Mechanistic Studies on AMD6221: A Ruthenium-Based Nitric Oxide Scavenger

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 ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. 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 stimulated RAW264 cells co-incubated with AMD6221, concomitant 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 nitric oxide synthase; ruthenium; septic shock.

Nitric oxide (NO) is an important mediator of many physiological and pathological processes (1). NO is produced 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, tetraAbbreviations used: nitric oxide (NO), lipopolysaccharide (LPS), interferon-␥ (IFN-␥), ethylenediamine tetraacetic acid (edta), diethylenetriamminepentaacetic acid (dtpa), L-N G-monomethyl-L-arginine (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-3phosphate dehydrogenase (G3PDH), thiazolyl blue (MTT), American Type Culture Collection (ATCC), hours (h), minutes (min), S-nitrosopenicillamine (SNAP), Actinomycin D (Act. D), cardiopulmonary bypass surgery (CBP). 1 To whom correspondence should be addressed. Fax: 604-5300976. E-mail: [email protected].

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 produced 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 immune 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, psoriasis and asthma (1). One therapeutic strategy for the intervention of NO-mediated disease is via the inhibition of NOS. In order to maintain the physiologically essential effects of NO, whilst attenuating the deleterious effects, isoform selective inhibitors are required and the identification of iNOS selective inhibitors has received much attention (4, 7–9). An alternative strategy is to scavenge or remove excess NO produced during pathological processes. To this end we have investigated a series of ruthenium(III) polyaminocarboxylate complexes as scavengers of NO (10). The polyaminocarboxylate ligand acts as a pentadentate ligand leaving one coordination site available for reaction with NO. Ruthenium(III) polyaminocarboxylate complexes 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 absorbance peak at 1897 cm ⫺1, characteristic of a linear Ru-(II)-NO bond, confirming the formation of a ruthenium(II) mononitrosyl (12).

519

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 macrophage cell line AMD1226, its aqua derivative AMD6245 [Ru(Hedta)(H 2O)] (12), and the dtpa analogue 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) polyaminocarboxylates 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 compounds 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).

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-Cl2H] ⫺. IR (CsI) ␯ (cm ⫺1) 1726 (CO 2H); 1667 (CO 2⫺).

MATERIALS AND METHODS

The cytotoxicity of L-NMMA, actinomycin D, AMD6221 and AMD3689 (Fig. 1) were determined in RAW264 cells using a modification of the MTT assay (12, 18).

FIG. 1.

Structure of (A) AMD6221 and (B) AMD3689.

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 precipitate 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

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 inhibitors 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 Lancaster and used without further purification. Potassium pentachlororuthenate, K 2[RuCl 5(OH 2)] 䡠 2H 2O was prepared according to literature 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 temperature. 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

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 bovine 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 compared 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

520

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(250 ␮M), actinomycin D (96.1 ␮M or 59.7 ␮M)). Nitric oxide production 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 measuring 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 detected 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); chymotrypsin (1 ␮g/mL) and aprotinin (5 ␮g/mL). The cells were lysed by

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 concentrations 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 processed as described above. The data was fitted to the MichaelisMenten 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 controls. 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 1100VWD2 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-

521

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Analysis of iNOS Transcription

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 prefiltered 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 coincubation 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 preliminary evidence that AMD6221 was capable of scavenging NO in a biological system.

In order to further investigate NO scavenging, larger 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 inhibitor 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 earlier observations. In separate experiments, AMD6221, AMD3689, NMMA and actinomycin D were shown to be non-cytotoxic by the MTT assay under the conditions of the experiment. After 18 h total RNA was isolated from these cultures 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 bromide (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. Figure 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). 522

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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 RAW264 cells induced for 18 h by Western blotting. Using a polyclonal iNOS antibody, a strong signal was 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) demonstrated that AMD6221 did not significantly affect iNOS protein levels. Moreover, in accordance with the results observed in Fig. 3, iNOS protein was not detected 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 a Vmax of 5 ⫻ 10 5 ⫾ 5.9 ⫻ 10 4 dpm/min L-citrulline

produced from a lysate from activated RAW264 cells. The Km is within the expected range of 0.1 ␮M to 100 ␮M as reported previously (25). Such a wide range of

FIG. 5. Km/Vmax determination of iNOS isolated from RAW264 cells under conditions of LPS/IFN-␥ (10 ␮g/mL/10 ng/mL).

523

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Identification of a Ruthenium-Nitrosyl Product in RAW264 Cell Culture

FIG. 6. Quantitation of 3H-L-citrulline produced from the reaction 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 preparation 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 presence of 5 mM EDTA resulted in no change in the enzyme activity. Thus, the activity observed and quantified 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 contrast, 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 preadjusted for total protein content prior to analysis.

Finally it was essential to positively confirm the production of the Ru-NO complex, AMD3689, after incubation 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 gradient system of acetonitrile and phosphate buffer as shown below in Figs. 8A and 8B. Under stimulated conditions, 100 ␮M AMD6221 was completely converted to AMD3689 as shown in Fig. 9A. By quantifying 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 filtration device that was used to remove serum components before HPLC injection. Taking this loss into consideration, 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 results 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 produced 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

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 compounds on iNOS, a crude preparation of iNOS isolated from RAW264 cells stimulated with LPS/IFN was incubated 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 inhibit the activity of iNOS in a direct competition assay.

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).

524

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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 corresponds 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.

Both down- and up-regulation of nitric oxide responses are involved in disparate disease states. Downregulation has been implicated in both essential and secondary hypertension (27). Increase in NO production 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 psoriasis 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 inhibitors 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 subsequently 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-

tion with NO. Chemical modification of the scavenger molecule can control distribution and pharmacokinetics. 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 promoted. This is in contrast to the NOS inhibitors which

526

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are independent of NO concentration and would therefore inhibit NO synthesis equally in regions of high and low NO synthesis (10). We have identified a class of compounds, ruthenium(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 ruthenium(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 authentic 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 compounds were subsequently shown to have pharmacological activity in a variety of biological models including: 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 LPSinduced hypotension in a rodent model (12) and a porcine model of septic shock (13), prolongation of graft survival in a rodent model of cardiac transplant rejection [Pieper, 2001 #13; Roza, 2001 #27], and reduction of tumour growth with concommitant reduction of tumour vasculature in a rodent tumour model (14). However though we had demonstrated that the molecules could scavenge NO in a “chemical” environment, we considered it essential to demonstrate that the observed 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 activity. Circumstantial evidence for NO scavenging as the mechanism was the lack of effect of the rutheniumnitrosyl of the dtpa complex AMD3689 to reduce nitrite accumulation in the cell culture supernatant of LPS/ IFN-␥ stimulated RAW264 macrophages. Initial attempts were made to demonstrate formation of the nitrosyl in the RAW264 cell culture medium after incubation with AMD6221 using IR spectroscopy to identify 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

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 investigated 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 activity, the two possible alternative mechanisms to NO scavenging. The final experiment was to demonstrate formation of the nitrosyl adduct AMD3689 after coincubation 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 complete stoichiometric conversion of AMD6221 to AMD3689 in the presence of stimulated NO-producing macrophages. This is compatible with the stoichiometry 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

527

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NO production) in the cell culture supernatant of RAW264 macrophages. AMD6221 did not affect translation of iNOS mRNA as shown by RT-PCR, neither did it inhibit iNOS protein expression as shown by Western 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 coincubated 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 enzyme activity. These data demonstrate that the ruthenium(III) polyaminocarboxylates can not only scavenge 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) polyaminocarboxylates 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 mediators (33) and proinflammatory cytokines and have been implicated as mediators of the myocardial dysfunction occurring after CPB (34). Inducible nitric oxide synthase was found to be elevated in the heart tissue and coronary artery in a canine model of CPB (35). Continuous intravenous administration of the NO scavenger AMD6221 in this canine model of CPB, whilst ameliorating some of the hemodynamic and inflammatory effects of cardiopulmonary bypass surgery, had no effect on the elevation of iNOS activity indicating that the pharmacological mechanism was not inhibiti on of iNOS (36). In another pharmacological model, administration of AMD6221 to allogeneic cardiac transplanted 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 demonstrate 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

their pharmacological effect by scavenging NO. Their low toxicity and activity in a variety of disease models indicates their potential as therapeutic agents for diseases where overproduction of nitric oxide has been implicated as a component of the disease pathophysiology. REFERENCES 1. Moncada, S., and Higgs, A. (1993) The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329, 2002–2012. 2. Marletta, M. A. (1993) Nitric oxide synthase structure and mechanism. J. Biol. Chem. 268, 12231–12234. 3. Knowles, R. G., and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249 –258. 4. Kerwin, J. F., Jr., Lancaster, J. R., Jr., and Feldman, P. L. (1995) Nitric oxide: A new paradigm for second messengers. J. Med. Chem. 38, 4343– 4362. 5. Szabo, C. (1995) Alterations in nitric oxide production in various forms of circulatory shock. New Horizons 3, 2–32. 6. Parratt, J. R. (1997) Nitric oxide. A key mediator in sepsis and endotoxaemia? J. Physiol. Pharmacol. 48, 493–506. 7. Marletta, M. A. (1994) Approaches toward selective inhibition of nitric oxide synthase. J. Med. Chem. 37, 1899 –1907. 8. Babu, B. R., and Griffith, O. W. (1998) Design of isoformselective inhibitors of nitric oxide synthase. Curr. Opin. Chem. Biol. 2, 491–500. 9. Mayer, B., and Andrew, P. (1998) Nitric oxide synthases: Catalytic function and progress towards selective inhibition. Naunyn Schmiedebergs Arch Pharmacol. 358, 127–133. 10. Fricker, S. P. (1999) Nitrogen Monoxide-Related Disease and Nitrogen Monoxide Scavengers as Potential Drugs, Dekker, New York. 11. Davies, N. A., Wilson, M. T., Slade, E., Fricker, S. P., Murrer, B. A., Powell, N. A., and Henderson, G. R. (1997) Kinetics of nitric oxide scavenging by ruthenium(III) polyaminocarboxylates: Novel therapeutic agents for septic shock. Chem. Commun. 47– 48. 12. Fricker, S. P., Slade, E., Powell, N. A., Vaughan, O. J., Henderson, G. R., Murrer, B. A., Megson, I. L., Bisland, S. K., and Flitney, F. W. (1997) Ruthenium complexes as nitric oxide scavengers: A potential therapeutic approach to nitric oxidemediated diseases. Br. J. Pharmacol. 122, 1441–1449. 13. Baggs, A. G., Fricker, S. P., Abrams, M., Lee, C., and Fink, M. P. (1997) A novel ruthenium-based nitric oxide scavenger ameliorates acute lung injury in porcine endotoxemia. Surg. Forum. 48, 84 – 86. 14. Pritchard, R., Flitney, F. W., Darkes, M. A., and Fricker, S. P. (1999) Ruthenium-based nitric oxide scavengers inhibit tumour growth by reducing tumour vasculature. Clin. Exp. Metast. 17, 776. 15. Pieper, G. M., Roza, A. M., Adams, M. B., Johnson, M., Hilton, G., Felix, C. C., and Fricker, S. P. (2001) A ruthenium(III) polyaminocarboxylate complex, a novel nitric oxide scavenger, enhances graft survival and decreases nitrosylated heme protein in models of acute and delayed cardiac transplant rejection. J. Heart Lung Transplant. 20, 157. 16. Pieper, G. M., Roza, A. M., Adams, M. B., Hilton, G., Johnson, M., Felix, C. C., Kampalath, B., Darkes, M., Wanggui, Y., Cameron, B., and Fricker, S. P. (2002) A ruthenium(III) polyaminocarboxylate complex, a novel nitric oxide scavenger, enhances graft survival and decreases nitrosylated heme protein in models of acute and delayed cardiac transplant rejection. J. Cardiovasc. Pharmacol., in press.

528

Vol. 292, No. 2, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

17. Mercer, E. E., and Buckley, R. R. (1965) Hexaaquoruthenium(III). Inorg. Chem. 4, 1692–1695. 18. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55– 63. 19. Raschke, W. C., Baird, S., Ralph, P., and Nakoinz, I. (1978) Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15, 261–267. 20. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. 21. Gilliam, M. B., Sherman, M. P., Griscavage, J. M., and Ignarro, L. J. (1993) A spectrophotometric assay for nitrate using NADPH oxidation by aspergillus nitrate reductase. Anal. Biochem. 212, 359 –365. 22. Schmidt, H. H. H., and Kelm, M. (1996) Determination of nitrite and nitrate by the Griess reaction, John Wiley and Sons, Chichester, UK. 23. Marzinzig, M., Nussler, A. K., Stadler, J., Marzinzig, E., Barthlen, W., Nussler, N. C., Beger, H. G., Morris, S. M., and Bruckner, U. B. (1997) Improved methods to measure end products of nitric oxide in biological fluids: Nitrite, nitrate, and s-nitrosothiols. Nitric Oxide 1, 177–189. 24. Han, B., Dubois, D. C., Boje, K. M. K., Free, S. J., and Almon, R. R. (1999) Quantification of iNOS mRNA with reverse transcription polymerase chain reaction directly from cell lysates. Nitric oxide Biol. Chem. 3, 281–291. 25. Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M. F., and Nathan, C. F. (1991) Purification and characterization of the cytokineinduced macrophage nitric oxide synthase: An FAD- and FMNcontaining flavoprotein. Proc. Natl. Acad. Sci. USA 88, 7773–7777. 26. Leatherbarrow, R. (1989) Grafit 4.0.12, Erithacus Software Ltd., Staines, UK. 27. Calver, A., Collier, J., and Vallance, P. (1993) Nitric oxide and cardiovascular control. Exp. Physiol. 78, 303–326. 28. Grover, R., Zaccardelli, D., Colici, G., Guntapalli, K., Watson, D., and Vincent, J.-L. (1999) An open label dose escalation study of

29.

30.

31.

32.

33.

34.

35.

36.

37.

529

the nitric oxide synthase inhibitor, NG-methyl-L-arginine hydrochloride (546C88), in patients with septic shock. Crit. Care Med. 27, 913–922. Grover, R., Lopez, A., Lorente, J., Bakker, J., Willatts, S., McLuckie, A., and Takala, J. (1999) Multi-center, randomized, placebo-controlled, double blind study of the nitric oxide synthase inhibitor 546C88: Effect on survival in patients with septic shock. Crit. Care Med. 27(Suppl. A), 33A. Cheshire, D. R. (2001) Use of nitric oxide synthase inhibitors for the treatment of inflammatory disease and pain. IDrugs 4, 795– 802. Cobb, J. P. (1999) Use of nitric oxide synthase inhibitors to treat septic shock: The light has changed from yellow to red. Crit. Care Med. 27, 855– 856. Cameron, B. R., Bridger, G. J., Davies, N. A., Fricker, S. P., Abrams, M. J., Rose, D. J., Wilson, M. T., and Zubieta, J. (1998) Ruthenium complexes as scavengers of nitric oxide. XXXIII International Conference on Coordination Chemistry. Downing, S. W., and Edmunds, L. H., Jr. (1992) Release of vasoactive substances during cardiopulmonary bypass. Ann. Thorac. Surg. 54, 1236 –1243. Gorson, J., Diana, P., Lee, J., Katz, W., and Hattler, B. (1994) Reversible diastolic dysfunction after successful coronary artery bypass surgery. Assessment by transesophageal doppler echocardiography. Chest 106, 1364 –1369. Mayers, I., Salas, E., Hurst, T., Johnson, D., and Radomski, M. W. (1999) Increased nitric oxide synthase activity after canine cardiopulmonary bypass is suppressed by s-nitrosoglutathione. J. Thorac. Cardiovasc. Surg. 117, 1009 –1016. Mayers, I., Hurst, T., Radomski, A., Johnson, D., Fricker, S. P., Bridger, G. J., Cameron, B., Darkes, M., and Radomski, M. W. (2002) Increased matrix metalloproteinase activity following canine cardiopulmonary bypass is suppressed by a nitric oxide scavenger. Submitted for publication. Yasuda, N., MacFarland, R., Darkes, M., and Fricker, S. P. (2000) Pharmacokinetics and tissue distribution of a rutheniumbased nitric oxide scavenger in the rat. 21st Annual Meeting of the American College of Toxicology.