Catalase protects cardiomyocytes via its inhibition of nitric oxide synthesis

Nitric Oxide 14 (2006) 189–199 www.elsevier.com/locate/yniox

Catalase protects cardiomyocytes via its inhibition of nitric oxide synthesis Han-Jung Chae b,1, Ki-Chan Ha b,1, Do-Sung Kim b, Gi-Seup Cheung a, Yong-Geun Kwak b, Hyung-Min Kim c, Young-Myeong Kim d, Hyun-Ock Pae e, Hun-Taeg Chung e, Soo-Wan Chae b, Hyung-Ryong Kim a,¤ a

Department of Dental Pharmacology and Wonkwang Biomaterial Implant Research Institute, School of Dentistry, Wonkwang University, Chonbuk 570-749, Republic of Korea b Department of Pharmacology and Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju, 560-180, Republic of Korea c Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, Seoul, Republic of Korea d Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chunchon, 200-701, Republic of Korea e Department of Microbiology and Immunology, School of Medicine, Wonkwang University, Chonbuk 570-749, Republic of Korea Received 18 November 2004; revised 27 October 2005 Available online 5 January 2006

Abstract Nitric oxide (NO) has been reported to play an important role as an eVector molecule in cytokine signal transduction in cardiomyocytes. A treatment of neonatal rat ventricular cardiomyocytes with interleukin-1 beta (IL-1), tumor necrosis factor-alpha (TNF-), and interferon-gamma (IFN-) induces apoptosis via an NO-dependent pathway. However, cardiomyocytes were more resistant to NOdependent cell death in the presence of catalase, while producing inducible nitric oxide synthase. This paper reports that catalase stimulates the NF-B-binding aYnity. However, the NO synthase activity is abolished by the addition of catalase, suggesting that H2O2 is involved in NO synthesis in a posttranslation state. The catalase-induced inhibition of NO was partially but signiWcantly reversed by H4B, an important cofactor of NO synthesis. Treatment of myocytes with IL-1, TNF-, and IFN- induced a signiWcant increase in the formation of peroxynitrite, and a pretreatment with catalase was found to quench the production of peroxynitrite. This paper shows that the catalase activity was signiWcantly down-regulated by H4B in a concentration-dependent manner. The treatment of H4B induced reactive oxygen species (ROS) release in cardiac cell system. These results suggest that catalase interferes with NO and peroxynitrite production as well as with the related apoptosis of cardiomyocytes. This study also shows that the catalase-induced inhibition of NO release may be reversed by H4B by the release of ROS. © 2005 Elsevier Inc. All rights reserved. Keywords: Nitric Oxide; Cardiomyocyte; Catalase; Peroxynitrite

ProinXammatory cytokines are a class of secretory polypeptides that are synthesized and released locally by macrophages, leukocytes, and endothelial cells in response to an injury [1,2]. Nitric oxide (NO)2 plays an important role in a *

variety of cell types as an eVector molecule in the cytokine signal transduction [3]. NO is formed from the amino acid, L-arginine, by a distinct family of NO synthase (NOS) [4]. ProinXammatory cytokines induce a third isoform of this

Corresponding author. Fax: +82 63 854 0285. E-mail address: [email protected] (H.-R. Kim). 1 These individuals contributed equally to the results of this paper. 2 Abbreviations used: NO, nitric oxide; iNOS, inducible nitric oxide synthase; H4B, tetrahydrobiopterine; TNF-, tumor necrosis factor-alpha; IFN-, interferon-gamma; IL-1, interleukin-1 beta. 1089-8603/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.11.008

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enzyme (inducible NOS, iNOS) in many cell types, including cardiac myocytes [5]. The NOS isoforms are dependent on a number of cofactors, including NADPH, Xavine-adenine dinucleotide (FAD), and tetrahydrobiopterin (H4B) [6,7]. The activity of these enzymes can be speciWcally inhibited by various L-arginine structural analogs such as L-NG-monomethyl arginine (L-NGMMA). The up-regulation of iNOS can be an important mechanism whereby the myocardium protects itself from ischemia. The cytoprotective function of iNOS in the heart has been reported [8]. However, NO produced by iNOS can be a major pathophysiological mediator of septic shock [9], and has been shown to mediate a large part of the negative inotropic eVects of cytokines [10]. The eVects of NO in inhibiting the myocyte contractility are well known, and can mediate the negative inotropy observed in states such as sepsis [11] and cardiac transplant rejection [12]. Furthermore, the role of NO in the death of myocytes has been suggested by a recent report showing that cytokine mixtures including interleukin-1 beta (IL-1) can induce apoptosis in neonatal cardiomyocytes [13]. Mittal [14] reported that the rat brain NOS activity is abolished by exogenous catalase, suggesting that H2O2 and superoxide are involved in NO synthesis. One of the more intriguing aspects of the NO activity is its ability to react with superoxide to form the toxic species, peroxynitrite, which is more reactive than NO [2]. Therefore, this study focused on the lethal eVects of iNOS-produced NO and its derivative, peroxynitrate, on cardiac myocytes. This paper report that catalase inhibits cardiomyocyte apoptosis, and this correlates with the reduction of NO and peroxynitrite synthesis. These results suggest that catalase may interfere with the production of NO and peroxynitrate and might interfere with the related cytotoxicity of cardiomyocytes. Materials and methods Materials Dulbecco’s modiWed Eagle’s medium, fetal bovine serum (FBS), collagenase (type II), pancreatin, and trypsin were purchased from Life Technologies (Gaithersburg, MD, USA). Catalase, H4B, europium (III) chloride, tetracyclin, and the monoclonal anti--sarcomeric actin antibody were obtained from Sigma (St. Louis, MO, USA). The antinitrotyrosine antibody was acquired from Upstate Biotechnology (Lake Placid, NY, USA). The anti-iNOS antibody (clone N-20) was purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK). Interferon-gamma (IFN-), tumor necrosis factor-alpha (TNF-), IL-1, and Hoechst 33258 were obtained from PharMingen (San Diego, CA, USA). Isolation and cultures of neonatal cardiomyocytes The primary cultures of cardiac myocytes were prepared from the ventricles of 1- to 2-day-old Wistar rats, as

previously described [15]. The ventricles were separated from the atrial tissue and washed brieXy in a digestion solution [116 mM NaCl, 20 mM Hepes, 1 mM NaH2PO4, 5.5 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4 (pH 7.35), collagenase (95 U/mL), and pancreatin (0.6 mg/mL)]. The myocytes were dissociated in a fresh digestion buVer and collected by centrifugation. The isolated cells, which were a mixture of myocytes and nonmyocyte Wbroblasts, were suspended in plating media (Dulbecco’s modiWed Eagle’s medium) and plated onto 150-mm diameter noncoated culture dishes for 1 h in order to reduce contamination from cardiac Wbroblasts. The myocytes were puriWed by Percoll gradient, replated at a density of 1 £ 105 in 25-mm diameter etched coverslips precoated with 1% collagen and grown in plating media. After 24 h, more than 98 % of the cells were conWrmed to be myocytes, as determined by cell morphology and sarcomeric actin staining. Measurement of nitrite production NO production was determined by measuring the amount of nitrite in the medium based on the Griess reaction [16]. An aliquot of the spent medium was mixed with an equal volume of a 1:1 mixture of 1 % sulfanilamide in water and 0.1% N-1-naphthyl-ethylenediamine dihydrochloride in 5% phosphoric acid. The absorbance was then read at 570 nm. Sodium nitrite dissolved in the culture medium was used as the standard. Western blot analysis The cell extracts (50 g) were separated by SDS–PAGE and blotted onto polyvinylidene diXuoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA), 1% milk powder in 10 mM Tris–HCl containing 150 mM NaCl, and 0.5% Tween 20 for 1 h and incubated overnight with the appropriate primary antibodies. After extensive washing with Tris-buVered saline Tween 20, the bands were detected using the enhanced chemiluminescence method. MTT cell viability assay The cell viability was determined as previously described [17]. BrieXy, after 48-h incubation, 10 L MTT was added to the 96-well microplates for 3 h, and the absorbance was read at 540 nm on a Titertek multiscan MicroELISA reader. The cell viability was calculated as the ratio of optical densities (OD) in the wells. Trypan blue cell viability assay Twenty-four hours after treatment, the cells were trypsinized and counted by trypan blue exclusion. BrieXy, 20 L of the cell suspension was combined with 180 L trypan blue (Gibco), and the cells were counted using a hemacytometer. The number of cells was determined by averaging

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the number of cells in four squares and multiplying this number by a dilution factor [10]. Quantitation of DNA fragmentation by gel electrophoresis The myocyte-membrane integrity was measured by determining the ability of the cells to exclude trypan blue. DNA was isolated from neonatal myocytes to determine the extent of DNA fragmentation using gel electrophoresis. The culture medium was removed and centrifuged at 3000g for 5 min to collect the detached myocytes. The adherent cells were lysed with a hypotonic lysis buVer [10 mM Tris– HCl (pH 7.4), 10 mM EDTA, 0.5% Triton X-100] and then pooled with a pellet made from detached cells. The cells were incubated at 4 °C with 0.1 vol. of 5 M NaCl and 1 vol. of isopropanol, electrophoresed on a 1.5 % agarose gel, and visualized under UV light after staining with ethidium bromide.

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1 mM dithiothreitol was added to each well. The cells were harvested using a disposable rubber policeman and subjected to three freeze–thaw cycles. The samples were then centrifuged at 100,000g for 30 min at 4 °C, and incubated with a 125 L assay mixture (50 mM Hepes buVer, 1.25 mM CaCl2, 1 mM EDTA, 0.5 mM reduced NADPH, 10 M FAD, 5 M Xavine mononucleotide, 10 M tetrahydrobiopterin, 10 g/mL calmodulin, 5 M L-[3H]arginine) at 37 °C for 1 h. Each sample was then applied to the Dowex 50W X-8 columns. The elutes from each column were collected, and the remaining [3H]citrulline was eluted with 2 mL of H2O. Aliquots were taken for liquid scintillation counting. Electrophoretic mobility shift assay

The cell lysates were generated using a buVer consisting of 1% Nonidet P-40, 50 mM Hepes (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl Xuoride, and 100 mM sodium Xuoride. Equal amounts of the lysates were subjected to SDS–10% PAGE and then transferred to Immobilon-P membranes (Millipore) in a transfer buVer [25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol]. The membranes were Wrst rinsed in Tris-buVered saline [TBS: 10 mM Tris (pH 7.4), 150 mM NaCl] and blocked in TBS–5% BSA overnight at room temperature. The antiiNOS antibody was used at a dilution of 1:500 in TBS–5% BSA. The anti-nitrotyrosine antibody was used at a dilution of 1:300 in TBS–5% BSA. The antibody–antigen complexes were detected using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (BioRad) and a chemiluminescent substrate development kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA).

The cardiomyocytes were washed twice with ice-cold PBS and lysed with a hypotonic buVer (10 mM Hepes, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.2 mM phenylmethylsulfonyl Xuoride (PMSF); 0.5 mM dithiothreitol; 10 g/ mL aprotinin; 20 M pepstatin A; 100 M leupeptin). After centrifugation at 1000g, the nuclear pellets were resuspended in an extraction buVer [20 mM Hepes (pH 7.9), 25 %(v/v) glycerol, 0.4 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM dithiothreitol] and incubated on ice for 10 min. The nuclear proteins in the supernatant were recovered after centrifugation at 15,000g, quantiWed using a BCA protein assay kit (Sigma), and used to carry out electrophoretic mobility shift assay (EMSA). The oligonucleotide probes of NF-B containing the IgG chain binding site (NF-B: 5⬘-CCG GTT AAC AGA GGG GGC TTT CCG AG-3⬘) were used to measure the level of NF-B activity. Two complementary strands of the oligonucleotide were annealed and labeled with the [-32P]dCTP using a random primer labeling kit (Rediprime, Amersham Life Science, Amersham, England). The nuclear extracts (5 g) were reacted with 2–5 ng of the radiolabeled NF-B probes (50,000– 100,000 cpm/ng). The reaction was performed in the presence of 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, and 4% glycerol (Wnal volume: 25 L) at room temperature for 30 min. The reaction products were subjected to 4% polyacrylamide gel electrophoresis in a 0.5£TBE buVer (50 mM Tris–HCl, pH 8.5; 50 mM borate; and 1 mM EDTA). The gels were dried under vacuum for 1 h. The DNA-binding activity for NFB was measured by using a PhosphoImage analyzer (BAS, Fuji, Tokyo, Japan).

Assay for NOS activity

Immunohistochemical detection of nitrotyrosine

The myocytes in the culture medium were distributed on 12-well plates at 5 £ 106 cells/well and incubated for 6 h at 37 °C and in 5% CO2. The nonadherent cells were removed by washing before adding the reagents. The cultures (2 mL/well) were incubated for 24 h, the supernatant was removed, and 250 L of 0.1 M Hepes (pH 7.4) with

The Wxed and permeabilized cells on the slides were blocked with 5% FBS and 1% BSA-containing PBS–Tween (0.1%) for 45 min. The slides were incubated with the polyclonal anti-tyrosine antibody (1:200 dilution) at room temperature for 1 h. The cells were then incubated with 1:200 dilution of the Xuorescein isothiocyanate-conjugated goat

Detection of chromatin condensation with Hoechst 33258 The neonatal myocytes were also stained with Hoechst 33258 in order to detect the chromatin condensation that is characteristic of apoptosis. The Wxed cells were stained for 30 min in PBS containing 10 M Hoechst 33258 as well as anti-sarcomeric actin to stain the actin in the myocytes. Immunoblotting

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anti-rabbit IgG secondary antibody in PBS for 1 h at room temperature.

Results Catalase protects from IL-1-, TNF--, and IFN--induced death in rat ventricular cardiomyocytes

Catalase activity assay The catalase activity was determined by comparing the ratio of destroying H2O2 with catalase alone or in the presence of various H4B concentrations using a spectrophotometer as previously described [18]. The catalase activity was also conWrmed by the kinetic change of the Xuorescence using a europium (III) ion-derived Xuorescent probe [19]. Statistical treatment of data The statistical diVerences were evaluated by analysis of variance (ANOVA) in the dose–response experiments as well as by two-tailed Student’s t tests.

The eVect of the cytokine mixture on cell viability was investigated using MTT assays in order to determine the ability of the combined cytokines to induce cell death. The incubation of IL-1 in the presence of TNF- (2 ng/mL) and IFN- (50 U/mL) caused signiWcant decrease in cell viability in a dose-dependent manner (Fig. 1A). A single treatment with TNF- (2 ng/mL), IL-1 (20 ng/mL), or IFN (50 U/mL) had no eVect on the viability of cardiomyocytes. The cells were then pretreated with diVerent catalase concentrations tested for cytotoxicity with 20 ng/mL of IL-1  in the presence of TNF- and IFN- to determine if catalase modiWes IL-1/TNF-/IFN--induced apoptosis in D

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Fig. 1. EVects of catalase on cell death in the IL-1-, TNF--, and IFN--treated cardiomyocytes. (A) The cells were treated with 2 ng/mL TNF- and 50 U/mL IFN- under various IL-1 concentrations. After 48-h incubation, the cell viability was assayed using an MTT assay, as described under Materials and methods. The results of the four experiments are expressed in the means § SEM. ¤ P < 0.05 versus control. (B) The cells were treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of 5 mM NGMMA, various concentrations of catalase (0, 100, 200, 400, and 800 U/ mL) or heat-inactivated catalase (800 U/mL), SNAP (1 mM), and SNP (1 mM). After 48-h incubation, the cell viability was determined using an MTT assay. The results of three experiments are expressed in the means § SEM. ¤P < 0.05 versus IL-1, TNF-, and IFN-. (N: NGMMA, H: heat-inactivated catalase). (C) The cells were treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of 5 mM NGMMA, 700 U/mL catalase or 700 U/mL heat-inactivated catalase. After 48-h incubation, the cells were harvested and assayed for DNA fragmentation (M: Marker, 1: control, 2: TNF- + IL-1 + IFN-, 3: TNF- + IL-1 + IFN- + NGMMA, 4: TNF- + IL-1+IFN- + catalase, 5: TNF- + IL-1 + IFN- + heat-inactivated catalase). (D) The cells were treated as described in (C). After 48-h incubation, the cells were Wxed with 4% paraformaldehyde and stained with anti-sarcomeric actin and Hoechst 33258 (2.5 g/mL in PBS). The nucleus condensation of the apoptotic cells was analyzed using Xuorescence microscopy (CON: control, T+I+I: TNF- + IL-1 + IFN-, CAT: catalase).

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cardiomyocytes. Catalase alone induced an increase in cell viability after 48-h incubation with the combined cytokines (Fig. 1B). This suggests that a catalase concentration >400 U/ mL can protect the combined cytokine-induced cell death in rat cardiomyocytes. Catalase did not aVect the viability of cells after 48 h of culture the concentration used in these experiments (200–800 U/mL). Therefore, the nonspeciWc toxicity of the agent could be excluded (data not shown). In addition, heat-inactivated catalase also did not have any protective eVects. Fig. 1B also showed that a treatment of G L-N MMA can inhibit cytotoxicity, conWrming that cardiomyocyte death occurs via an NO-dependent pathway [13]. In addition, exposing the cells to high concentrations of exogenous NO donors, S-nitroso-N-acetylpenicillamine (SNAP: 1 mM) and sodium nitroprusside (SNP: 1 mM), led to cell death. This suggests that a treatment with catalase protects the cardiomyocytes against cytotoxicity induced by IL-1/ TNF-/IFN- through reactive oxygen species (ROS) including NO. The levels of internucleosomal DNA fragmentation and nuclear morphology were examined using Hoechst 33258 in order to determine if the loss of viability caused by the combined cytokines-IL-1/TNF-/IFN- correlates with a biochemical feature that discriminates between apoptosis and necrosis. A major component of IL-1/TNF-/ IFN--induced cell death was attributable to apoptosis, as shown by DNA laddering (Figs. 1C and D). Both the DNA laddering and the apoptotic nuclear morphology were blocked by the presence of 700 U/mL catalase. It is evident that catalase, which is a H2O2 scavenger, repressed the combined cytokines-induced apoptosis in the neonatal rat ventricular cardiomyocyte, (>95% purity, as determined by immunostaining with cardiomyocyte-speciWc antibody–antisarcomeric actin; Fig. 1D, lower panel). Catalase regulates NO release, NOS expression, and NOS activity in IL-1-, TNF--, and IFN--treated cardiomyocytes The exposure of cardiomyocytes to IL-1 (20 ng/mL) resulted in a signiWcant increase in NO2 production at 48 h, as has been previously reported [13]. The exposure of myocytes to TNF- alone (from 1 to 20 ng/mL) had no eVect on NO2 production compared with the vehicle. The addition of TNF- to IL-1 resulted in a statistically signiWcant increase in NO2 production compared with IL-1 alone (data not shown). Furthermore, the addition of 50 U/mL IFN- to TNF- and IL-1 synergistically stimulated the release of NO in the cardiomyocytes (Fig. 2A). The synthesis of NO was reduced by catalase in a dose-dependent manner (Fig. 2B). A single treatment of catalase had no eVect on the cell viability (data not shown). The inhibitory eVects of the three diVerent batches of catalase were tested in order to rule out the possibility that the inhibitory eVect of catalase was due to the nonspeciWc action of contaminants present in the enzyme preparation. All three preparations had identical inhibitory activity based on the unit enzyme activity (data not shown). Furthermore, inactiva-

193

tion by heat completely abolished the inhibitory activity of catalase. Although no changes in H2O2 production were observed with the DCFDA Xuorescence assays, the results suggest that there is H2O2 formation in these cardiomyocytes. Increasing evidence has shown that cytokines including TNF- or IL-1 can stimulate the production of ROS and induce lipid peroxidation in various cells [20–22]. These results indicate that in this paradigm, H2O2 plays an important role in NO synthesis and subsequent apoptosis. The eVect of catalase on NO production as well as the likelihood of a role for H2O2 in NO synthesis was determined by examining the eVect of catalase on the iNOS protein in the cardiomyocytes activated with combined cytokines. The blockade of NO synthesis and NOS activity was not correlated with the expression of the iNOS protein, as determined by Western blot analysis (Fig. 2C). Catalase can signiWcantly enhance the expression of iNOS protein in rat ventricular cardiomyocytes. This study then measured the alterations of NOS activity in IL-1/TNF-/IFN--treated cardiomyocytes in the presence or absence of catalase. After treating the cardiomyocytes with the combined cytokines in the presence or absence of catalase for 24 h, the cells were collected to determine their NOS activity by measuring the conversion of [3H] arginine to [3H] citrulline. As shown in Fig. 2D, catalase signiWcantly inhibited the combined cytokines-stimulated NOS activity in rat ventricular cardiomyocytes. Catalase enhances IL-1-, TNF--, and IFN--induced NF-B activity in rat ventricular cardiomyocytes The activation of the transcription factor, NF-B, has previously been shown to be essential for iNOS gene induction by various cytokines [23,24]. An experiment was performed to determine the NF-B DNA- binding activity in combined cytokines-exposed cardiomyocytes. The cells were treated with the cytokine mixture in the presence or absence of catalase. The results showed that IL-1/TNF-/ IFN- markedly activated the binding activity of the nuclear extract to the oligonucleotide probe of NF-B by 30 min and that catalase can stimulate the NF-B-binding aYnity (Fig. 3). This is consistent with our results showing that catalase stimulates the induction of the iNOS gene in rat ventricular cardiomyocytes. Inhibitory eVect of catalase on the release of NO can be reversed by H4B in rat ventricular cardiomyocytes This study examined the possibility that the activity of catalase may have resulted in the oxidization of the cofactor H4B, thereby rendering it unavailable for the synthesis of NO. H4B was added to the cultures of the IL-1-, TNF--, and IFN--treated cardiomyocytes in the presence or absence of catalase. Again, the production of NO and cell death were signiWcantly inhibited by catalase, but the inhibition of NO was progressively reversed by the increasing H4B concentrations and returned to 50% of the control levels with 300 M of H4B (Fig. 4A). H4B alone had no eVect on NO

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0 N 100 200 400 800 H CAT T+I+I Fig. 2. EVects of catalase on the release of NO, NOS expression, and activity in IL-1-, TNF--, and IFN--treated cardiomyocytes. (A) The cells were treated with 2 ng/mL TNF- and 50 U/mL IFN- under various concentrations of IL-1 (0, 2, 5, 10, or 20 ng/mL). After 48-h incubation, the culture media were mixed with the Griess reagent to estimate the release of NO. The results of three experiments are expressed as the means § SEM. ¤ P < 0.05 versus control. (B) The cells were treated with 20 ng/mL IL-1, 2 ng/ml TNF-, and 50 U/mL IFN- in the presence or absence of 5 mM NGMMA, catalase (0, 100, 200, 400, and 800 U/mL) and heat-inactivated catalase (800 U/mL). After 48-h incubation, the release of NO release was assayed as described under Materials and methods. The results of four experiments are expressed as the mean § SEM. ¤ P < 0.05 versus IL-1, TNF-, and IFN-. (C) The cells were treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of catalase (700 U/mL) or heat-inactivated catalase for 24 h. The cell extracts were subjected to Western blot analysis using the antibodies speciWc for iNOS. This blot is representative of three experiments with similar results (1, control; 2, TNF- + IL-1 + IFN-; 3, TNF- + IL-1 + IFN- + catalase; 4, TNF- + IL-1 + IFN- + heat-inactivated catalase). (D) The cells were treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of catalase (700 U/mL) for 24 h. The NO synthase activity was assayed at the culture media and cell extracts as described under Materials and methods. The results of three experiments are expressed in the means § SEM. Media: ¤ P < 0.05 versus IL-1/TNF-/ IFN-, cell lysate: # P < 0.05 versus IL-1/TNF-/ IFN-. (T+I+I: TNF- + IL-1 + IFN-, CAT: catalase, H-CAT: heat-inactivated catalase).

synthesis nor did it have any cytotoxic eVects on cardiomyocytes (data not shown). Considering that a large amount of NO, >70 M NO, is required for apoptosis in this system, the high amount of H4B (300 M) used was insuYcient to completely reverse the catalase-induced protective eVect (Fig. 4B). These results also showed that the maximum concentration of H4B used (300 M) only partially reversed the catalaseinduced inhibition of NO release (about 40%).

conWrm that the cytokine combination of IL-1, TNF-, and IFN- can induce increases in nitrotyrosine in neonatal rat ventricular cardiomyocytes, and the content of nitrotyrosine can be regulated in the presence of 700 U/mL catalase. The combined cytokines induced a signiWcant increase in nitrotyrosine formation in the myocytes, and the presence of catalase (700 U/mL) regulated the formation of nitrotyrosine (Figs. 5A and B).

Catalase can inhibit peroxynitrite release in cardiomyocytes

Catalase is regulated by H4B in vitro

Since peroxynitrite production has been observed in many inXammatory conditions and can also be a potent cytotoxic mediator [25], experiments were performed to

This study also tested the possibility that H4B may directly regulate the activity of catalase, rendering it unavailable for the inhibitory eVect of NO release. Cata-

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T+I+I (min)

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0 10 30 60 120 240 10 30 60 120 240

NF-κB

Fig. 3. EVect of catalase on the NF-B activity in IL-1-, TNF--, and IFN--treated rat ventricular cardiomyocytes. The cells were treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of catalase (700 U/mL) for the indicated periods. The nuclear proteins were analyzed in an EMSA with an [-32P]-label oligonucleotide encompassing the NF-B binding site. This autoradiograph is representative of two experiments with similar results. (T+I+I, TNF- + IL1 + IFN-; CAT, catalase).

lase (30 U/mL) was incubated in the presence or absence of H4B (50, 100, 200 or 300 M) in vitro. Consistent with this hypothesis, the H2O2 ratio was signiWcantly down-regulated by H4B in a dose-dependent manner (Fig. 6A). Fig. 6B shows the kinetic response of the europium–tetracyclin–hydrogen peroxide (EuTc-HP) complex system to various levels of catalase activity. The Xuorescence of the EuTc-HP system decreases with increasing catalase activity. Therefore, the slope reXects the catalase activity. As shown in Fig. 6B, H4B (100 M) inhibited the catalase activity in a similar manner to 3-amino-1,2,4-triazole(3AT) which is a speciWc catalase inhibitor. It is possible that H4B directly regulates catalase in vitro and that H4B releases H2O2 that cannot be quenched by 30 U/mL catalase. In order to conWrm the possibility of H4B-induced H2O2, experiments were performed to determine if H4B releases H2O2 in neonatal cardiac cells. Fig. 6C shows that H4B releases ROS including H2O2, which was inhibited by the addition of catalase. Discussion These results demonstrate that catalase can inhibit the production of NO and apoptosis induced by combined cytokines—IL-1, TNF-, or IFN-. Since catalase converts H2O2 to molecular oxygen, H2O2 may be involved in both NO synthesis and the resulting cell death in the combined cytokines-treated rat neonatal cardiomyocytes. Superoxide dismutase (SOD) that converts superoxide to H2O2 can eVectively enhance the vascular relaxation and half-life of NO [26,27]. This was interpreted as the result of the removal of O2¡ by SOD, which protects NO from breakdown as a result of an interaction with O2¡. There-

Fig. 4. EVect of H4B on catalase-induced inhibition of NO and the protective eVect in rat ventricular cardiomyocytes. (A) The cells were exposed to various concentrations of H4B (0, 50, 150, or 300 M) for 30 min before being treated with 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- in the presence or absence of 700 U/mL catalase for 48 h. The culture media were mixed with Griess reagent to estimate the amount of NO releases. The data are expressed means § SEM of four experiments (24 h: ¤ P < 0.05 versus IL-1, TNF-, and IFN- + catalase, 48 h: # P < 0.05 versus IL-1, TNF-, and IFN- + catalase). (B) The cells were pretreated with H4B (300 M) before 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- were added in the presence or absence of 700 U/mL catalase. After 48-h incubation, the cell viability was assayed using an MTT assay. The results of three experiments are expressed as the means § SEM (T+I+I, TNF + IL-1 + IFN-; CAT, catalase; H-CAT, heat-inactivated catalase).

fore, these results support the interpretation that SOD protects and enhances the half-life of NO. However, the mechanism for the inhibition of NO synthesis by catalase may be diVerent. The data reported here also demonstrates that the production of NO and apoptosis induced by combined cytokines can be inhibited by catalase. Because catalase converts H2O2 to molecular oxygen, these results suggest that H2O2 may be involved in both NO synthesis and proinXammatory cytokines-induced apoptosis. During all the processes in this study, we used exogenous catalase. Up to now, there is no consensus regarding the permeability of exogenous catalase. Studies on catalase have revealed that H2O2 may be produced within the mitochondria, and that the cellular uptake of exogenous catalase is indeed possible [28–31]. If exogenous catalase was unable to permeate through the cells, it would be diYcult to explain the antioxidant enzyme catalase-induced protection in various cells

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Fig. 5. The eVect of catalase on the formation of nitrotyrosine in rat ventricular cardiomyocytes. (A) The cells were pretreated with H4B (300 M) before 20 ng/mL IL-1, 2 ng/mL TNF-, and 50 U/mL IFN- were added in the presence or absence of 700 U/mL catalase. Twenty-four hours later, the cell extracts were subjected to Western blot analysis using antibodies speciWc to nitrotyrosine (1, control; 2, TNF- + IL-1 + IFN-; 3, TNF- + IL-1 + IFN + catalase). (B) The cells were treated as described in (A). After 24-h incubation, the cells were Wxed with 4% paraformaldehyde and stained with antinitrotyrosine and anti-sarcomeric actin. MagniWcation 400£. (T+I+I, TNF- + IL-1 + IFN-).

[31–33]. However, the uptake of catalase does not appear to work eYciently, compared with the expression of catalase gene. It is the reason why a very high concentration of catalase, 700 U/mL, was used in this study. The inhibition of NO synthesis and apoptosis by catalase is unlikely to be the result of the nonspeciWc action of contaminants in the catalase preparations. Several batches of catalase with diVerent degrees of purity gave identical eVects based on the unit enzyme activity. The heat-inactivated enzyme was devoid of inhibitory activity, which suggests that the eVect is unlikely to be mediated by transition metal contamination. Catalase rather enhanced the induction of NOS, but inhib-

ited NOS activity in our system. It was recently reported that catalase abolished the NOS activity as measured by NO-stimulated cyclic GMP accumulation, suggesting that H2O2 is involved in NO synthesis [14]. This conclusion is supported by the present study, where catalase altered the NOS activity and rather enhanced NOS expression in the IL-1-, TNF--, or IFN--treated cardiomyocytes. The discrepancy between the NOS activity and the protein expression may be due to catalase-induced posttranslational regulation in NO synthesis. Furthermore, the inhibitory eVect of catalase on the release of NO was reversed by the presence of H4B in a concentration-dependent manner

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197

Fig. 6. H4B-induced regulatory eVect on catalase activity in vitro. (A) Catalase (30 U/mL) was incubated with various concentrations of H4B (0, 50, 100, 200, or 300 M) and the subsequent catalase activity was assayed as described under Materials and methods. The data are expressed means § SEM of two independent experiments. ¤P < 0.05 versus catalase; ¤¤P < 0.001 versus catalase. (B) a—The activities of catalase are (from top) 0, 1, 3, 5, 10, and 30 U/mL, respectively. The concentration of H2O2 was 1 mM. b—Inhibition of catalase by 3-amino-1,2,4-triazole (3-AT, 10 mM) or H4B (100 M). The following concentrations were employed: H2O2, 1 mM; catalase, 30 U/mL. The europium–tetracycline–hydrogen peroxide system are used for measurement of kinetics of the decomposition of H2O2 by catalase. (C) The cells were pretreated with/without 700 U/mL catalase for 30 min and then with 300 M H4B. The cells were then incubated with the dye 2⬘,7⬘-dichloroXuorescin diacetate (100 M) and the Xuorescence intensities of 10,000 cells were analyzed using Xow cytometry (Partec PAS).

(Fig. 4A). The requirement for H4B as a cofactor in the generation of NO has been well documented [34]. In addition, H4B can readily penetrate the cellular membrane. Other reports have indicated that the synthesis of NO involves a number of cofactors besides H4B, including NADPH and FAD [25]. The present study suggests that the eVect of catalase on apoptosis may be due to the oxidation of this cofactor as a result of the enzymatic activity of catalase. In addition, the eVect of catalase on the release of NO can be

partially but signiWcantly reversed by H4B suggesting that the oxidation of H4B plays an important role on the inhibition of NO synthesis. In parallel with this result, H4B can induce the release of ROS, which was abrogated by a treatment with catalase (Fig. 6B). This result suggests that H4B-induced H2O2 interferes with the eVect of catalase in neonatal rat cardiomyocytes, which is inconsistent with another report [35]. Han et al. [36] suggested that antioxidant enzymes, including catalase, decreased the levels of

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iNOS protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor, L-NGMMA, as well as Hb increased the level of iNOS protein, indicating that the inhibition of NO rather increased iNOS protein expression. The eVect of Hb was similar to that of catalase in this study. The concentration of catalase, 120 U/mL, showed the maximum inhibitory eVect on the release of NO. However, the catalase dose had no eVect on the cardiac system. The addition of catalase increased the activity of antioxidant enzyme catalase at the intracellular level at high concentrations; >400 U/mL (data not shown). The treatment of catalase at 120 U/mL may be suYcient to nullify the intracellular increase of H2O2. Peroxynitrite was detected as nitrotyrosine (formed by the peroxynitrite-induced nitration of tyrosine residues on proteins) in the combination of cytokines-treated cells (Figs. 5A and B). Furthermore, the presence of catalase inhibited peroxynitrite formation, as measured by nitrotyrosine. The protective eVect of catalase against the cytokine-induced apoptosis is related to the prevention of both nitrotyrosine and NO formation. The combined cytokinesinduced H2O2 can form other ROS that can react with NO leading to the formation of peroxynitrite in rat ventricular cardiomyocytes. However, the H4B pretreatment does not have any eVect on the formation of peroxynitrite in this system, which is diVerent from the results for NO (data not shown). In order to conWrm the formation of nitrotyrosine, it might be necessary to use a high concentration of NO in this immunoblot and immunostaining system. This can also explain why H4B (300 M) did not reverse the catalaseinduced protective eVects in cardiomyocytes. These results show that peroxynitrite, in parallel with NO, can be another mediator of IL-1-, TNF--, or IFN--induced cell death, and that catalase abrogates the combined cytokinesinduced H2O2 and other free radicals including the superoxide anion and hydroxyl radicals in this ventricular myocyte system.

TNF-α TNF-χ IFN-γ IFN-ι IL-1β IL-1δ LPS LPS

NF-κB NF-kB iNOS iNOS Enzyme activation H4B

Catalase

H H22O O22 NO NO NO NO ONO ONO-

Death Death

ONO ONO-Death Death

Fig. 7. The scheme for the catalase-induced protective function.

Fig. 6 showed that H4B can regulate the catalase activity through an in vitro assay. A H4B-associated catalase assay was also performed on the other cofactors including FAD and NADPH. However, these factors did not have any regulatory eVect on catalase (data not shown). Although the H4B-induced inhibition of catalase activity may be due to its ability to interact with catalase directly, the increase of H2O2 by the H4B can be a key factor for the regulation of antioxidant. H4B releases H2O2 that cannot be quenched by 30 U/mL catalase. The Wnding that H4B liberates ROS including H2O2 is consistent with another report [34]. Although catalase-induced inhibition of NO release may be reversed by H4B via a direct interaction between catalase and H4B, the possibility that H4B-induced release of H2O2 can be an important factor to reduce the eVect of catalase cannot be ruled out (Fig. 7). Acknowledgments This work was supported by Korea Research Foundation Grant (KRF-2000-005-F00001). The authors wish thank Dr. John C. Reed (President of The Burnham Institute in San Diego, CA, USA) for his critical review. References [1] T. Takahashi, F. Hato, T. Yamane, H. Fukumasu, K. Suzuki, S. Ogita, Y. Nishizawa, S. Kitagawa, Activation of human neutrophil by cytokine-activated endothelial cells, Circ. Res. 88 (2001) 422–429. [2] M.C. Carreras, G.A. Pargament, S.D. Catz, J.J. Poderoso, A. Boveris, Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils, FEBS Lett. 341 (1994) 65–68. [3] A.R. Clemons-Miller, G.W. Cox, J. Suttles, R.D. Stout, LPS stimulation of TNF-receptor deWcient macrophages: a diVerential role for TNF-alpha autocrine signaling in the induction of cytokine and nitric oxide production, Immunobiology 202 (2000) 477–492. [4] N.S. Kwon, C.F. Nathan, C. Gilker, O.W. GriYth, D.E. Matthews, D.J. Stuehr, L-Citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen, J. Biol. Chem. 265 (1990) 13442–13445. [5] F. Jung, L.A. Palmer, N. Zhou, R.A. Johns, Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes, Circ. Res. 86 (2000) 319–325. [6] P.J. Andrew, B. Mayer, Enzymatic function of nitric oxide synthases, Cardiovasc. Res. 43 (1999) 521–531. [7] D.S. Bredt, C.D. Ferris, S.H. Snyder, Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identiWcation of Xavin and calmodulin binding sites, J. Biol. Chem. 267 (1992) 10976–10981. [8] D.A. Parks, F.M. Booyse, Cardiovascular protection by alcohol and polyphenols: role of nitric oxide, Ann. N.Y. Acad. Sci. 957 (2002) 115–121. [9] X. Wang, M. Lu, Y. Gao, A. Papapetropoulos, W.C. Sessa, W. Wang, Neuronal nitric oxide synthase is expressed in principal cell of collecting duct, Am. J. Physiol. 275 (1998) F395–F399. [10] H. Takano, T. Nagai, M. Asakawa, T. Toyozaki, T. Oka, I. Komuro, T. Saito, Y. Masuda, Peroxisome proliferator-activated receptor activators inhibit lipopolysaccharide-induced tumor necrosis factoralpha expression in neonatal rat cardiac myocytes, Circ. Res. 87 (2000) 596–602.

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