Biochemical and Biophysical Research Communications 292, 519 –529 (2002)
doi:10.1006/bbrc.2002.6685, available online at http://www.idealibrary.com on
Mechanistic Studies on AMD6221: A Ruthenium-Based
Nitric Oxide Scavenger
R. Mosi, B. Seguin, B. Cameron, L. Amankwa, M. C. Darkes, and S. P. Fricker 1
AnorMED Inc., 200-20353 64th Avenue, Langley, British Columbia, Canada V2Y 1N5
Received February 25, 2002
Nitric oxide is a mediator of many disease states.
Previous studies have demonstrated that rutheni-
um(III) polyaminocarboxylates can react with NO to
form stable complexes reducing the levels of nitrite in
the culture medium of stimulated RAW264 macro-
phages and reverse the NO-mediated hypotension in
animal models of septic shock. It was necessary to
confirm that these observations were due to NO scav-
enging and not inhibition of the NO metabolic path-
way. Using RAW264 cells it was confirmed that
[Ru(H 3dtpa)(Cl)] (AMD6221) was neither acting at the
level of iNOS induction, nor as an inhibitor of iNOS by
measuring iNOS mRNA by RT-PCR and protein by
Western blot and enzyme activity. Using HPLC,
the nitrosyl adduct of reaction of AMD6221,
[Ru(H 2dtpa)NO], was identified in the medium of stim-
ulated RAW264 cells co-incubated with AMD6221, con-
comitant with a stoichiometric reduction in nitrite/
nitrate levels, thus confirming that the ruthenium(III)
polyaminocarboxylates exert their pharmacological
effect by scavenging NO. © 2002 Elsevier Science (USA)
Key Words: NO scavenger; nitric oxide; inducible ni-
tric oxide synthase; ruthenium; septic shock.
Nitric oxide (NO) is an important mediator of many
physiological and pathological processes (1). NO is pro-
duced by nitric oxide synthase (NOS) which catalyses a
five electron oxidation of L-arginine to L-citrulline and
NO (2, 3). The enzyme requires haem, NADPH, tetra-
Abbreviations used: nitric oxide (NO), lipopolysaccharide (LPS),
interferon-␥ (IFN-␥), ethylenediamine tetraacetic acid (edta), dieth-
ylenetriamminepentaacetic acid (dtpa), L-N G-monomethyl-L-argi-
nine (L-NMMA), reverse transcriptase polymerase chain reaction
(RT-PCR), inducible nitric oxide synthase (iNOS), Flavin adenine
dinuclotide (FAD), ␤-nicotinamide adenine dinucleotide phosphate,
reduced form (NADPH), lactic dehydrogenase (LDH), glucose-3-
phosphate dehydrogenase (G3PDH), thiazolyl blue (MTT), American
Type Culture Collection (ATCC), hours (h), minutes (min), S-nitro-
sopenicillamine (SNAP), Actinomycin D (Act. D), cardiopulmonary
bypass surgery (CBP).
1
To whom correspondence should be addressed. Fax: 604-530-
0976. E-mail: sfricker@anormed.com.
519
hydrobiopterin, and calmodulin as cofactors. NO is a
vasorelaxant produced by vascular endothelial cells
where its role is to control vascular tone. It is also a
neurotransmitter both in the peripheral and central
nervous system. In both these cases the NO is pro-
duced in low levels by constitutive, calcium regulated,
nitric oxide synthase enzymes known as NOS III
(eNOS or ecNOS), and NOS I (nNOS, or bNOS and
enNOS) respectively (4). NO plays a role in the im-
mune response and has been shown to be cytotoxic at
high concentrations towards bacteria, parasites and
tumour cells. In this instance, NO is produced by the
transcriptionally regulated, inducible NOS II, or iNOS.
Overproduction of NO, primarily by iNOS, has been
implicated in a wide variety of disease states including
septic shock (5, 6), and inflammatory diseases such as
rheumatoid arthritis, inflammatory bowel disease, pso-
riasis and asthma (1). One therapeutic strategy for the
intervention of NO-mediated disease is via the inhibi-
tion of NOS. In order to maintain the physiologically
essential effects of NO, whilst attenuating the delete-
rious effects, isoform selective inhibitors are required
and the identification of iNOS selective inhibitors has
received much attention (4, 7–9). An alternative strat-
egy is to scavenge or remove excess NO produced
during pathological processes. To this end we have
investigated a series of ruthenium(III) polyaminocar-
boxylate complexes as scavengers of NO (10). The poly-
aminocarboxylate ligand acts as a pentadentate ligand
leaving one coordination site available for reaction
with NO. Ruthenium(III) polyaminocarboxylate com-
plexes react rapidly with NO to form stable, inert
Ru(II) nitrosyls. We have shown using stopped-flow
techniques that AMD1226 (K[Ru(Hedta)Cl]) can bind
NO rapidly with a 1:1 stoichiometry and a second order
rate constant of 2 ⫻ 10 7 M ⫺1s ⫺1 at 7°C, pH 7.4 to form
a stable Ru(II) nitrosyl (11). The infra red spectrum of
the reaction product of AMD1226 and NO had an ab-
sorbance peak at 1897 cm ⫺1, characteristic of a linear
Ru-(II)-NO bond, confirming the formation of a ruthe-
nium(II) mononitrosyl (12).
0006-291X/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
Vol. 292, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
These compounds have been shown to have activity
in a variety of biological models. When co-incubated
with the LPS/interferon-␥ stimulated RAW264 macro-
phage cell line AMD1226, its aqua derivative
AMD6245 [Ru(Hedta)(H 2O)] (12), and the dtpa ana-
logue AMD6221 [Ru(H 3dtpa)Cl], were all able to lower
nitrite levels, a measure of NO, in the cell culture
supernatant. Activity has been demonstrated in in vivo
pharmacological models of disease including reversal
of endotoxin induced hypotension in animal models
(rat and porcine) and septic shock (12, 13). In addition,
an improvement in lung function was observed in the
porcine model. This observation is clinically significant
as it is the multiple organ dysfunction that is the
ultimate cause of death in this disease state. More
recently we have shown that ruthenium(III) polyami-
nocarboxylates can inhibit tumour progression of a rat
tumour (P22) by apparent reduction of NO-mediated
tumour vascularisation (14) and prolong graft survival
in a rodent model of cardiac allograft rejection (15, 16).
Though we have shown that chemically the com-
pounds can react with NO as hypothesized we have set
out to prove that the effects observed in biological
systems were due to bona fide NO scavenging rather
than inhibition of either iNOS induction or enzyme
inhibition. The mechanism of action of AMD6221 was
investigated using LPS/IFN-␥ stimulated RAW264
murine macrophage cells. In this paper we report that
AMD6221 neither inhibits transcription of iNOS
mRNA, nor inhibits expression of iNOS protein, nor
inhibits iNOS activity in this system. On the contrary
we demonstrate that the observed decrease in nitrite is
concomitant with the production of the ruthenium(II)
nitrosyl [Ru(H 2dtpa)NO] (AMD3689).
MATERIALS AND METHODS
Materials
LPS, IFN-␥, L-NMMA, FAD, NADPH, LDH, sodium pyruvate,
nitrate reductase, and goat anti-rabbit IgG conjugated to alkaline
phosphatase were from Sigma. Actinomycin D and protease inhibi-
tors were from Roche Molecular biochemicals. The centrifugal filter
devices were from Amicon Bioseparations. The rabbit polyclonal IgG
antibody specific for iNOS was from Chemicon Inc. The commercial
preparation of crude iNOS was from Calbiochem as was the iNOS
synthase assay kit. The [2,3- 3H]-L-arginine was from ICN. The
RAW264 cell line was obtained from the ATCC.
Diethylenetriamminepentaacetic acid was purchased from Lan-
caster and used without further purification. Potassium pentachlo-
roruthenate, K 2[RuCl 5(OH 2)] 䡠 2H 2O was prepared according to lit-
erature procedures (17).
Methods
Preparation of AMD6221 and AMD3689
AMD6221. Diethylenetriamminepentaacetic acid (142.0 g, 360
mmol) was dissolved in HCl (1 mM, 1 L) by heating to reflux tem-
perature. After 30 min the ligand was completely dissolved, at which
time K 2[RuCl 5(OH 2)] (135.0 g, 360 mmol) dissolved in HCl (1 mM, 1.2
520
FIG. 1. Structure of (A) AMD6221 and (B) AMD3689.
L) was added and stirred with the aid of a mechanical stirrer. The
reaction mixture was heated at reflux for 2 h (within 1 h the solution
had turned yellow and a precipitate began to form). The yellow
solution was filtered while hot and the collected precipitate was
washed with ice cold water, ethanol, and finally diethyl ether. The
yellow solid was dried in vacuo overnight (100.5 g, 51%). Anal. Calcd
for C 14H 21ClN 3O 10Ru 䡠 1.OH 2O: C, 30.80; H, 4.25; N, 7.70; Cl, 6.49.
Found: C, 30.68; H, 4.34; N, 7.70; Cl, 6.49. ES-MS m/z 491 [M-Cl-
2H] ⫺. IR (CsI) ␯ (cm ⫺1) 1726 (CO 2H); 1667 (CO 2⫺).
AMD3689. AMD6221 (20.0 g, 36.6 mmol) was added to a nitrogen
purged solution of H 2SO 4 (0.1 M, 400 mL). The reaction mixture was
stirred under a N 2 atmosphere with the aid of a mechanical stirrer.
Sodium nitrite (10.1 g, 146.5 mmol) was added to the solution and
the reaction mixture was heated to reflux. After 20 min the solution
turned a deep red colour. After 2 h the reaction mixture was removed
from the heat and cooled to room temperature. A light purple pre-
cipitate formed which was collected by filtration and washed with ice
cold water, ethanol and diethyl ether. The solid was then dried in
vacuo at room temperature (9.9 g, 49%). Anal. Calcd for
C 14H 20N 4O 11Ru 䡠 0.3H 2O: C, 31.92; H, 3.94; N, 10.64; Cl, 0. Found: C,
31.96; H, 3.92; N, 10.68; Cl, 0. ES-MS m/z 523 [M ⫹ H] ⫹. IR (CsI) ␯
(cm ⫺1) 3450 (H 2O); 1913 (NO); 1730 (CO 2H); 1680 (CO 2⫺).
Cytotoxicity Assay
The cytotoxicity of L-NMMA, actinomycin D, AMD6221 and
AMD3689 (Fig. 1) were determined in RAW264 cells using a modi-
fication of the MTT assay (12, 18).
Nitric Oxide Production by the RAW264 Macrophage
Cell Line Measured Using the Greiss Assay
The RAW264 murine macrophage cell line was maintained in
Eagles minimal essential medium supplemented with 10% fetal bo-
vine serum, 100 U/mL penicillin and 100 ␮g/mL streptomycin (19).
For the nitrite assay, cells were cultured on 24 well plates, 2 ⫻ 10 6
cells per well, in 2 mL of Eagle minimal essential medium without
phenol red. The cells were stimulated with 10 ␮g/mL LPS and 10
ng/mL IFN-␥. Nitric oxide production was measured after 18 h by
assaying for nitrite in the cell culture medium using the Greiss assay
(20); 1 mL aliquots of the cell culture medium were added to 2 mL of
the Greiss reagent (1 mL of 1% sulfanilamide in 5% phosphoric acid
followed by 1 mL of 0.1% naphthylethylenediamine dihydrochloride),
and absorbance was measured at 540 nm (12). Samples were com-
pared against a calibration curve prepared using nitrite standards.
In subsequent studies the RAW264 cells were cultured on 6 cm petri
plates, 3 ⫻ 10 6 cells per dish, in 2 mL of Eagle minimal essential
medium supplemented with 10% FBS in duplicate. The cells were
stimulated with 10 ␮g/mL LPS and 10 ng/mL IFN-␥ and incubated in
the presence of the appropriate compound at the following final
concentrations (AMD6221 (100 ␮M), AMD3689 (100 ␮M), L-NMMA
Vol. 292, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(250 ␮M), actinomycin D (96.1 ␮M or 59.7 ␮M)). Nitric oxide produc-
tion was measured after 6 or 18 h using the Greiss assay.
Measurement of Nitrate/Nitrite
Total nitrate and nitrite was measured by enzymatic reduction of
nitrate to nitrite followed by nitrite analysis using the Griess assay
to give a measure of total NO produced by the RAW264 cells. The
assay was adapted to a microtitre plate format as follows. Using 50
␮L of sample, 13 ␮L of solution A [nitrate reductase (1 U/mL), FAD
(14 ␮M) in 50 mM sodium phosphate buffer, pH 7.4] and 11.5 ␮L of
solution B [NADPH (1.3 mM) in 50 mM sodium phosphate buffer, pH
7.4] were added and the mixture was incubated at 25°C for 45 min in
the dark. A volume of 25 ␮L of solution C [LDH (55 U/ml), sodium
pyruvate (1.8 mM) in 50 mM sodium phosphate buffer, pH 7.4] was
added and the mixture was incubated a further 10 min at 25°C in the
dark. A volume of 200 ␮L of the Greiss reagent was added as
discussed above and the plate was read at 540 nm. The final nitrite
was quantified by comparison to a standard curve. By first measur-
ing the nitrite originally present before enzymatic conversion, the
amount of nitrate produced can be calculated (21–23).
RT-PCR Analysis
The 1 ⫻ 10 6 RAW264 cells were grown up for 6 or 18 h after
induction as discussed above and the total RNA was isolated using
the RNAeasy isolation kit (Qiagen) and resuspended in 50 ␮L of
diethyl-pyrocarbonate-treated water. The iNOS transcripts were de-
tected by reverse transcriptase polymerase chain reaction (RT-PCR)
using THERMOSCRIPT reverse transcriptase from GibcoBRL. Heat
denatured RNA (2 ␮g) was annealed to an oligo dT primer at 55°C for
45 min in 50 mM Tris acetate (pH 8.4), 75 mM potassium acetate, 8
mM magnesium acetate, 2 mM dNTPs, 40 U RNaseOUT, and 15 U
THERMOSCRIPT RT. A 2 ␮L aliquot of the resulting cDNAs was
directly amplified with 5 U Platinum Taq DNA polymerase, 1.87 mM
MgCl 2, 1 mM dNTPs, and 10 ␮M iNOS specific primers (24): sense 5⬘
GTC AAC TGC AAG AGA ACG GAG AAC 3⬘; antisense 5⬘ GAG CTC
CTC CAG AGG GTA GG 3⬘. Cycle conditions were followed according
to the procedure of Han et al. First cycle, 95°C for 2 min, 55°C for 1
min, and 72°C for 1 min; followed by 25 cycles of 95°C for 1 min, 55°C
for 1 min, and 72°C for 1 min and a final extension at 72°C for 7 min.
Duplicate RNA samples were amplified under the same conditions
with primers for glyceraldehyde phosphate dehydrogenase (G3PDH):
sense 5⬘ TGA AGG TCG GTG TGA ACG GAT TTG GC 3⬘; reverse 5⬘
CAT GTA GGC CAT GAG GTC CAC CAC 3⬘ to control the amount of
input RNA. The PCR products were resolved on a 1.2% agarose gel
and visualized by ethidium bromide staining.
Western Blot Analysis
Total RAW264 cell extract samples (⬃30 ␮g) were analyzed by
SDS–PAGE on a 7.5% gel. After transfer to a PVDF membrane, the
blots were probed with rabbit polyclonal IgG antibodies specific for
iNOS at 1/1000 dilution (v/v). Bound antibodies were detected with
1/5000 (v/v) goat anti-rabbit IgG conjugated to alkaline phosphatase.
The blots were visualised on a FluorChem Imager.
Preparation of Crude iNOS Lysates
RAW264 macrophages were stimulated with LPS/IFN-␥ (10 ␮g/
mL/10 ng/mL) and incubated either in the presence or absence of the
ruthenium compounds (AMD6221 (100 ␮M) and AMD3689 (100
␮M)), or actinomycin D (96.1 nM) or L-NMMA (250 ␮M)). After 18 h
the cells were harvested according to the method of Stuehr et al.,
1991 (25). Briefly, 3 ⫻ 10 6 cells were centrifuged and re-suspended in
cold PBS containing 25 mM glucose. Cells were re-pelleted and
re-suspended in cold H 2O containing pepstatin (5 ␮g/mL); chymo-
trypsin (1 ␮g/mL) and aprotinin (5 ␮g/mL). The cells were lysed by
521
three freeze-thaw cycles. The lysate was centrifuged at 100,000g for
90 min at 4°C. The supernatant was concentrated 10 fold using
Centricon centrifugal filter devices (molecular weight cut off 30
KDa).
iNOS Assay
The biochemical conversion of L- 3H-arginine to L- 3H-citrulline was
used to assay iNOS activity. The crude iNOS lysates prepared as
described above were incubated in the presence of 3H-arginine in 25
mM Tris–HCl buffer pH 7.4 containing 3 ␮M tetrahydrobiopterin, 1
␮M FAD, 1 ␮M FMN, 0.125 mM NADPH for 30 min at 37°C (total
reaction volume was 40 ␮L). The amount of L- 3H-arginine added per
40 ␮L reaction was 2.52 ⫻ 10 6 dpm or 0.6 ␮M. After incubation, the
reactions were stopped by the addition of 400 ␮L of 50 mM HEPES
buffer, pH 5.5 containing 5 mM EDTA. An aliquot of 100 ␮L of
equilibrated resin was added and the entire sample was transferred
to spin cups for separation of the L-citrulline from unreacted
L-arginine as provided by the Calbiochem nitric oxide synthase kit.
The flow through was quantified for the amount of L- 3H-citrulline
produced. The resin was further treated with 400 ␮L of 0.5 M NH 4Cl
in order to release and quantify the unreacted arginine. In both
cases, the entire volume of each sample (⬇440 ␮L ⫹ 3.5 mL Ultima
Gold scintillation cocktail (Packard)) was counted in a LKB 1209
RackBeta liquid scintillation counter.
A Km/Vmax determination was completed for the iNOS isolated
from RAW264 cells induced with LPS/IFN-␥. A range of concentra-
tions of 3H-L-arginine from 0.06 ␮M to 6 ␮M was used and the
reaction was allowed to proceed for 30 min at 37°C and then pro-
cessed as described above. The data was fitted to the Michaelis-
Menten equation using the software Grafit 4.0.12 (26).
The percentage of iNOS activity inhibited by the presence of
AMD6221, AMD3689 or L-NMMA was determined by incubating
enzyme and compound in the presence of buffer as described above
for 30 min at 37°C and then quantifying enzyme activity by the
conversion of L-arginine to L-citrulline. The IC 50 of the inhibitor was
measured where appropriate.
HPLC Analysis of the Products of the Reaction
of 6221 and 3689 in the Presence
of Induced RAW264 Cells
The quantity of AMD6221 and AMD3689 was measured using
HPLC analysis after induction of RAW264 cells. The cell culture
conditions were the same as described above. Namely, the RAW264
cells were cultured on 6 cm petri plates, 3 ⫻ 10 6 cells per dish, in 2
mL of Eagle minimal essential medium supplemented with 10% FBS
in duplicate and incubated for 48 h. The cells were stimulated with
10 ␮g/mL LPS and 10 ng/mL IFN-␥ and incubated in the presence of
the appropriate compound at the following final concentrations
(AMD6221 (100 ␮M) and AMD3689 (100 ␮M)). Unstimulated cells
were incubated in the presence of AMD6221 or AMD3689 as con-
trols. Media alone (not containing cells) was also incubated with
AMD6221 or AMD3689 or the two together ⫾ LPS-IFN-␥. Nitric
oxide production was measured after 18 h by measuring total
nitrate/nitrite using the Greiss assay as described above. Samples
for HPLC were also collected after 18 h and measured directly.
The HPLC instrumentation used was a Hewlett-Packard HP 1100-
VWD2 equipped with an Eclipse XDB C8 column (150 mm ⫻ 4.6 mm,
3.5 ␮m, 100 A). A two component mobile phase of A (80% 10 mM
KH 2PO 4, 0.05% Nonylamine, pH 6.6 and 20% acetonitrile) and B
(100% acetonitrile) was used in the following gradient set-up: 100%
A from 0 to 8 min, 100% A to 60% A from 8 to 20 min, 60% A from 20
to 25 min and 60% A to 100% A from 25 to 26 min. An ambient
temperature of 25°C was maintained on the column. Standards and
samples were analyzed in duplicate. Quantitation of AMD6221 and
AMD3689 in the test samples was performed by comparison of the
areas of AMD6221 and AMD3689 peaks in each sample chromato-
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Analysis of iNOS Transcription

In order to further investigate NO scavenging, larger

FIG. 2. Quantification of NO produced from RAW264 cells under
various conditions after 6 or 18 h of incubation as measured by the
amount of NO 2⫺ using the Greiss assay. (1) LPS/IFN-␥ (10 ␮g/mL/10
ng/mL), (2) LPS/IFN-␥ ⫹ AMD6221 (100 ␮M), (3) LPS/IFN-␥ ⫹
AMD3689 (100 ␮M), (4) LPS/IFN ⫹ L-NMMA (250 ␮M), (5) LPS/
IFN-␥ ⫹ Act. D (96 ␮M), (6) LPS/IFN-␥ ⫹ Act. D (59.7 nM), (7)
untreated control.
gram to that of the corresponding peak areas in chromatograms of
standard solutions of AMD6221 and AMD3689. Samples were pre-
filtered with a 22 ␮m filter in order to remove serum particulates
before injection onto the HPLC.
RESULTS
Nitric Oxide Production by Activated RAW264
Macrophages
In a preliminary experiment RAW264 macrophages
cultured on 24 well plates were activated by LPS and
IFN-␥ as described under Materials and Methods and
co-incubated with either 250 ␮M L-NMMA, or 100 ␮M
AMD6221 or AMD3689. Both L-NMMA and AMD6221
reduced nitrite levels from 50.2 ⫾ 1.5 ␮M to 13.0 ⫾ 0.8
␮M and 12.6 ⫾ 0.5 ␮M respectively, whereas co-
incubation with the nitrosyl adduct AMD3689 had no
effect on nitrite levels (48.8 ⫾ 0.9 ␮M). All compounds
were shown, using the MTT assay, to be non-cytotoxic
in separate experiments. These results provided pre-
liminary evidence that AMD6221 was capable of scav-
enging NO in a biological system.
scale cultures of RAW264 cells were plated on 6 cm
tissue culture dishes. Under these conditions typically
concentrations over 80 ␮M nitrite were obtained in the
culture medium from LPS/IFN-␥ stimulated RAW264
cells after 18 h. The nitric oxide synthase inhibitor
L-NMMA inhibited nitrite production by 55% percent
at 250 ␮M (Fig. 2). Addition of the transcription inhib-
itor actinomycin D at concentrations of either 59.7 nM
or 96.1 nM decreased nitrite production by 41.3% and
70%, respectively. The addition of 100 ␮M of AMD6221
resulted in a decrease of final nitrite production by
42.8% while the control compound, AMD3689, did not
significantly decrease the production of nitrite from the
level of the induced control sample confirming the ear-
lier observations. In separate experiments, AMD6221,
AMD3689, NMMA and actinomycin D were shown to
be non-cytotoxic by the MTT assay under the condi-
tions of the experiment.
After 18 h total RNA was isolated from these cul-
tures and analysed by RT-PCR in the presence of iNOS
specific primers. A 445 bp iNOS amplicon was readily
observed in an agarose gel stained with ethidium bro-
mide (Fig. 3). No significant changes in band intensity
were visible between treated and untreated samples.
Further analysis by densitometry (data not shown)
clearly demonstrated that iNOS mRNA expression was
not altered when induced RAW264 cells were treated
with AMD6221. To confirm the RNA input was the
same in all samples, G3PDH was also amplified. Fig-
ure 3 shows that G3PDH mRNA was expressed at
similar levels in all samples while iNOS message was
not present in uninduced samples or samples treated
with actinomycin D.
Expression of iNOS Protein
To further support the role of AMD6221 as a nitric
oxide scavenger rather than an inhibitor of translation,

FIG. 3. Ethidium bromide stained 1.2% agarose gel of 445 bp iNOS RT-PCR product. The mRNA from RAW264 cells was reverse
transcribed according to the procedure of GibcoBRL THERMOSCRIPT RT-PCR System obtained from the following conditions: (1)
LPS/IFN-␥ (10 ␮g/mL/10 ng/mL), (2) LPS/IFN-␥ ⫹ AMD6221 (100 ␮M), (3) LPS/IFN-␥ ⫹ AMD3689 (100 ␮M), (4) LPS/IFN-␥ ⫹ L-NMMA (250
␮M), (5) LPS/IFN-␥ ⫹ Act. D (96.1 nM), (6) untreated control, (7) no RNA input (-ctrl).

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FIG. 4. (A) Western blot of iNOS protein isolated from RAW264 cells. The replicate 7.5% PAGE/SDS gel was transferred onto a PVDF
membrane and probed using a rabbit polyclonal anti-iNOS antibody followed by detection with an alkaline-phosphatase conjugated
secondary antibody. (B) Coomassie Blue stain of 7.5% PAGE/SDS gel of crude lysate. 3 ⫻ 10 6 RAW264 macrophages were induced with
LPS/IFN-␥. Approximately 30 ␮g of lysate was loaded onto duplicate 7.5% PAGE/SDS gels. The first gel was stained with Coomassie Blue.
(1) LPS/IFN-␥ (10 ␮g/mL/10 ng/mL), (2) LPS/IFN-␥ ⫹ AMD6221 (100 ␮M), (3) LPS/IFN-␥ ⫹ AMD3689 (100 ␮M), (4) LPS/IFN-␥ ⫹ L-NMMA
(250 ␮M), (5) LPS/IFN-␥ ⫹ Act. D (96.1 nM), (6) untreated control, (7) ⬃30 ␮g of commercial iNOS crude lysate (Calbiochem) [positive control
for blot].

we analysed crude protein lysates prepared from produced from a lysate from activated RAW264 cells.
RAW264 cells induced for 18 h by Western blotting. The Km is within the expected range of 0.1 ␮M to 100
Using a polyclonal iNOS antibody, a strong signal was ␮M as reported previously (25). Such a wide range of
detected which corresponded to the reported molecular
weight of iNOS (130,000 Da) (Fig. 4A). As a further
control, a commercial preparation of iNOS extract
(⬃30 ␮g) displayed the same electrophoretic mobility
as our crude iNOS preparations. As expected, results
in Fig. 4B and densitometry analysis (not shown) dem-
onstrated that AMD6221 did not significantly affect
iNOS protein levels. Moreover, in accordance with the
results observed in Fig. 3, iNOS protein was not de-
tected in uninduced samples or upon treatment with
actinomycin D.
In order to further demonstrate that AMD6221 was
not inhibiting translation to enzyme product, crude
lysates of activated RAW264 cells co-incubated with
AMD6221, AMD3689, L-NMMA and actinomycin D
were assayed for iNOS activity. As shown in Fig. 5, a
Km for NOS activity of 4.9 ⫾ 1.0 ␮M was obtained with FIG. 5. Km/Vmax determination of iNOS isolated from RAW264
a Vmax of 5 ⫻ 10 5 ⫾ 5.9 ⫻ 10 4 dpm/min L-citrulline cells under conditions of LPS/IFN-␥ (10 ␮g/mL/10 ng/mL).

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FIG. 6. Quantitation of 3H-L-citrulline produced from the reac-
tion of iNOS and L-arginine (1 ␮Ci/reaction). iNOS was isolated from
RAW264 cells grown under the following conditions: (1) LPS/IFN (10
␮g/mL/10 ng/mL), (2) LPS/IFN-␥ ⫹ AMD6221 (100 ␮M), (3) LPS/
IFN-␥ ⫹ AMD3689 (100 ␮M), (4) LPS/IFN-␥ ⫹ L-NMMA (250 ␮M),
(5) LPS/IFN-␥ ⫹ Act. D (96.1 nM), (6) untreated control.
substrate affinities appears to be related to the prepa-
ration and the source of iNOS. It is unlikely that this
activity is due to either nNOS or eNOS since both
require calcium for proper function and calcium was
not added to our assay solutions. In addition, the pres-
ence of 5 mM EDTA resulted in no change in the
enzyme activity. Thus, the activity observed and quan-
tified was exclusively attributed to iNOS.
There was no difference in the activity of crude iNOS
regardless of whether it was isolated from RAW264
cells stimulated with LPS/IFN-␥ in the presence of
AMD6221 or AMD3689. This was measured by the
amount of L- 3H-arginine converted to L- 3H-citrulline
for each sample compared to that of a control sample of
iNOS isolated from stimulated cells (Fig. 6). In con-
trast, the percent activity for iNOS isolated from
RAW264 cells in the presence of L-NMMA was 44%
while iNOS isolated from RAW264 cells incubated in
the presence of actinomycin D (96.1 nM) was 12.9%
compared to the control. The background activity of
iNOS from uninduced control RAW264 cells was 19%
of that from stimulated cells. All samples were pre-
adjusted for total protein content prior to analysis.
Inhibition of iNOS
The above results also suggest that the ruthenium
compounds do not inhibit iNOS activity. To further
investigate the potential inhibitory effect of the com-
pounds on iNOS, a crude preparation of iNOS isolated
from RAW264 cells stimulated with LPS/IFN was in-
cubated directly with either AMD6221 or AMD3689 at
concentrations of 0.1 mM and 1 mM in the enzyme
assay mix. No inhibition of iNOS activity was noted
(Fig. 7) therefore, neither AMD6221 nor AMD3689 in-
hibit the activity of iNOS in a direct competition assay.
524
Identification of a Ruthenium-Nitrosyl Product
in RAW264 Cell Culture
Finally it was essential to positively confirm the
production of the Ru-NO complex, AMD3689, after in-
cubation of AMD6221 in a culture of NO producing
RAW264 cells. Using a C8 column, it was possible to
clearly separate AMD6221 from AMD3689 using a gra-
dient system of acetonitrile and phosphate buffer as
shown below in Figs. 8A and 8B. Under stimulated
conditions, 100 ␮M AMD6221 was completely con-
verted to AMD3689 as shown in Fig. 9A. By quantify-
ing the peak areas as described in the Materials and
Methods section, it was determined that 100 ␮M of
AMD6221 was converted to 83.85 ␮M (83%) AMD3689.
In contrast, AMD3689 remained unchanged in the cell
supernatant even after 18 h of incubation (Fig. 9B).
From the initial amount of AMD3689 (100 ␮M), a total
of 87.81 ␮M (87%) was quantified in the final solution.
The compound loss (13%) can be attributed to the fil-
tration device that was used to remove serum compo-
nents before HPLC injection. Taking this loss into con-
sideration, the conversion of AMD6221 to AMD3689 is
approximately 96%. AMD6221 and AMD3689 remain
unchanged when incubated in media alone or in the
presence of RAW264 cells in the absence of stimulation
(data not shown). Furthermore, by combining the re-
sults from a nitrite/nitrate assay, a quantification can
be made of the total NO produced in solution under the
induction conditions used assuming that total NO pro-
duced equals nitrate plus nitrite plus any scavenged by
AMD compound added to the media. The total nitrate/
nitrite measured in the LPS/IFN stimulated control
cells was 164 ␮M. In the presence of 100 ␮M AMD3689,
a total of 168 ␮M nitrate/nitrite was measured. In
contrast, in the presence of AMD6221, only 87 ␮M of
nitrate/nitrite was measured in the combined assay.
The amount of nitrosyl, AMD3689, produced under
these conditions (83.4 ␮M), indicates that a total of 171
␮M of NO was produced (83.4 ␮M scavenged by
FIG. 7. Activity of iNOS incubated in the presence of various
compounds: (1) iNOS control, (2) iNOS ⫹ L-NMMA (250 ␮M), (3)
iNOS ⫹ AMD6221 (1 mM), (4) iNOS ⫹ AMD6221 (100 ␮M), (5)
iNOS ⫹ AMD3689 (1 mM), (6) iNOS ⫹ AMD3689 (100 ␮M).
Vol. 292, No. 2, 2002
FIG. 8. (A) HPLC chromatogram of unstimulated cells with 100 ␮M AMD6221; (B) HPLC chromatogram of unstimulated cells with 100
␮M AMD3689.
AMD6221 to form AMD3689 and 87 ␮M remaining in
the media and measured as nitrate/nitrite) which cor-
responds well to the total NO produced in the control.
DISCUSSION
It is not surprising that, because of the ubiquitous
and essential nature of nitric oxide, perturbation of NO
metabolism and the L-arginine/NO/cGMP metabolic
pathway has been implicated as a contributory factor
to the pathophysiology of a number of disease states.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Both down- and up-regulation of nitric oxide responses
are involved in disparate disease states. Down-
regulation has been implicated in both essential and
secondary hypertension (27). Increase in NO produc-
tion leading to excess levels of NO has been shown to
play a role in diseases such as septic shock, rheumatoid
arthritis, inflammatory bowel disease, diabetes psori-
asis and asthma (1). This overproduction of NO has
been attributed to the inducible nitric oxide synthase.
One therapeutic strategy is to utilize selective NOS
inhibitors which preferentially inhibit iNOS (4, 7–9).
525
Vol. 292, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 9. (A) HPLC chromatogram of stimulated cells (⫹LPS/IFN-␥) with AMD6221; (B) HPLC chromatogram of stimulated cells
(⫹LPS/IFN-␥) with AMD3689.

Intense effort has been put in to this area. Numerous
selective inhibitors have been identified, and NOS in-
hibitors have been tested in the clinic (28 –30) but as
yet a NOS inhibitor has not been approved for a clinical
indication (31).
We have adopted an alternative approach which is to
scavenge excess nitric oxide with a metal compound
which can tightly bind the NO and then be subse-
quently excreted. The selectivity of scavengers for the
nitric oxide responsible for causing pathological effects
is not based on specificity for a particular enzyme, but
rather on compartmental localisation and rate of reac-
526
tion with NO. Chemical modification of the scavenger
molecule can control distribution and pharmacokinet-
ics. A large molecule and/or a hydrophilic molecule
would be unable to cross cell membranes and would
therefore be restricted to extracellular compartments
such as the blood, and by extravasation, interstitial
fluids. The rate of NO scavenging, assuming a second
order process, would also be dependent upon both the
concentration of nitric oxide and the scavenger. This
means that when NO concentrations are elevated, as in
a number of disease states, scavenging would be pro-
moted. This is in contrast to the NOS inhibitors which
Vol. 292, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are independent of NO concentration and would there-
fore inhibit NO synthesis equally in regions of high and
low NO synthesis (10).
We have identified a class of compounds, rutheni-
um(III) polyaminocarboxylates, that can bind NO
rapidly, forming stable metal-nitrosyl complexes. We
have previously demonstrated using K[Ru(Hedta)Cl)]
(AMD1226), where the polyaminocarboxylate ligand is
ethylenediamine tetraacetic acid (EDTA), that the ru-
thenium(III) centre can be reduced upon reaction with
either authentic NO, or NO derived from a NO donor
molecule like S-nitrosopenicillamine (SNAP), to Ru(II)
with the formation of a linear Ru-NO bond. This has
been confirmed by IR spectroscopy of the product which
has a spectrum with a peak at 1897 cm ⫺1 characteristic
of a linear Ru(II)-NO bond (12). Stopped-flow analysis of
the reaction of the EDTA complexes K[Ru(Hedta)Cl)]
(AMD1226), [Ru(Hedta)H 2O] (AMD6245) with authen-
tic NO has demonstrated the rapid reaction with NO
with second order rate constants of 2–3 ⫻ 10 ⫺7 M ⫺1s ⫺1
at 8°C (11) and for [Ru(H 3dtpa)Cl] (AMD6221) of 2.5 ⫻
10 5 M ⫺1s ⫺1 (32) with the formation of a 1:1 adduct
between the NO and the Ru(III) complex. These com-
pounds were subsequently shown to have pharmaco-
logical activity in a variety of biological models includ-
ing: reduction of nitrite accumulation in the cell
culture supernatant of LPS/IFN-␥ stimulated RAW264
macrophages, attenuation of isolated rat tail artery
relaxation in response to SNAP, reversal of LPS-
induced hypotension in a rodent model (12) and a por-
cine model of septic shock (13), prolongation of graft
survival in a rodent model of cardiac transplant rejec-
tion [Pieper, 2001 #13; Roza, 2001 #27], and reduction
of tumour growth with concommitant reduction of tu-
mour vasculature in a rodent tumour model (14). How-
ever though we had demonstrated that the molecules
could scavenge NO in a “chemical” environment, we
considered it essential to demonstrate that the ob-
served pharmacological activity of these molecules was
unequivocally due to their acting as NO scavengers in
a biological environment.
Alternative mechanisms that could produce the
same pharmacological effects are either inhibition of
induction of iNOS, or inhibition of NOS enzyme activ-
ity. Circumstantial evidence for NO scavenging as the
mechanism was the lack of effect of the ruthenium-
nitrosyl of the dtpa complex AMD3689 to reduce nitrite
accumulation in the cell culture supernatant of LPS/
IFN-␥ stimulated RAW264 macrophages. Initial at-
tempts were made to demonstrate formation of the
nitrosyl in the RAW264 cell culture medium after in-
cubation with AMD6221 using IR spectroscopy to iden-
tify the presence of the characteristic Ru(II)-NO peak.
These studies were able to give an indication of the
formation of the Ru(II)-NO bond (data not shown) but
owing to the poor sensitivity of the method this did not
give the unequivocal demonstration of NO scavenging
527
that we sought. We therefore embarked upon a series
of experiments to examine the effect of [Ru(H 3dtpa)Cl]
(AMD6221) on induction of iNOS, inhibition of NOS
enzyme activity, and identification of the formation of
the corresponding nitrosyl complex [Ru(H 2dtpa)NO]
(AMD3689) using HPLC.
The mechanistic studies were carried out using the
well characterised LPS/IFN-␥ RAW264 macrophage
cell culture system. Induction of iNOS was investi-
gated by assaying for iNOS mRNA using RT-PCR, and
iNOS protein expression by Western blot and enzyme
activity. The RAW264 macrophages were co-incubated
with LPS/IFN-␥ and either AMD6221 or actinomycin D
for 18 h. Under these conditions nitrite accumulation
was decreased in the cell culture supernatant of both
AMD6221 and actinomycin D treated cells. Incubation
with AMD6221 however had no effect on either iNOS
mRNA production, or iNOS protein expression,
whereas the control compound actinomycin D reduced
both iNOS mRNA transcription and subsequent iNOS
protein expression.
A crude preparation of iNOS was isolated from LPS/
IFN-␥ stimulated RAW264 cells and enzyme activity
assayed by monitoring the conversion of 3H-L-arginine
to 3H-L-citrulline. The non-specific NOS inhibitor
L-NMMA, an arginine analogue, inhibited the activity
of the enzyme preparation with complete inhibition at
250 ␮M L-NMMA, and had an IC 50 of 1.0 ␮M under the
conditions described under Materials and Methods. On
the contrary neither AMD6221 nor AMD3689 had an
effect on enzyme activity at concentrations of either 0.1
mM or 1 mM.
The above data strongly indicates that the AMD6221
does not inhibit either induction of iNOS or iNOS ac-
tivity, the two possible alternative mechanisms to NO
scavenging. The final experiment was to demonstrate
formation of the nitrosyl adduct AMD3689 after co-
incubation of AMD6221 with stimulated RAW264
cells. RAW264 cells were incubated with AMD6221
either with or without the LPS/IFN-␥ mix. The cell
culture supernatant was assayed by HPLC for the
presence of AMD6221 and AMD3689 after 18 h. The
major peak identified in the cell culture supernatant of
unstimulated RAW264 cells was AMD6221, whereas
conversely the major peak identified in the cell culture
supernatant from stimulated RAW264 cells was the
nitrosyl adduct AMD3689. The formation of AMD3689
was concomitant with a quantitative reduction in
nitrite/nitrate concentration, indicating almost com-
plete stoichiometric conversion of AMD6221 to
AMD3689 in the presence of stimulated NO-producing
macrophages. This is compatible with the stoichiome-
try of NO scavenging predicted by the chemical studies
(11).
The data presented here therefore shows that the
ruthenium(III) polyaminocarboxylate [Ru(H 3dtpa)Cl]
AMD6221 reduced nitrite accumulation (a measure of
Vol. 292, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NO production) in the cell culture supernatant of
RAW264 macrophages. AMD6221 did not affect trans-
lation of iNOS mRNA as shown by RT-PCR, neither did
it inhibit iNOS protein expression as shown by West-
ern blot or enzyme activity assay. In addition,
AMD6221 was unable to directly inhibit iNOS activity
as shown by inhibition studies with iNOS prepared
from stimulated RAW264 cells. The formation of the
nitrosyl adduct of AMD6221, AMD3689, in the cell
culture medium from stimulated RAW264 cells co-
incubated with AMD6221 was demonstrated by HPLC.
These results therefore provide unequivocal evidence
that the ruthenium(III) polyaminocarboxylate AMD6221
[Ru(H 3dtpa)Cl] acts to reduce nitrite accumulation
in RAW264 cell culture by scavenging NO, and not
by inhibition of either iNOS induction or iNOS en-
zyme activity. These data demonstrate that the ru-
thenium(III) polyaminocarboxylates can not only scav-
enge NO in a “chemical” system but also in a more
complex biological milieu.
Recent data goes further to confirm that this is also
the mechanism by which the ruthenium(III) polyami-
nocarboxylates exert their pharmacological effect in
vivo. Cardiopulmonary bypass surgery (CBP), which
can be considered as a form of inflammatory injury, has
been associated with release of inflammatory media-
tors (33) and proinflammatory cytokines and have been
implicated as mediators of the myocardial dysfunction
occurring after CPB (34). Inducible nitric oxide syn-
thase was found to be elevated in the heart tissue and
coronary artery in a canine model of CPB (35). Contin-
uous intravenous administration of the NO scavenger
AMD6221 in this canine model of CPB, whilst amelio-
rating some of the hemodynamic and inflammatory
effects of cardiopulmonary bypass surgery, had no ef-
fect on the elevation of iNOS activity indicating that
the pharmacological mechanism was not inhibiti on of
iNOS (36). In another pharmacological model, admin-
istration of AMD6221 to allogeneic cardiac trans-
planted rats was shown to prolong the lifetime of graft
survival. There was also a reduction in heme-nitrosyl
formation at post-operative day 6 in treated animals as
shown by e.p.r. and a decrease in plasma nitrite levels
immediately post-treatment. In addition, formation of
the ruthenium nitrosyl adduct AMD3689 in the plasma
of treated animals was demonstrated by HPLC [Pieper,
2001 #13; Roza, 2001 #27]. The data from these in vivo
pharmacological models of disease further demon-
strate that the ruthenium-based compounds do not
affect either induction or activity of iNOS but lower NO
levels by scavenging NO with concommitant formation
of the stable ruthenium nitrosyl. We have also shown
that AMD6221 is not only well tolerated in vivo but is
also rapidly cleared from plasma and excreted in the
urine (37).
These data therefore present conclusive evidence
that the ruthenium(III) polyaminocarboxylates exert
528
their pharmacological effect by scavenging NO. Their
low toxicity and activity in a variety of disease models
indicates their potential as therapeutic agents for dis-
eases where overproduction of nitric oxide has been
implicated as a component of the disease pathophysi-
ology.
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