating evidence arising from different in
vitro cellular systems that the generation of PN occurs during
cytokine stimulation leading to cellular injury and death (20-23).
Both apoptotic and necrotic pathways for cell death have been invoked as consequences of exposure to PN (24-27). In this report, we test the
hypothesis that by pharmacologically manipulating the effective concentration of either exogenously added or endogenously produced PN,
we can protect cells from injury. The peroxynitrite decomposition catalysts FeTMPS, FeTPPS and FeTMPyP (28, 29) were cytoprotective in both experimental paradigms, demonstrating that the catalytic shunting of PN to an innocuous form, i.e. nitrate, may
have therapeutic utility in reducing tissue damage during
inflammation.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
RAW 264.7 cells and the human adenocarcinoma
cell line, DLD-1, were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum at 5% CO2 and
37 °C. Dissociated cultures of rat hippocampal neurons were grown on
a glial feeder layer as described previously (30, 31) with the
following modifications. Briefly, 1-day old Sprague-Dawley (Charles
River, MA) rat pups were sacrificed, and their hippocampi were
surgically removed and then dissociated both mechanically and
enzymatically by papain digestion. Debris was removed by low speed
centrifugation. Cells were plated at a density of 50,000 cells per well
on a 96-well plate containing a confluent layer of glia (initial
plating of approximately 3100 cells per well 5-6 days previously).
Cultures were grown for 7 days in MEM (minus phenol red) supplemented
with 0.5% glucose, 0.22% NaHCO3, 2 mM
L-glutamine, and 10% NU-Serum I (Becton Dickinson). No
anti-mitotic agents were used. A mixture of cytokines was then added to
induce iNOS in this culture system: lipopolysaccharide (0111:B4) at 100 to 200 ng/ml; interferon
(200 to 400 units/ml); tumore necrosis
factor
(150 to 300 units/ml); interleukin 1
(5-10 ng/ml). Five
to seven days following the initiation of cytokine treatment, the
cultures were analyzed for nitrite production and cell viability.
Rat Aortic Ring Preparation and Assay for the Interaction of NO
with PN Catalysts--
Four aortic rings were prepared from each of
five rats. Briefly, thoracic aortas were removed from adult
Sprague-Dawley rats anesthetized by injection with 10 mg/kg xylaxine
and 50 mg/kg ketamine. Connective tissue was carefully trimmed to avoid
damage to the endothelium. Rings were cut into 3-mm lengths and placed into a 10-ml tissue bath. In some experiments, the endothelium was
removed by gentle rubbing of the ring preparation. Successful removal
of the endothelium was confirmed by the lack of a relaxation response
to acetylcholine (10 µM). Aortic rings were maintained at
37 °C in Krebs bicarbonate buffer, pH 7.2: 130 mM NaCl,
15 mM NaHCO3, 5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM
MgSO4, 2.5 mM CaCl2, 1.2 mM D-glucose, and 0.1 mM ascorbic
acid bubbled with 5% CO2, 95% O2.
Rings were preloaded with 1 g of tension and equilibrated for 30 min with 2-3 buffer changes. After stabilization of the base line, the
rings were contracted with 0.1 µM phenylephrine, producing 90-100% contraction. Nitric oxide-mediated relaxation of
the precontracted, endothelium-intact rings was produced by cumulative
increases in the concentration of exogenously added acetylcholine (1 nM to 10 µM) or by treatment of
endothelium-denuded rings with the NO donor, sodium nitroprusside (1 nM to 10 µM). Test compounds were added to
the tissue bath 10 min prior to acetylcholine or sodium nitroprusside
addition. Isometric tension was recorded, and relaxation was determined
as the percentage of maximum tone developed to phenylephrine.
PN-mediated Tyrosine Nitration--
Peroxynitrite and PN
decomposition catalysts (28, 29) were synthesized as described
previously. The PN concentration was determined prior to use by
measuring the absorbance at 302 nm and using an extinction coefficient
of 1.67 mM
1 cm
1. Bovine serum
albumin (BSA) was dissolved at a concentration of 5 mg/ml in 100 mM potassium phosphate buffer, pH 7.4, that had been
vigorously degassed with nitrogen. A 2 mM NO solution was
prepared by several minutes of bubbling pure NO gas into the same
buffer at ambient temperature.
To test the relative nitration efficiency of NO and PN in the presence
and absence of PN decomposition catalysts, the stock BSA solution was
injected via syringe into the NO solution while stirring. After a
10-min open air incubation, aliquots were collected for analysis.
Peroxynitrite was similarly added to BSA in the presence or absence of
the PN decomposition catalyst, FeTMPS. Formation of nitrotyrosine
residues on the BSA target molecule was monitored by Western blot in
each reaction. Briefly, samples (2.5 µg) were solubilized in
SDS-Laemmli buffer and heated for 10 min before loading onto a 7.5%
polyacrylamide gel.The gel was transferred to nitrocellulose (32) and
subsequently probed for 18 h at 4 °C with a 1:500 dilution of a
rabbit polyclonal anti-nitrotyrosine antiserum (raised to PN-treated
keyhole limpet hemocyanin, KLH) containing 10 mM
L-tyrosine (to reduce nonspecific staining). To demonstrate
the specificity of staining for nitrotyrosine-like immunoreactivity, a
duplicate blot was similarly incubated with 10 mM
3-nitro-L-tyrosine. Following extensive washing, the blots were probed with a 1:3000 dilution of a goat anti-rabbit alkaline phosphatase conjugate and developed using the Immun-Lite system (Bio-Rad) for detection of antigen by enhanced chemiluminescence.
PN-mediated Cell Injury Assay--
RAW 264.7 cells were plated
onto 96-well plates at approximately 2 × 105 cells
per well and then grown in Earle's MEM (minus phenol red) containing
10% fetal calf serum and 2 mM L-glutamine.
Before the addition of PN, the plates were washed several times with
Dulbecco's phosphate-buffered saline to remove medium components such
as protein and amino acids that might react with the PN pulse. The PN
was then added to each well of cells, containing 200 µl of Dulbecco's phosphate-buffered saline with or without test compound, followed by gentle swirling. After 15 min at ambient temperature, cells
were washed once with medium and incubated with 200 µl per well of
10% Alamar Blue in complete medium for 1-2 h at 37 °C and 5%
CO2 to assess cellular viability. At the end of the assay, 100 µl was removed from each well and read on a Pandex fluorescent plate reader at an excitation of 545 nm and an emission wavelength of
575 nm with a 1% gain setting. The mitochondrial generation of the
fluorescent product of Alamar Blue was linear over a 2-h period and
correlated well with cellular viability and number (data not shown).
Viability is expressed as the percent of untreated control following
subtraction of the nonspecific dye background.
Cytokine-driven Cytotoxicity Assays--
RAW 264.7 cells were
treated with 10 µg/ml of LPS in Earle's MEM (minus phenol red)
containing 10% fetal bovine serum and 2 mM
L-glutamine at 37 °C and 5% CO2.
Twenty-four h after LPS addition, the conditioned medium was removed,
cells were washed, and cellular viability was assessed using Alamar
Blue as described above.
Primary cultures of neuronal cells and glia (predominantly astrocytes)
were treated with cytokines as described above for 5-7 days. To assess
viability, representative fields of phase bright cells with neuronal
morphology and excluding trypan blue were counted in each of four
separate wells. In most cases, a radioimmunoassay for neuron-specific
enolase (described below) was also performed to confirm the cell counts
since the value obtained for the Alamar Blue assay was a better
indicator of glial viability than of neuronal health (glia were present
in larger numbers than neurons by the end of each experiment).
Neuron-specific Enolase Quantitation--
In order to assess
neuronal viability, release of neuron-specific enolase (NSE) from
injured and dying neurons into the culture medium was quantitated by
radioimmunoassay as described by the manufacturer, Amersham Pharmacia
Biotech. Briefly, 150 µl of conditioned medium was assayed for the
isozyme of neuron-specific enolase, which, unlike lactate
dehydrogenase, is not present in glia. However, serum contains a small
amount of this enzyme, the levels of which were taken into account by
measuring its presence in the conditioned medium of untreated
controls.
Radioimmunoassay for Nitrotyrosine--
The radioimmunoassay for
protein nitrotyrosine content was a modification of the method of
Ischiropoulos et al. (33). Samples were denatured by boiling
in SDS and then applied to nitrocellulose. After blocking, rabbit
polyclonal anti-serum to nitrotyrosine (1:500 dilution) was incubated
with the blot in the presence of blocking buffer (5% non-fat dry milk,
2% fetal bovine serum, 0.25% Tween 20, and 10 mM tyrosine
in Tris-buffered saline). The blot was extensively washed and then
incubated with 30 µCi/ml of radioiodinated donkey anti-rabbit IgG
(protein concentration of approximately 10 µg/ml, Amersham Pharmacia
Biotech). After further washing to remove unbound secondary antibody,
the blot was exposed for 5 min or more on a PhosphorImager (Molecular
Dynamics). PN-treated BSA was used as the nitrotyrosine standard to
determine the level of protein-bound nitrotyrosine. The sensitivity of
this assay was approximately 200 pg of nitrotyrosine. In most assays,
the arbitrary PhosphorImager units obtained for the PN-treated control were normalized to 100% in order to assess the concentration
dependence of PN decomposition catalyst activity.
Immunocytochemical Staining--
Immunocytochemical staining was
performed as described previously (34) with the following
modifications. Briefly, cells were lightly fixed in 1% formaldehyde in
Dulbecco's phosphate-buffered saline for 5 min at room temperature
followed by cold 100% ethanol for 5 min. Nonspecific staining was
blocked with 3% normal goat serum in 0.5 M Tris-HCl, pH
7.4, containing 0.5% Triton X-100 for 1 h at room temperature.
All subsequent incubations were carried out in this buffer containing
10 mM L-tyrosine. For detection of
nitrotyrosine immunoreactivity, cells were incubated for 16 h at
4 °C with a 1:1000 dilution of either preimmune serum, an anti-nitrotyrosine polyclonal rabbit serum generated to nitrated keyhole limpet hemocyanin, or the anti-nitrotyrosine serum containing 10 mM nitrotyrosine to eliminate specific staining,
followed by sequential incubations with biotinylated anti-rabbit IgG
and an avidin-biotin-glucose oxidase complex (ABC Vector Laboratories, Inc., Burlingame, CA) for 2 h each. The reaction product was
visualized using tetranitro blue tetrazolium for approximately 10 min.
Measurement of Nitrite and Nitrate--
Nitrite concentrations
were measured as described previously using a fluorescent assay (35).
When indicated, nitrate was converted to nitrite by nitrate reductase
before analysis (35).
Materials--
Tissue culture media and sera were from Life
Technologies, Inc. (Grand Island, NY), NO gas was from Matheson Gas
Products, Inc. (St. Louis, MO), Alamar Blue was from BioSource
International (Camarillo, CA), and all other chemicals and reagents
were from Sigma, unless otherwise indicated.
 |
RESULTS |
Reduction in Protein Nitrotyrosine Content As a Measure of PN
Decomposition Catalyst Activity--
To assess the activity of
compounds as PN decomposition catalysts, FeTMPS was tested for its
ability to reduce the level of protein nitrotyrosine content of bovine
serum albumin that had been treated with synthetic peroxynitrite. Fig.
1A shows a Western blot for
nitrotyrosine content of BSA treated with either 2 mM nitric oxide (lane 1) or 1 mM peroxynitrite
(lane 4) in the presence (lane 5) or absence of
PN catalyst in PBS. As expected, the nitrotyrosine content of
PN-treated BSA (lane 4) was greatly elevated over BSA alone
(lane 7), NO-treated BSA (lane 1), or PN allowed
to decompose in PBS for 15 min prior to addition (lane 8).
The nitrotyrosine content of the PN-treated BSA was decreased in the
presence of the active catalyst, FeTMPS (10 µM,
lane 5), while TMPS (an inactive compound) failed to reduce
the detectable signal (lane 6). Antiserum specificity for
nitrotyrosine was demonstrated either by competition with excess
nitrotyrosine (10 mM) or by conversion of nitrotyrosine to
aminotryrosine with 1 M dithionite (data not shown). Fig.
1B shows the results using a direct radioimmunoassay for
nitrotyrosine to analyze the same samples that had been spotted onto
nitrocellulose. This quantitative measure for nitrotyrosine confirmed
the qualitative results using the Western analysis and was not limited
to only protein that had entered the gel. It is interesting to note
that the free ligand TMPS, lacking an iron center, produced an apparent increase in the measurable levels of nitrotyrosine, a result that may
be explained by the observation that trace contamination with metals
such as iron, especially in the presence of potential chelating agents
such as TMPS, can catalyze PN-mediated nitration of tyrosine (36).

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Fig. 1.
Inhibition by FeTMPS of PN-mediated nitration
of tyrosyl residues in BSA. A, Western blot analysis
for protein-bound nitrotyrosine formation in bovine serum albumin
treated as follows: lane 1, BSA + NO (2 mM);
lane 2, BSA + NO (2 mM) + FeTMPS (10 µM); lane 3, BSA + FeTMPS; lane 4,
BSA + PN (1 mM); lane 5, BSA + PN + FeTMPS (10 µM); lane 6, BSA + PN + TMPS (10 µM); lane 7, BSA alone; lane 8,
decomposed PN + BSA. B, direct radioimmunoassay for
nitrotyrosine content of PN-treated BSA. Each sample was individually
applied to nitrocellulose. All immunoreactivity of antisera to
nitrotyrosine was competed in the presence of 10 mM
nitrotyrosine (data not shown).
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Fig. 2 illustrates the
concentration-dependent reduction of the nitrotyrosine
content of BSA treated with PN (1 mM) in the presence of
increasing amounts of the active catalyst FeTMPS. This contrasted with
the profile obtained using ascorbate, a known PN scavenger, where
concentrations equimolar (or higher) to the PN challenge were required
to see an effect. TMPS, on the other hand, did not reduce the level of
protein-bound nitrotyrosine, indicating that it is neither a PN
catalyst nor a scavenger of PN (data not shown).

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Fig. 2.
Reduction in PN-mediated protein tyrosine
nitration by the PN decomposition catalyst, FeTMPS.
Concentration-response curve for FeTMPS ( ) and ascorbate ( )
against 1 mM PN addition to a 2 mg/ml solution of fatty
acid-free BSA dissolved in 200 µl of PBS. Nitrotyrosine formation is
measured as described under "Experimental Procedures" with
peroxynitrite-treated BSA representing the 100% value of arbitrary
PhosphorImager units.
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Immunocytochemical Detection of Nitrotyrosine on Cells Treated with
PN Is Decreased by the Presence of Active Catalyst--
To investigate
the ability of PN catalysts to reduce the PN-mediated modification of
proteins in a cellular milieu, immunohistochemical staining of cells in
culture for nitrotyrosine was performed. The human adenocarcinoma cell
line DLD-1 showed surprising resistance to PN-mediated cellular damage
(data not shown). When cells were treated with increasing
concentrations of PN, a corresponding increase in the number of cells
that stained with nitrotyrosine-specific rabbit antiserum (Fig.
3A) was observed. When the
final concentration of PN was kept constant at 200 µM,
cell staining was decreased in a concentration-dependent
manner using the active catalyst FeTMPS (Fig. 3B). All
positive staining was competed by incubation with excess nitrotyrosine
(data not shown).

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Fig. 3.
Treatment of DLD-1 cells with peroxynitrite
in the presence of the PN catalyst FeTMPS reduces the
immunocytochemical detection of nitrotyrosine. A,
peroxynitrite-mediated nitrotyrosine staining of DLD-1 cells in
culture. Increasing pulses of peroxynitrite (20, 50, 100, and 200 µM) were added to each well of cells. PN (200 µM) was allowed to decompose for 15 min in PBS and then
added to the cells. Excess nitrotyrosine (10 mM) was used
to compete the positive nitrotyrosine staining resulting from
treatment with 200 µM PN. B, reduction in
nitrotyrosine staining of DLD-1 cells treated with peroxynitrite (200 µM) in the presence of increasing concentrations of
FeTMPS (20, 50, 100, 200 µM) and the absence of an effect
by TMPS (50 µM).
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PN Decomposition Catalysts Protect Cells from PN-mediated
Injury--
Cellular injury is a very real consequence of exposure to
PN, a powerful oxidant of not only protein but also nucleic acid and
lipid. A typical concentration-response curve for cellular injury to
RAW 264.7 cells by exogenously added peroxynitrite is shown in Fig.
4A. Cellular viability was
measured by monitoring the reduction of the electron acceptor Alamar
Blue to a fluorescent product by the mitochondrial electron transport
chain. Fig. 4, B and C, illustrates the
cytoprotection afforded by FeTMPyP and FeTPPS, respectively, against
exogenously added synthetic PN.

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Fig. 4.
The PN catalysts FeTMPyP and FeTPPS protect
RAW cells from PN-mediated cellular injury. A, cellular
viability with increasing concentrations of exogenously added PN.
B, concentration-response curve of FeTMPyP ( ) and TMPyP
( ) to an exogenous pulse of PN (300 µM). C,
concentration-response curve for FeTPPS ( ) and TPPS ( ) against a
200 µM PN pulse. Viability was determined by the capacity
of cells to metabolize Alamar Blue to a fluorescent product. Each value
is the average of measurements from four wells of cells ± S.E.
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Nitrotyrosine is a relatively specific and stable biochemical marker
for the generation of PN. The mechanism of action of PN decomposition
catalysts should favor a reduction in the nitrotyrosine content of
cellular proteins exposed to PN. When the level of protein-bound
nitrotyrosine following a PN pulse was measured by radioimmunoassay
analysis of cellular contents either released into the medium (Fig.
5) or remaining on cells (data not
shown), there was a strong correlation between nitrotyrosine level and the size of the PN pulse (Fig. 5A). In both cases,
the level of nitrotyrosine was reduced in a
concentration-dependent manner by the active compound
FeTPPS (Fig. 5B).

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Fig. 5.
Effect of FeTPPS on the cellular release of
nitrotyrosine-labeled proteins into the medium resulting from PN
treatment. A, increasing PN pulses result in larger
amounts of nitrotyrosine-labeled proteins released into the medium.
PhosphorImager units are expressed as AU (arbitrary units).
B, FeTPPS reduces the PN-mediated (200 µM)
release of nitrated cellular proteins into the medium as compared with
PN-treated control. Values are the average of two wells of cells ± S.D.
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Fig. 6 illustrates the difference in the
concentration-response profile for a PN catalyst compared with a
typical free radical scavenger such as ascorbate which reacts
stoichiometrically with the oxidant until its reactive moieties are
depleted. There was an essentially complete protection of RAW cells
from a 200 µM PN challenge in the presence of 10 µM FeTMPS. This is in sharp contrast to ascorbic acid
which was fully protective at a 2-fold molar excess to PN, and to TMPS
which was inactive at all concentrations tested. Nitrotyrosine content
of released intracellular proteins was used as a marker for the extent
of PN-mediated injury in these cells. FeTMPS reduced the level of
detectable protein-bound nitrotyrosine release (Fig.
6B).

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Fig. 6.
Comparison of the cytoprotective capacity of
the PN catalyst FeTMPS and the antioxidant ascorbate.
A, differential protection from PN-induced cellular injury
of RAW cells by the PN catalyst FeTMPS ( ) and the PN scavenger
ascorbate ( ) while TMPS ( ) was inactive. Values represent the
average of four wells ± S.E. B, FeTMPS ( ) and
ascorbate ( ) reduce the level of nitrated cellular proteins (NT)
released into the medium following PN treatment (200 µM).
Values are the average of measurements from two wells of cells ± S.D.
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There was no cytoprotection observed when FeCl3 was used or
when catalyst was added after PN application (data not shown). The free
ligands TPPS, TMPyP, and TMPS are inactive as decomposition catalysts
and were unable to protect from PN-mediated damage.
Cytokine-mediated Injury Is Attenuated by Treatment with PN
Decomposition Catalysts--
We next examined the cytoprotective
capacity of PN decomposition catalysts against endogenously produced
PN. The murine monocyte-macrophage cell line, RAW 264.7, can be
stimulated to express iNOS with endotoxin. Enzyme activity was
monitored by measuring nitrite accumulation in the conditioned medium.
As shown in Fig. 7B, the
induction of iNOS markedly reduced cell viability (
20%).
Inhibition of cellular iNOS activity with L-NMA or the
selective iNOS inhibitors aminoguanidine (data not shown) and
L-NIL (32, 39, 40) resulted in a
concentration-dependent increase in cellular viability
accompanied by a corresponding decrease in accumulated nitrite (Fig. 7)
when compared with the endotoxin control.

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Fig. 7.
L-NIL protects LPS-treated RAW
cells from loss of cellular viability. A,
L-NIL inhibition of nitrite production by LPS ( )
-treated RAW cells. Uninduced ( ) cells did not produce nitrite.
B, L-NIL protects cells from endotoxin-induced
injury ( ) while L-NIL treatment of uninduced cells
showed no apparent toxicity ( ). C, inverse relationship
of nitrite production with cellular viability as assessed by
mitochondrial reduction of Alamar Blue. Each value is the average of
determinations from four wells of cells ± S.E.
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As shown in Fig. 8, co-incubation of the
PN decomposition catalysts with endotoxin increased cellular viability
when examined 24 h after the beginning of treatment. Fig. 8
illustrates the cytoprotective profile for the active catalysts FeTMPS
(Fig. 8A) and FeTPPS (Fig. 8B). The free ligands
(i.e. TMPS, TPPS) afforded cytoprotection at much higher
concentrations than the active catalysts. To rule out metabolic
conversion of the free ligand to an active form, the inactive analog
ZnTMPS and the weak catalyst MnTPPS were tested (Fig. 8B).
Neither compound showed protective effects at the highest
concentrations applied.

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Fig. 8.
Effect of PN catalysts on LPS-mediated
cellular injury. A, RAW cells treated for 20 h
with LPS(10 µg/ml) were assayed for cell viability as described under
"Experimental Procedures." Cells were induced in the presence of
either FeTMPS ( ) or TMPS ( ). B, cells induced with LPS
were treated as follows: FeTPPS ( ), TPPS ( ), MnTPPS ( ), ZnTMPS
( ). Each determination is the average of values from eight wells of
cells ± S.E.
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We next extended our studies utilizing cell lines to primary cultures
of neurons and glia, cells known to be susceptible to cytokine-mediated
injury. In fact, cytokine-mediated injury accompanied by expression of
iNOS and cyclooxygenase-2 may contribute significantly to the neural
damage associated with human disorders such as stroke. Accordingly, we
examined the cytoprotection afforded by PN decomposition catalysts on
mixed cultures of dissociated hippocampal neurons and glia
(predominantly astrocytes) treated with a mixture of cytokines and
endotoxin. Cellular viability was assessed using several biochemical
criteria: for neurons, by counting phase bright trypan blue impermeant
cells exhibiting neuronal morphology and by measuring neuron-specific
enolase release from injured and dying cells; and for glia (comprising
the majority of the cells per well), by measuring the release of
lactate dehydrogenase from injured and dying cells or by assessing the
cellular capacity to convert Alamar Blue to its fluorescent product by
functional mitochondria. Primary cultures were exposed to cytokines and
endotoxin for 5-7 days at the end of which viability and NOS activity
were quantitated. Fig. 9 summarizes the
results that we obtained using FeTMPS. Although there was typically no
concentration-dependent decrease in accumulated nitrite and
nitrate by the cells (data not shown), there was a profound protection
of both neurons and glia by treatment with either FeTMPS (Fig. 9) or
FeTPPS (data not shown) when compared with the inactive analogue,
ZnTMPS. The pharmacological response profile differed between neurons
(Fig. 9, A and B) and glia (Fig. 9C),
with glial cells being more amenable to cytoprotection. Nevertheless,
PN catalyst protection was evident in either case. The only obvious
difference was morphological, with glial cells maintaining their
morphology while neurons largely withdrew their processes from the
glial feeder layer at the higher concentrations of catalyst used (10 and 20 µM). This result was in sharp contrast to
L-NIL treatment which preserved neuronal morphology
with a coincident drop in nitrite production (data not shown) and
neurotoxicity (Fig. 9).

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Fig. 9.
Effect of PN catalysts on cytokine-mediated
cellular injury in mixed cultures of rat hippocampal neurons and
glia. A, hippocampal neurons prepared from postnatal
day 1 rat pups were treated for 5 days with a cytokine/endotoxin
mixture as described under "Experimental Procedures." At the end of
this period, phase-bright cells excluding trypan blue were counted in
representative fields in each of four replicate wells for each
condition on the 96-well plate. 100% viability is equal to 490 cells/mm2 ± 49 (S.E.). The cytoprotective capacity of
FeTMPS ( ) and ZnTMPS ( ) was examined. B, release of
neuron-specific enolase from cytokine-treated cultures was examined
using L-NIL (100 µM), FeTMPS (5 µM), and ZnTMPS (5 µM). C,
metabolism of Alamar Blue was used to assess the mitochondrial
integrity of all cells in the mixed culture of neurons and glia.
Because of the absence of an anti-mitotic, the greater number of glia
contributed largely to the signal obtained. The results obtained with
L-NIL (100 µM), FeTMPS (5 µM),
and ZnTMPS (5 µM) are the average values from four wells
of cells ± S.E.
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Lack of Interaction between NO and PN Catalysts--
To rule out
the catalytic removal of NO by the PN catalysts as a possible
explanation for their cytoprotective action, we examined their effect
on guanylate cyclase-mediated relaxation of rat aortic rings. The
effect of FeTMPS and FeTMPyP on endothelium-dependent (endogenously produced NO) and -independent (exogenously added NO)
relaxation in rat aortic rings was examined. Acetylcholine-mediated relaxations are dependent upon the release of nitric oxide from the
endothelium while its removal eliminates the vasorelaxant effect of
this vasodilator (1). Fig.
10A demonstrates that
acetylcholine (1-100 nM) -mediated relaxation of
phenylephrine (0.1 µM) -preconstricted aortic rings was
not affected by preincubation with either FeTMPS or FeTMPyP (300 µM). In contrast, hemoglobin (5 µM), which
is known to bind to and completely inactivate NO, abolished the
acetylcholine-mediated relaxation. To further illustrate that
acetylcholine was producing its effects by stimulating NO generation,
the NOS inhibitor L-NAME (100 µM) was added
to the organ bath. Like hemoglobin, L-NAME completely
inhibited the relaxations.

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|
Fig. 10.
Lack of PN catalyst interaction with
NO. Acetylcholine-mediated relaxation of precontracted rat aortic
rings (top panel) was unaltered by the presence of either
FeTMPS or FeTMPyP while hemoglobin (Hb) and the NOS
inhibitor L-NAME were completely effective in abolishing
relaxation. A similar experiment using the NO donor SNP was conducted
on endothelium-denuded rings (bottom panel). PN
decomposition catalysts (300 µM) had no effect while
hemoglobin (5 µM) completely abolished the NO-mediated
relaxation.
|
|
The relaxation of precontracted endothelium-denuded aortic rings
following the exogenous addition of sodium nitroprusside (SNP), an NO
donor, at concentrations ranging from 1 nM to 1 µM was also unaffected by the presence of FeTMPS or
FeTMPyP (300 µM). In contrast to the catalysts,
hemoglobin (5 µM) eliminated the relaxation response of
the denuded rings (Fig. 10B). Thus, regardless of the source
of NO, PN catalysts produced no detectable inhibition of the guanylate
cyclase-mediated relaxation of vascular smooth muscle. Conversely,
because SOD and SOD-mimics clearly potentiate the activation of
guanylate cyclase by NO (1, 37), the lack of a PN catalyst-mediated
increase in the NO-driven relaxation response of aortic rings suggests
that any SOD mimetic activity associated with the catalysts is
neglibible.
 |
DISCUSSION |
A greater understanding of the role played by peroxynitrite in the
pathogenesis of human disease will aid in the design of rational
therapies for pharmacological intervention. Peroxynitrite is thought to
possess a dual free radical nature capable of hydroxyl radical-like
lipid peroxidation and NO2·-driven
nitration of tyrosine. Stability of PN is pH-dependent, with protonation resulting in either decomposition into predominantly nitrate or the initiation of oxidative processes including lipid peroxidation and the nitration of tyrosine. Thus, a compound capable of
diverting PN into a "non-destructive" pathway would increase nitrate while reducing free radical-initiated damage to critical molecular targets within the cell. The chemical synthesis and proposed
mechanism of action of such compounds have recently been described (28,
38) as has their efficacy in in vivo models of inflammation
(29).The purpose of this study was to more fully characterize the
cytoprotective properties of this novel class of compounds, the
peroxynitrite decomposition catalysts.
Inherent in the transient nature of many free radical species is the
difficulty in measuring their presence in more complex living systems.
While lipid peroxidation can result from Fenton/Haber-Weiss chemistry,
tyrosine nitration represents a stable biochemical marker, which with a
few noteworthy exceptions (19), is relatively specific for
peroxynitrite generation (4). We have presented evidence for a
reduction in protein nitrotyrosine content when synthetic PN is added
to BSA in the presence of the active catalyst FeTMPS; the inactive
compound TMPS, which lacks the iron center of active catalysts, did not
reduce PN-mediated tyrosine nitration, indicating that it is neither a
catalyst nor a scavenger of PN. To assess the activity of the catalysts
on a broader array of protein targets within a cellular context, human
adenocarcinoma cells (DLD-1) treated with PN showed positive
immunostaining for nitrotyrosine. As was observed with BSA, PN
decomposition catalysts caused a concentration-dependent
reduction in PN-generated nitrotyrosine staining of cellular
protein.
Because the physiological consequences of peroxynitrite generation are
associated with tissue damage, we next tested the utility of
peroxynitrite decomposition catalysts in preventing PN-mediated cellular injury and death. Peroxynitrite, the product of the
diffusion-limited reaction of superoxide anion with nitric oxide, has
been shown to participate in both apoptotic and necrotic cellular
injury (24-26). Diseases in which PN has been implicated include
ischemia-reperfusion injury, stroke, Alzheimer's disease, ALS,
multiple sclerosis, and rheumatoid arthritis (14-18). In fact, there
is compelling evidence for PN generation by cytokine and LPS-treated
cells. It includes analysis of predicted nitrite/nitrate ratios (20, 41, 42) as well as the direct measurement of nitrotyrosine formation by
HPLC/mass spectroscopy (43), the detection of catalase-insensitive but
superoxide dismutase/NOS inhibitor-sensitive 1,2,3-rhodamine formation
(21), and immunohistochemical staining of primary cultures of
cytokine-stimulated glia (22).
We have utilized the monocyte-macrophage line, RAW 264.7, both as a
target for exogenously added PN and as a cellular source of
endogenously produced PN. To determine the efficacy of PN catalysts, we
initially used a defined pulse of synthetic PN in our studies. FeTMPS,
FeTPPS, and FeTMPyP were cytoprotective at concentrations 30-50 fold
below the size (final concentration) of the PN challenge. The inactive
(free ligand) analogues did not protect. This protection depended on
the prior presence of catalyst and was not apparent with
FeCl3 alone. Perhaps most compelling was the difference
between concentration-response (cytoprotection) curves for FeTMPS and ascorbate. Ascorbate followed a scavenger activity profile
necessitating a 2-fold molar excess of the anti-oxidant over the size
of the PN pulse in order to be totally effective. This requirement for a molar excess of free radical scavenger (or reactive groups contained within such an antioxidant) is consistent with that observed for uric
acid and cysteine when exposed to PN (44, 45). PN-generated nitrotyrosine formation in cellular proteins was also reduced in a
concentration-dependent manner by PN catalyst, a result
comparing favorably with its EC50 value for cytoprotection
(approximately 5 µM for FeTPPS).
Endotoxin and cytokines have been used to induce iNOS and to produce a
sustained elevation of superoxide anion in cells of monocyte-macrophage
origin (20-22, 41). The resulting peroxynitrite formation was
correlated with a decrease in cellular viability as measured by
mitochondrial metabolism of MTT (21). In our study, RAW cells treated
for 24 h with LPS showed a greatly reduced capacity for converting
Alamar Blue to its fluorescent metabolite, an indicator of
mitochondrial oxidative metabolism. This, in turn, could be prevented
by NOS inhibition, suggesting NO-driven mitochondrial dysfunction, a
well documented effect on mitochondrial electron transport (10, 46).
Treatment of these cells with catalyst also resulted in cytoprotection
by this criterion, an observation consistent with
peroxynitrite-mediated cell injury. Uninduced cells incubated overnight
with catalyst did not show any significant loss in viability (data not
shown), although compound seemed to be taken up into cells as evidenced
by the visible reddish-brown tint of the cell layer after washing.
Neurons of the central nervous system, especially those of the
hippocampus, are very susceptible to cytokine-mediated damage especially in the context of the later stages of stroke (47-52). Glial
cells, on the other hand, seem to be more resistant to such injury
(11). In vitro generation of PN by cytokine-activated glia
has recently been demonstrated (22). Primary cultures of human
astrocytes stained positively for nitrotyrosine after several days of
treatment with cytokines. This staining correlated well with the
expression of iNOS (22). We decided to test the cytoprotective capacity
of PN decomposition catalysts on primary cultures of rat hippocampal
neurons and glia subjected to cytokine insult for 5-7 days. FeTMPS was
more cytoprotective than FeTPPS and compared favorably with
L-NIL, a selective iNOS inhibitor. We found the astrocytic
feeder layer more amenable to protection by these compounds when
compared with their neuronal counterparts. Accumulated levels of
nitrite were usually unaffected, suggesting that NOS inhibition is not
part of the mechanism of action of PN decomposition catalysts.
In summary, our data demonstrate the utility of PN decomposition
catalysts as cytoprotective agents against PN-mediated cellular injury
and death. Our data are also consistent with their proposed chemical
mechanism of action by revealing a measurable reduction in protein
nitrotyrosine content of PN-treated cells in culture. Moreover, the
preservation of cellular viability from PN-mediated damage is critical
to any consideration of the potential therapeutic value of PN
catalysts. Where peroxynitrite fits into the pathogenesis of diseases
such as Alzheimer's and multiple sclerosis and whether it is a
causative agent or merely a marker for a pathological faite accompli
remain very much open questions. Answers to questions such as these
will define the clinical utility of drugs capable of intercepting PN
and catalyzing its conversion into innocuous metabolites such as
nitrate.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Discovery Pharmacology,
Searle R&D, 800 N. Lindbergh Blvd., St. Louis, MO 63167. Tel.:
314-694-8954; Fax: 314-694-8949.
1
The abbreviations used are: iNOS, inducible
nitric-oxide synthase; FeTMPS,
5,10,15,20-tetrakis(2,4,6-trimethyl-3,3-disulfonatophenyl)porphyrinato iron (III); FeTPPS,
5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron (III);
FeTMPyP,
5,10,15,20-tetrakis(N-methyl-4
-pyridyl)porphyrinato iron
(III); PN, peroxynitrite; NO, nitric oxide; ALS, amyotrophic lateral
sclerosis; L-NMA, N-METHYL-L-arginine;
L-NAME, L-nitro-arginine-methyl ester;
L-NIL, N-iminoethyl-L-lysine; SOD,
superoxide dismutase; SNP, sodium nitroprusside; BSA, bovine serum
albumin; LPS, lipopolysaccharide.
2
Cross, A. H., Manning, P. T., Keeling, R. M., Schmidt, R. E., and Misko, T. P. (1998) J. Neuroimmunol., in press.
 |
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