Cardiovascular Research

Cardiovascular Research 1997 36(2):205-215; doi:10.1016/S0008-6363(97)00137-5
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase

Basilia Zingarelli, Salvatore Cuzzocrea, Zsuzsanna Zsengellér, Andrew L. Salzman and Csaba Szabó^*

Children's Hospital Medical Center, Division of Critical Care, 3333 Burnet Avenue, Cincinnati OH 45229, USA

^* Corresponding author. Tel.: +1-513-636-8714; fax: +1-513-636-4892; e-mail: csaba.szabo@chmcc.org

Received 10 February 1997; accepted 7 May 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Peroxynitrite and hydroxyl radical, reactive oxidants produced during reperfusion, are potent triggers of DNA single strand breakage. DNA injury triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), which contributes to cellular energetic depletion. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of myocardial reperfusion injury in a rat model. Methods and results: Occlusion of the left main coronary artery (one hour) followed by reperfusion (one hour) in the anesthetized rat caused severe cardiac necrosis, neutrophil infiltration, and increased plasma creatine phosphokinase activity. There was significant peroxynitrite production during reperfusion, as indicated by a massive increase in nitrotyrosine in the necrotic myocardium. Reperfusion was also associated with a significant loss of myocardial ATP. In vivo administration of the PARS inhibitor 3-aminobenzamide (10 mg/kg i.v.) to rats subjected to myocardial ischemia and reperfusion, reduced myocardial infarct size and blunted the increase in plasma creatine phosphokinase activity and myeloperoxidase activity in infarcted hearts. In addition, 3-aminobenzamide partially preserved the myocardial ATP levels. In vitro, pharmacological inhibition of PARS also ameliorated peroxynitrite-induced cytotoxicity in rat cardiac myocytes and human endothelial cells. Conclusion: 3-aminobenzamide has significant protective effects in myocardial reperfusion injury. We hypothesize that activation of PARS activation plays a role in the pathophysiology of acute myocardial infarction.

KEYWORDS Peroxynitrite; DNA damage; Nitrotyrosine; Nitric oxide; Oxygen radicals

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme which, when activated by DNA single-strand breaks, initiates an energy consuming, inefficient repair cycle by transferring ADP ribose units to nuclear proteins. The results of this process is a rapid depletion of the intracellular NAD⁺ and ATP energetic pools which slows the rate of glycolysis and mitochondrial respiration leading to cellular dysfunction and death [1, 2]. Reactive oxygen-centered radicals (superoxide, hydroxyl radicals, singlet oxygen and hydrogen peroxide) and the nitrogen-centered radical nitric oxide (NO) have been recently proposed as powerful triggers of DNA single strand breakage, and consequent activation of the cell suicide cycle by PARS in various cell types in vitro. Under this oxidative stress and energetic substrate deficiency pharmacological inhibition of PARS has been shown to exert beneficial effects against free radicals mediated cell injury [3–7]. Recently we have demonstrated that peroxynitrite, a potent oxidant formed by the reaction of superoxide and NO, is a potent activator of PARS in vitro in murine macrophages and rat vascular smooth muscle cells [8–10]. PARS activation, most likely due to peroxynitrite production, is also responsible of the cellular energetic derangement and vascular contractile failure observed during endotoxic shock [11, 12].

The role of reactive oxygen-centered radicals is well established in the pathogenesis of myocardial ischemia and reperfusion injury. Experimental evidence suggests that during early reperfusion, adhesion, activation and extravasation of neutrophils results in a burst of primary oxygen free radicals, including superoxide, hydrogen peroxide and hydroxyl radicals which are responsible for the massive cellular damage and dysfunction of the myocardium [13–16]. An early endothelial injury, and consequent increased adhesion of neutrophil granulocytes to the intima has been implicated as an important early step in reperfusion injury [13–16].

Our recent studies have also implicated peroxynitrite, formed from superoxide and constitutive NO synthase derived NO, in the pathogenesis of cardiovascular shock caused by systemic low oxygen supply such as hemorrhagic shock or local ischemia and reperfusion injury after splanchnic artery occlusion [17, 18]. Furthermore, the presence of peroxynitrite in the reperfused heart after ischemia has also been confirmed by electron paramagnetic resonance spectroscopy and increased formation of the peroxynitrite-mediated nitration product nitrotyrosine [19]. The formation of peroxynitrite appears to be an important contributor to development of reperfusion damage, since inhibition of NO, and consequently of peroxynitrite formation is protective in in vitro myocardial anoxia and reoxygenation injury [20, 21].

Considering the potential importance of PARS activation during oxidative stress, the current study was designed to investigate the effect of pharmacological blockade of PARS activity by 3-aminobenzamide in a rat model of myocardial ischemia and reperfusion. Since our results demonstrated that 3-aminobenzamide treatment reduced the amount of peroxynitrite detected during reperfusion, in in vitro studies we have also investigated whether 3-aminobenzamide is a scavenger of peroxynitrite, or an inhibitor of endothelial NO synthase. Furthermore, in order to characterize the cellular sites of PARS activation, experiments were carried out to study the role of PARS activation in the cytotoxicity elicited by peroxynitrite and hydrogen peroxide in rat myoblasts and human umbilical vein endothelial cells in vitro.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Myocardial ischemia and reperfusion
Male normotensive Wistar rats (Charles River Laboratories, Wilmington, MA) weighing 250–275 g were used. The animals were anesthetized with thiopentone sodium (120 mg/kg, intraperitoneally). The right common carotid artery was cannulated to measure mean arterial blood pressure (MAP) and heart rate by a pressure transducer connected to a Maclab A/D converter (AD instruments, Milford, MA, USA). The left jugular vein was cannulated for drug administration. The animals were also instrumented for electrocardiographic recordings (S-T segment, II lead). The trachea was cannulated and artificial respiration was maintained by a respirator with a frequency of 60 strokes/min and a tidal volume of 1 ml/100 g. Myocardial ischemia and reperfusion was performed as previously described by opening the chest at the fourth and fifth intercostal space, and by occluding the left main coronary artery by a 4.0 silk ligature [22]. The occlusion was released after 1 h, and the ischemic period was followed by a 1 h long reperfusion period. Elevation of S-T segment and subsequent decrease were considered indicative of coronary occlusion and reperfusion, respectively. At the end of reperfusion hearts were taken out for quantification of myocardial injury and biochemical studies.

2.2 Experimental groups
In the treated group of animals, 3-aminobenzamide, an inhibitor of PARS, was given as a intravenous bolus 10 min before reperfusion (10 mg/kg) followed by infusion of 10 mg/kg/h during the period of reperfusion (MIR+3-aminobenzamide group). In a vehicle-treated group of rats, vehicle (saline) was given instead of 3-aminobenzamide (MIR group). In separate groups of rats, surgery was performed in its every aspect identical to the one in the MIR group, except that the blood vessels were not occluded (time-controlled sham group; Sham). In an additional group of animals, sham surgery was combined with the administration of 3-aminobenzamide (dose as above) (Sham+3-aminobenzamide). In one set of experiments, n = 6 animals were used for each group for the determination of cardiovascular parameters, infarct size, creatin phosphokinase and myeloperoxidase. In a different set of experiments, hearts were processed for nitrotyrosine immunohistochemistry (n = 5–6 hearts for each experimental group). In an additional, distinct set of experiments, animals were treated as above, and whole hearts were removed and frozen for ATP measurement (n = 6 in each group). No animals were excluded from the statistical analysis.

The reason for the selection of the timing of the administration of this inhibitor was that PARS is activated in response to DNA injury, and the production of the oxidants and free radicals that may potentially injure DNA occurs in the reperfusion phase, rather than during the ischemic period. The current dosage regimen of 3-aminobenzamide has been previously shown to be effective in reducing the development of endotoxin-induced vascular hypocontractility and cellular energetic alterations in anesthetized rats [11, 12].

2.3 Nitrotyrosine immunohistochemistry
Tyrosine nitration, a specific "footprint" of peroxynitrite-induced damage, was measured in myocardial sections by immunohistochemistry [23, 24]. After reperfusion hearts were fixed in 10% buffered formalin and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in phosphate buffered saline for 20 min. Non specific adsorption was minimized by incubating the section in 2% normal goat serum in phosphate buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with 1:1000 dilution of primary anti-nitrotyrosine antibody or control solutions. Controls included buffer alone or non specific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex. To quantitate the degree of nitrotyrosine staining, a 0–4 grading system was used: 0 indicated no staining, 4 indicated extensive staining, and 1–3 indicated increasing degrees of intermediate staining. In each experimental group, n = 5–6 sections were evaluated by an investigator blinded to the treatment protocol. The pictures shown in the Results section are representative sections for each experimental group.

2.4 Biochemical measurements
Serum levels of creatine phosphokinase were evaluated as an index of cardiac cellular damage by using commercial kit (Sigma, St. Louis, MO, USA). To measure ATP levels in whole hearts by HPLC, tissue were homogenized in 2 N perchloric acid, neutralized with 1 M KH₂PO₄ and centrifuged at 3 500xg at 4°C. Supernatants were then assayed using a Beckman Model 231 HPLC instrument equipped with a Waters Wisp autoinjector. For determinations of ATP aliquots of the samples were analyzed on a Whatman SAX-10 ion exchange column, utilizing a mobile phase consisting of 0.16 mM KH₂PO₄ and 0.1 M KCl in deionized water (pH 6.5) at a flow rate of 1.5 ml/min. ATP was detected by ultraviolet absorption spectrophotometry at 254 nm and values were expressed as nmoles/mg protein. Myeloperoxidase activity was determined in whole hearts as index of neutrophil accumulation [25]. Cardiac tissues were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20 000xg at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H₂O₂. The rate of change in absorbance was measured by a spectrophotometer at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of peroxide/min at 37°C and was expressed in units per 100 mg weight of tissue.

2.5 Quantification of cardiac damage
Necrotic and perfused areas were determined by the triphenyl tetrazolium chloride-Evans blue technique [26]. At the end of the reperfusion period the ligature around the left main coronary artery was retightened; 2 ml of 5% Evans blue dye was injected into the jugular vein to stain the area of the myocardium perfused by the patent coronary arteries. The area at risk was therefore determined by negative staining. The atria, right ventricle, and major blood vessels were removed from the heart. The left ventricle was sliced into 3 sections 3 mm thick parallel to the atrioventricular groove. The unstained portion of myocardium (i.e., the area at risk) was separated from the stained portion (i.e., the area not at risk). The unstained portion was then sliced into 1 mm thick sections and incubated in a 1% solution of triphenyl-tetrazolium chloride (TTC) stain in phosphate buffer 20 mmol/L pH 7.4 and 37°C for 20 min. The area at risk of infarction was colored brick red due to formation of a precipitate that results from reaction of TTC with dehydrogenase enzymes [27]. Loss of these enzymes from infarcted myocardium prevents formation of the precipitate; thus the infarcted area within the risk region remains pale yellow (i.e. necrotic area). Samples from the various portions of the left ventricle were weighed. Three areas were compared in the control and the 3-aminobenzamide-treated animals: (1): non-ischemic; (2): area at risk (which includes the ischemic non-necrotic as well as the necrotic area) and (3) the necrotic area (infarct size). Infarct size was then normalized to the area at risk.

2.6 Effect of 3-aminobenzamide on peroxynitrite-induced oxidative processes in vitro
To study potential peroxynitrite scavenging effects of the PARS inhibitor used in our study, the peroxynitrite-dependent oxidation of cytochrome c²⁺, oxidation of dihydrorhodamine 123 to rhodamine 123, and hydroxylation of benzoate were measured as described [18, 28–30]. These assays detect oxidative processes triggered by various forms of peroxynitrite, namely, oxidation of cytochrome c²⁺, which follows a second-order reaction triggered by ground state peroxynitrite, hydroxylation of benzoate, which follows a first-order reaction triggered by activated peroxynitrous acid and oxidation of dihydrorhodamine 123, which detects the combination of first- and second-order processes [28, 29]. In all assays, glutathione, a known scavenger of peroxynitrite, was used as a positive control.

For the measurement of the peroxynitrite-dependent oxidation of cytochrome c²⁺, cytochrome c was reduced by sodium dithionite immediately before use and purified by chromatography on Sephadex G-25 using 100 mM potassium phosphate plus 0.1 mM DTPA, pH 7.2 as the elution buffer. Cytochrome c²⁺ oxidation (50 µM) yields upon addition of peroxynitrite (25 µM initial concentration after mixing) were assessed by incubation of reaction mixtures in 100 mM potassium phosphate plus 0.1 mM DTPA, pH 7.2 at 22°C for 3 min in the absence or presence of various concentrations of 3-aminobenzamide or glutathione. Oxidation of cytochrome c²⁺ was followed at 550 nm using a Beckman DU 640 spectrophotometer (Fullerton, CA). For the measurement of the peroxynitrite-dependent hydroxylation of benzoate, peroxynitrite (100 µM initial concentration after mixing) was added into a buffer containing 1 mM sodium benzoate in 100 mM potassium phosphate plus 0.1 mM DTPA, pH 7.2 at 25°C, in the absence or presence of various concentrations of 3-aminobenzamide or glutathione. After a 3 min incubation at 22°C, fluorescence was measured using a Perkin-Elmer fluorimeter (Model LS50B; Perkin-Elmer, Norwalk, CT) at an excitation wavelength of 300 nm, emission wavelength of 410 nm (slit widths 2.5 and 3.0 nm, respectively). For the peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine 123, [18], peroxynitrite (5 µM initial concentration after mixing) was added into phosphate-buffered saline containing 10 µM dihydrorhodamine 123, in the absence or presence of 3-aminobenzamide or glutathione. After a 10 min incubation at 22°C, the fluorescence of rhodamine 123 was measured using the Perkin-Elmer fluorimeter at an excitation wavelength of 500 nm, emission wavelength of 536 nm (slit widths 2.5 and 3.0 nm, respectively).

2.7 Effect of 3-aminobenzamide on endothelial NO synthase activity in vitro
For the investigation of potential effects of 3-aminobenzamide on the activity of endothelial NO synthase activity, we measured the effect of 3-aminobenzamide on the conversion of L-arginine to L-citrulline in endothelial cell homogenates. We have also measured the potential effects of 3-aminobenzamide on the acetylcholine-elicited, endothelium-dependent relaxations in precontracted rat thoracic aortic rings. In these experiments, we have used L-NMA, a prototypical NO synthase inhibitor as positive control.

Calcium-dependent conversion of L-arginine to L-citrulline in cell homogenates obtained from the scraped intimal surface of fresh bovine aortae served as a model of endothelial NO synthase activity [31]. Cells were homogenized in a buffer composed of 50 mM Tris·HCl, 0.1 mM EDTA and 1 mM phenylmethylsulphonyl fluoride (pH 7.4) on ice. Conversion of [³H]-L-arginine to [³H]-L-citrulline was measured in the homogenates as described [31]. Briefly, homogenates (30 µl) were incubated in the presence of [³H]-L-arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM) and calcium (2 mM) for 20 min at 22°C. Reactions were stopped by dilution with 0.5 ml of ice cold HEPES buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W (Na⁺ form) columns and the eluted [³H]-L-citrulline activity was measured by a scintillation counter.

For the measurement of isometric force in vascular rings [11], thoracic aortae from rats were cleared of adhering periadventitial fat and cut into rings of 3–4 mm width. Rings were mounted in organ baths (5 ml) filled with warmed (37°C), oxygenated (95% O₂/5% CO₂) Krebs' solution (pH 7.4) consisting of (mM): NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25 and glucose 11.7. Experiments were performed in the presence of indomethacin (10 µM). Isometric force was measured with isometric transducers (Kent Scientific Corp. Litchfield, CT, USA), digitized using a Maclab A/D converter (AD Instruments, Milford, MA, USA), and stored and displayed on a Macintosh personal computer. A tension of 1 g was applied and the rings were equilibrated for 60 min. Fresh Krebs' solution was provided at 15 min intervals. To investigate changes in endothelium-dependent relaxant responsiveness, rings were precontracted with noradrenaline (10^–6 M), and then concentration-response curves to acetylcholine (10^–8–10^–5 M) were obtained. These vascular studies were performed in three groups of rings: in control rings, in rings pretreated with 3-aminobenzamide (1 mM for 10 min) and in control rings pretreated with L-NMA (3 mM, 10 min).

2.8 Studies in cell culture
Rat heart myoblasts (H9C2) and human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (Rockville, MD). H9C2 cells were cultured in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum; HUVEC cells were cultured in F12K nutrient medium containing 10% fetal calf serum, heparin (100 µg/ml) and endothelial cell growth supplement (30 µg/ml) in 96 well plates or 12 well plates. When cells reached 90–100% confluence culture medium was replaced by fresh medium and the cells were incubated with exogenous peroxynitrite (500 µM) for 1 h in the presence or absence of 3-aminobenzamide (1 mM).

Mitochondrial respiration was assessed by the mitochondrial-dependent reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan as described [9]. For measurement of PARS activity, cells were incubated for 5 min at 37°C with 0.5 ml of 56 mM HEPES buffer (pH 7.5) containing 28 mM KCl, 28 mM NaCl, 2 mM MgCl₂, 0.01% digitonin, and 125 nM NAD⁺ spiked with 0.25 µCi [³H]-NAD⁺ [9]. The proteins that were ribosylated with [³H]-NAD⁺ were precipitated with 200 µl of 50% TCA. After two washes with TCA, the protein pellet was solubilized in 2% SDS in 0.1 M NaOH, incubated at 37°C overnight, and the radioactivity was determined by scintillation counting.

2.9 Materials
Cell culture medium, heparin and fetal calf serum were obtained from Gibco (Grand Island, NY). Perchloric acid was obtained from Aldrich (St. Louis, MO). [³H]-NAD⁺ was purchased from DuPont/NEN (Boston, MA, USA). Primary anti-nitrotyrosine antibody was purchased by Upstate Biotech (Saranac Lake, NY). Reagents, secondary and non specific IgG antibodies for immunohistochemical analysis were from Vector Laboratories Inc. (Burlingame, CA). Dihydrorhodamine 123 was from Molecular Probes (Eugene, OR). Peroxynitrite was synthesized and kindly provided by Dr. H. Ischiropoulos (University of Pennsylvania). All other chemicals were from Sigma/Aldrich (St. Louis, MO).

2.10 Data analysis
All values in the figures and text are expressed as mean ± standard error of the mean of n observations, where n represents the number of rats (n = 6 animals for each group) in the in vivo experiments or wells in the in vitro cell culture experiments (6–9 wells from 2–3 independent experiments). The results were examined by one- and two-way analysis of variance and individual group means were then compared with Bonferroni's test. For the quantification of the nitrotyrosine staining, Mann–Whitney's non-parametric test was used to determine statistical differences between the various groups. A p-value less than 0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Peroxynitrite production during myocardial ischemia and reperfusion
Nitrotyrosine (a specific marker of peroxynitrite-induced tyrosine nitration process) was detected by immunohistochemical analysis in the reperfused myocardium. Sections of control hearts did not reveal any immunoreactivity for nitrotyrosine within the normal architecture (Fig. 1A). In sections of reperfused hearts after ischemia a strong positive brown staining for nitrotyrosine was observed within injured myocytes in the infarcted area (Fig. 1B). In infarcted hearts from 3-aminobenzamide-treated rats nitrotyrosine staining was markedly reduced (Fig. 1C). On the 0–4 grading scale, the intensity of nitrotyrosine staining was 0; 0; 3.5±0.2 and 0.8±0.3 in the control, control+3-aminobenzamide; ischemia-reperfusion and ischemia-reperfusion +3-aminobenzamide groups, respectively (n = 5–6). Thus, there was a significant (p<0.01) reduction in the degree of nitrotyrosine staining by 3-aminobenzamide during ischemia-reperfusion.

View larger version (98K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Immunohistochemical staining of nitrotyrosine in heart sections. (A) Section from a sham rat showing the normal architecture of myocardial tissue and absence of nitrotyrosine staining. (B) Section from a rat subjected to myocardial ischemia and reperfusion treated with vehicle showing massive derangement of myocyte structure and nitrotyrosine positive staining (arrows). (C) Section from a rat subjected to myocardial ischemia and reperfusion treated with 3-aminobenzamide showing a reduction of myocardial damage and absence of nitrotyrosine staining. Magnification:x62.5. Figures show representative immunohistochemical patterns. A similar pattern was seen in n = 4–6 different tissue sections in each group.

 
3.2 PARS inhibition reduces myocardial injury and neutrophil infiltration
Myocardial ischemia and reperfusion resulted in a marked cellular injury, as measured by an increase of plasma creatine phosphokinase activity (Fig. 2A) and by depletion of cellular high energetic phosphate stores of ATP (Fig. 2B). Analysis of tissue myeloperoxidase activity indicated a marked neutrophil accumulation in the infarcted hearts (Fig. 2C). The infarcted area corresponded 56.2±6.4 of total area at risk. Inhibition of PARS activity by 3-aminobenzamide reduced the extent of myocardial injury after reperfusion as indicated by a reduction of creatine phosphokinase levels in the plasma, and partially preserved tissue ATP levels (Fig. 2A, B). Administration of 3-aminobenzamide also resulted in a suppression of the increase in cardiac myeloperoxidase activity in response to reperfusion (Fig. 2C). Infarct size (normalized to the area at risk) was reduced to 36.0±0.4% by 3-aminobenzamide treatment (p<0.05, n = 6). Area at risk was equal in both groups (49.8±2.5% in vehicle-treated rats and 47.5±4.3% in the 3-aminobenzamide-treated rats). The pressure-rate index decreased shortly after occlusion of the main left coronary artery and further declined upon reperfusion reflecting an impaired myocardial function. Pressure-rate index (expressed as mmHgxbeats/min/1000) decreased from control value (62.8±4.2) to 27.5±3.6 at the end of the occlusion and to 23.6±3.2 at the end of reperfusion in vehicle-treated animals. In the 3-aminobenzamide-treated animals there was a similar decrease in the pressure-rate index (35.3±4.5 at the end of the occlusion and 30.4±4.6 at the end of reperfusion, n = 6).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of in vivo treatment with 3-aminobenzamide or vehicle on plasma levels of creatine phosphokinase (A), myocardial levels of ATP (B) and myocardial myeloperoxidase activity (C). All values are expressed as mean±standard error of the mean of 6 animals in each group. *P<0.01 versus sham; #P<0.01 indicates protective effect of 3-aminobenzamide versus vehicle treatment.

 
3.3 3-aminobenzamide does not scavenge peroxynitrite and does not inhibit NO synthase
The reduction in myocardial nitrotyrosine immunoreactivity in response to reperfusion raised the question whether the PARS inhibitor would also act as a direct scavenger of peroxynitrite and thus would directly interfere with peroxynitrite-induced oxidative processes. We have therefore tested the effect of 3-aminobenzamide in multiple, peroxynitrite-related oxidative assays. The known peroxynitrite scavenger glutathione at 1 µM–3 mM, caused a dose-dependent suppression of peroxynitrite-induced oxidation of cytochrome c²⁺, hydroxylation of benzoate and oxidation of dihydrorhodamine 123 (Fig. 3). On the other hand, 3-aminobenzamide did not inhibit these processes, indicating that the inhibitor does not act as a scavenger of peroxynitrite (Fig. 3).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of 3-aminobenzamide (open circles) or glutathione (closed circles) on the peroxynitrite-induced (a) oxidation of cytochrome c2+, hydroxylation of benzoate (b) and oxidation of dihydrorhodamine 123 (c). 100% represents values in the presence of peroxynitrite, and in the absence of the pharmacological agents tested. Data are expressed as means±s.e.m. of n = 4–6 determinations.

 
The question also arises whether 3-aminobenzamide may act as a direct inhibitor of NO synthase. In in vitro experiments, we have demonstrated that 3-aminobenzamide up to 3 mM does not inhibit endothelial NO synthase activity, whereas the positive control compound used (L-NMA) does (Fig. 4a). Moreover, L-NMA, but not 3-aminobenzamide, inhibited the endothelium-dependent relaxations elicited by acetylcholine in precontracted rat thoracic aortic rings (Fig. 4b).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (a) Effect of 3-aminobenzamide (open circles) or L-NMA (closed circles) on the conversion of L-arginine to L-citrulline in endothelial cell homogenates. (b) Effect of 3-aminobenzamide (1 mM) or L-NMA (3 mM) on the acetylcholine-induced, endothelium-dependent relaxations in precontracted rat thoracic aortic rings. Data are expressed as means±s.e.m. of n = 4–6 determinations.

 
3.4 PARS inhibition suppresses oxidant-mediated cytotoxicity in rat myoblasts and human umbilical vein endothelial cells
Based on the above-described data demonstrating that 3-aminobenzamide protects against the myocardial injury and against the neutrophil infiltration into the reperfused myocardium, we hypothesized that the cardiac myocytes and the vascular endothelium may be important sites of protection by the PARS inhibitor. Therefore, we investigated the effect of inhibition of PARS against oxidant injury in cultured cardiac myoblasts and endothelial cells. Both in cultured rat myoblast H9C2 cells, and in human umbilical vein endothelial cells (HUVEC), exogenous peroxynitrite (500 µM) caused a significant suppression of mitochondrial respiration and simultaneous activation of PARS (Fig. 5). Pretreatment of H9C2 and HUVEC cells with 3-aminobenzamide (1 mM) resulted in the prevention of PARS activation and protection against the cytotoxic effect of peroxynitrite (Fig. 5).

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of peroxynitrite (ONOO–, 500 µM) on PARS activity (A, C) and mitochondrial respiration (B, D) in H9C2 rat myocytes (A, B) and human umbilical vein endothelial cells (HUVEC) (C, D) in the presence or absence of the PARS inhibitor 3-aminobenzamide (3-AB, 1 mM). **P<0.01 represents significant changes elicited by ONOO– when compared to unstimulated controls; ##P<0.01 represents significant protection by 3-AB against the peroxynitrite-induced alterations. Data are expressed as means±standard error of the mean of n = 6–9 wells.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 3-aminobenzamide, an inhibitor of PARS, reduces myocardial ischemia-reperfusion injury: potential mechanisms of action
In the experiments described here, 3-aminobenzamide, an inhibitor of PARS, exerted cardioprotective effects in a rat model of myocardial infarction by occlusion and reperfusion of the left coronary artery. The following functional parameters were measured: (1) infarct size; (2) histological damage; (3) plasma creatine phosphokinase activity; (4) cardiac myeloperoxidase; (5) cardiac ATP levels and (6) pressure-rate index. The reduction in the infarct size, plasma creatine phosphokinase activity and the improved histological picture of the reperfused myocardium is all consistent with an amelioration of the myocardial injury by 3-aminobenzamide in this model. The reduced cardiac myeloperoxidase activity after 3-aminobenzamide treatment is consistent with reduced infiltration of neutrophil granulocytes into the reperfused myocardium. The modest preservation of the myocardial ATP levels may be related to the reduced infarct size, since the ATP determinations were performed in whole hearts. Alternatively, the increased ATP may be related to improved cellular energetic status of the myocardium.

Clearly, the clarification of the mechanism of 3-aminobenzamide's protective action requires further investigation. 3-aminobenzamide is a prototypical PARS inhibitor which has been used in a large number of investigations to inhibit the catalytic activity of PARS, when DNA single strand breakage was triggered by oxyradicals or by peroxynitrite. In these studies, 3-aminobenzamide provided cytoprotective effects but did not interfere with the development of DNA strand breakage, supporting the view that the agent, at 1–3 mM, does not scavenge oxyradicals or peroxynitrite [3, 5, 7, 9, 10, 33–35]. The benzamide structure does nevertheless have some potential for hydroxyl scavenging [36, 37], and, thus, hydroxyl scavenging may represent an additional mode of 3-aminobenzamide's action. In a recently completed set of parallel investigations, Thiemermann and co-workers (1997) tested a range of structurally distinct PARS inhibitors, as well as 3-aminobenzoic acid (the latter being an agent structurally related to 3-aminobenzamide, without inhibitory effect on PARS) in a rabbit model of myocardial reperfusion injury [38]. Since several distinct PARS inhibitors had protective effects, whereas 3-aminobenzoic acid did not, these investigators concluded that the protection is likely related to inhibition of the catalytic activity of PARS [38].

In the current studies, we directly demonstrated the ability of peroxynitrite to activate PARS and to cause a related suppression of mitochondrial respiration in endothelial cells and cardiac myocytes. One could argue that the concentration of peroxynitrite required in vitro to elicit PARS activation and cytotoxicity is rather high. However, as discussed previously [9, 11, 39, 40], when peroxynitrite is added to the culture medium, the vast majority of the oxidant will be neutralized due to interactions with culture medium constituents, and then due to interactions with the membrane, and cytosolic scavengers. For instance, when cells are exposed to peroxynitrite in phosphate buffered saline, rather than culture medium containing 10% serum, the concentrations of peroxynitrite required to elicit DNA single strand breakage are markedly reduced [40]. Moreover, the pharmacological depletion of cellular glutathione pools causes a 10-fold enhancement of the cytotoxic effect of exogenously added peroxynitrite [41]. The other important issue is related to the half-life of peroxynitrite: in the in vitro studies, a single "injection" of peroxynitrite (half-life<1 s) is used, as opposed to a more prolonged production of peroxynitrite in vivo. Theoretical considerations implicate that the addition of 250 µM peroxynitrite is equivalent to an in vitro steady-state production of 1 µM peroxynitrite for only 7 min [39]. Because of the complexity of the reaction kinetics of peroxynitrite in vivo, and the presence of endogenous antioxidants, it is difficult to estimate the concentration of peroxynitrite in vivo. However, the nitrotyrosine staining clearly shows the presence of nitrotyrosine in the reperfused heart, in accordance with a recent study by Wang and Zweier [19]. It is conceivable that peroxynitrite, produced under such conditions, contributes to the cellular injury. Such a conclusion is also strengthened by a number of pharmacological studies, where treatment with NOS inhibitors reduced myocardial injury after ischemia and reperfusion [19–21].

4.2 Inhibition of PARS reduces neutrophil accumulation and peroxynitrite formation during reperfusion
Although we have demonstrated that 3-aminobenzamide is not a direct scavenger of peroxynitrite and does not inhibit the synthesis or action of its precursors (see above), we found that, unexpectedly, there was a remarkable reduction in the degree of tyrosine nitration in the reperfused myocardium in the 3-aminobenzamide-treated animals. These observations suggest that inhibition of PARS, in an indirect way, influences the amounts of reactive peroxynitrite produced. Although the mechanism of this action clearly requires further work, we propose a hypothesis related to self-amplifying, positive feedback circles. Myocardial ischemia and reperfusion is associated with neutrophil accumulation with subsequent burst of oxygen free radical production, and ultimately, cell death [13–15, 42]. In our study, we found that inhibition of PARS by 3-aminobenzamide prevented the neutrophil infiltration into the reperfused myocardium, as demonstrated by a significant reduction in myeloperoxidase activity. Peroxynitrite and hydroxyl radical are known to exert cytotoxic effects to various tissues, including the vascular endothelium, and the mechanism of this injury, at least in part, is mediated by PARS activation [4, 34–36, 43, 44]. Endothelium-derived NO is a potent inhibitor of both neutrophil aggregation and adherence [45, 46]. Therefore, an improvement of endothelial function by 3-aminobenzamide would be expected to reduces the infiltration of neutrophils during reperfusion, thereby resulting in reduced tissue injury. In other words, we propose the following positive feedback cycle in myocardial ischemia-reperfusion: early hydroxyl radical and peroxynitrite production >> PARS-related endothelial injury >> neutrophil infiltration >> more hydroxyl and peroxynitrite production. Inhibition of PARS would interrupt this cycle at the level of endothelial injury. It is presumable that various oxyradical scavengers, which also have been found to block the increase in myocardial myeloperoxidase activity during reperfusion [47, 48], interrupt a similar positive feedback cycle.

4.3 Conclusion and implications
During the completion of the current studies, a report demonstrating the reduction by several classes of PARS inhibitors (including 3-aminobenzamide) of the infarct size in a rabbit model of myocardial ischemia reperfusion has been published [38]. Taken together, based on the results of this latter study and on the results of the current study, it can be proposed that pharmacological inhibitors of PARS may be useful for improving the outcome of myocardial ischemia/reperfusion injury. Nevertheless, the direct proof for the role of PARS in the pathogenesis of shock or reperfusion injury is still lacking, and we believe that definite proof for the involvement of PARS can only be reached by the use of genetically engineered animals lacking PARS [49].

Based on the results of the current study, and also based on a number of other in vivo and in vitro studies [3–12, 24, 33–35, 38, 44]it appears to be of importance to further investigate the therapeutic potential of strategies based on the inhibition of PARS in shock, inflammation and reperfusion injury.

Time for primary review 31 days.

	Acknowledgements

 
This work was supported by the American Heart Association (Ohio Section) to Dr. Zingarelli and by a Grant from the Children's Hospital Medical Center of Cincinnati ("Trustees Grant") to Dr. Szabó. The authors would like to thank Michael O'Connor and Alvin Denenberg for technical assistance.

	References

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