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Am J Physiol Heart Circ Physiol 283: H69-H76, 2002. First published February 28, 2002; doi:10.1152/ajpheart.00817.2001
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Vol. 283, Issue 1, H69-H76, July 2002

Nitrate tolerance does not increase production of peroxynitrite in the heart

Tamás Csont1, Csaba Csonka1, Annamária Ónody1, Anikó Görbe1, László Dux1, Richard Schulz2, Gary F. Baxter3, and Péter Ferdinandy1

1 Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, H-6720, Hungary; 2 Cardiovascular Research Group, Departments of Pharmacology and Pediatrics, University of Alberta, Edmonton, Alberta, Canada T6G 2R7; and 3 The Royal Veterinary College, University of London, London NW1 0TU, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.

nitroglycerin; nitric oxide; reactive oxygen species; myocardium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC HEART DISEASE is a leading cause of death in developed countries and the quality of life of patients suffering from ischemic heart disease is significantly impaired. Organic nitrates including nitroglycerin have been used successfully in the treatment and prevention of ischemic heart disease for >100 years. However, development of tolerance to the vascular effects of nitrates due to long-term administration limits their clinical application (27). Moreover, recent clinical studies (17, 25) suggest that long-term nitrate treatment does not improve and may even worsen mortality due to cardiovascular reasons. Although the background of nitrate-induced increase in cardiovascular mortality is still not known, the possible role of nitrate tolerance has been suspected (17, 25).

Experimental evidence on the cellular mechanism of some of the adverse effects of nitrates have been described only in the past few years. Development of experimental vascular nitrate tolerance due to nitroglycerin treatment has been shown to increase the formation of reactive oxygen species (10, 24) and peroxynitrite in the vasculature, which leads to vascular dysfunction (9, 21). In light of these observations, treatment of patients with organic nitrate compounds may lead to side effects possibly due to generation of reactive oxygen species in the vasculature.

Most of the studies describing the possible oxidative effect of nitrates have looked at their vascular effects. There have been no attempts made so far to evaluate the effect of nitrates on the generation of reactive oxygen species directly in the heart. However, this may be the key missing information relevant to the unwanted effects of nitrates.

The reaction of nitric oxide (NO) with superoxide radical leads to the formation of peroxynitrite. The rate of this reaction is determined by the amount of NO and superoxide and the activity of superoxide dismutase (SOD) in the cellular microenvironment. Endogenous formation of peroxynitrite and reactive oxygen species is known to exert detrimental effects on myocardial function (1, 13, 14, 32). These findings suggest that similar to that in the vascular system, treatment with organic nitrates, especially during the development of nitrate tolerance, may result in oxidative stress and contractile dysfunction in the heart.

On the other hand, many studies (2, 6, 7, 15, 16, 26, 34, 37) show that NO donor compounds including nitroglycerin exert direct cardiac protective effects. We (7) have shown that nitroglycerin exerts a direct myocardial anti-ischemic effect in isolated rat hearts that is independent of the vascular effects of the drug and it is not diminished even in the presence of vascular nitrate tolerance. Furthermore, nitroglycerin and other NO donors have been shown to be cardioprotective by inducing delayed (or late) preconditioning via a possible free radical-mediated trigger mechanism (2, 26, 37).

The chemical nature of reactive oxygen species including peroxynitrite makes them very difficult to measure. This technical difficulty may result in the controversial findings in the literature as to whether nitrate treatment leads to enhanced oxidative stress and subsequent organ dysfunction or exerts protective effects in the cardiovascular system. Therefore, in the present study we systematically analyzed whether nitroglycerin treatment, leading to the development of vascular nitrate tolerance, influences formation of cardiac reactive oxygen species and thus myocardial contractile performance. We therefore measured cardiac levels of NO, superoxide, their reaction product peroxynitrite, and hydroxyl radical, a major toxic derivative of peroxynitrite and superoxide. We also determined activities of major enzymatic sources for NO and superoxide, i.e., NO synthases and xanthine oxidoreductase (11), activity of the major antioxidant enzyme SOD, as well as several parameters of myocardial contractile function.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All investigations conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985), and was approved by the ethics committee at University of Szeged.

Nitrate treatment and induction and verification of vascular nitrate tolerance. Male Wistar rats (300-360 g) were given nitroglycerin (100 mg/kg sc) or its vehicle lactose 3 times a day for 3 days (9 repetitive injections) to induce vascular tolerance to nitroglycerin, as described in earlier studies (7, 16, 35). All of these animals were used for isolated heart preparations and biochemical measurements on the fourth day, ~12 h after the last injection. Development of vascular tolerance to nitroglycerin was confirmed by testing aortic rings for isometric tension as described (16, 36). Rats were deeply anesthetized with diethylether and the thoracic aorta was then carefully removed and cleaned. Rings (4 mm long) were exposed to increasing concentrations of norepinephrine (10-9-10-5 M) until a maximum contractile response was obtained. After being washed, rings were precontracted with an EC50 concentration of norepinephrine in addition to a resting tension of 20 mN. Rings were then exposed to cumulative concentrations of nitroglycerin in half-log increments.

In separate experiments, rats were injected subcutaneously with a single dose of 100 mg/kg nitroglycerin to test whether acute treatment with a single dose of nitrate without the development of tolerance affects systemic peroxynitrite formation. Rats were anesthetized with diethylether 1, 5, and 12 h after nitroglycerin treatment (n = 6 in each group). The thorax was then quickly opened and immediately before the heart was excised; ~0.5-1 ml blood was drawn from the vena cava inferior for determination of serum nitrotyrosine levels. Hearts were used for isolated heart preparations.

Measurement of cardiac function in isolated rat hearts. Rats were anesthetized with diethylether and given 500 U/kg heparin intravenously. Hearts were then isolated and perfused at 37°C in a working mode with recirculating Krebs-Henseleit bicarbonate buffer containing (in mmol/l) 118 NaCl, 4.3 KCl, 2.4 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, and 11.1 glucose, gassed with 95% O2-5% CO2, and supplemented with 0.3 mmol/l L-tyrosine (7, 13). Preload (1.7 kPa) and afterload (9.8 kPa) were kept constant throughout the experiments. After a 10-min normoxic, normothermic perfusion, cardiac mechanical functional and hemodynamic parameters including heart rate, coronary flow, aortic flow, left ventricular developed pressure and its first derivatives (±dP/dtmax), and left ventricular end-diastolic pressure were monitored as described (5, 7, 13). Coronary effluent and cardiac tissue were sampled and frozen in liquid nitrogen for further biochemical measurements.

Measurement of cardiac NO, superoxide, and hydroxyl radical formation. In separate experiments, NO content of ventricular tissue was measured using electron spin resonance (ESR) spectroscopy after loading the heart with the NO-specific spin trap Fe2+-N-methyl-D-glucosamine-dithiocarbamate (MGD) as described (5, 7). The spin trap for NO was prepared freshly before each experiment. MGD (175 mg) and 50 mg FeSO4 dissolved in distilled water (pH 7.4, volume 6 ml) was infused into the aortic cannula under Langendorff perfusion (constant pressure at 9.8 kPa) for 5 min at a rate of 1 ml/min to measure basal cardiac NO content. Tissue samples from the apex of the heart (~150 mg) were collected at the end of the infusion of Fe2+ (MGD)2 and placed into quartz ESR tubes and frozen in liquid nitrogen. ESR spectra of NO-Fe2+-(MGD)2 adducts were recorded with a spectrometer (model ECS106, Bruker; Rheinstetten, Germany) (ESR parameters: X band, 100 kHz modulation frequency, 160 K temperature, 10 mW microwave power, 2.85 G modulation amplitude, 3,356 G central field) and analyzed for NO signal intensity as described (5, 6).

Superoxide production in freshly minced ventricles was assessed by lucigenin-enhanced luminescence as described (13). Approximately 150 mg of the heart apex were placed in 1 ml of air-equilibrated Krebs-Henseleit solution containing 10 mmol/l HEPES-NaOH (pH 7.4) and either 250 or 5 µmol/l lucigenin (13). Chemiluminescence was measured in a liquid scintillation counter with single active photomultiplyer positioned in out-of-coincidence mode in the presence or absence of the superoxide scavenger nitro blue tetrazolium (200 µmol/l). Nitro blue tetrazolium-inhibitable chemiluminescence was considered an index of cardiac superoxide generation.

To estimate cardiac hydroxyl radical generation, 2,5-dihydroxybenzoic acid formation was determined by high-performance liquid chromatography (HPLC) from coronary effluents following Langendorff perfusion (9.8 kPa) of the hearts with Krebs-Henseleit buffer containing 1 mmol/l salicylic acid in separate experiments (40). Twenty microliters of filtered coronary effluents were injected to a LiChrospher 100 RP-18 cartridge (model 50734, Merck; Darmstadt, Germany). The HPLC system consisted of a Gilson 307 pump, an ESA model 5011 analytical cell, and an ESA Coulochem II electrochemical detector. 2,5-Dihidroxybenzoic acid was eluted by a buffer containing 50 mM sodium acetate, 50 mM citric acid, and 25% methanol (pH 2.5) at a flow rate of 0.5 ml/min. The detection potential was maintained at 750 mV. Peaks were integrated and compared with values obtained with serial concentrations of standard 2,5-dihydroxybenzoic acid.

Measurement of cardiac NO synthase, xanthine oxidoreductase, and SOD activities. To estimate endogenous enzymatic NO production, Ca2+-dependent and Ca2+-independent NO synthase activities in ventricular homogenates were measured by the conversion of L-[14C]arginine to L-[14C]citrulline as previously described (13). Powdered frozen ventricular tissue was placed in four volumes of ice-cold homogenization buffer (composition given in Ref. 33) and homogenized with an Ultra-Turrex disperser using three strokes of 20-s duration each. The homogenate was centrifuged (1,000 g for 10 min) at 4°C and the supernatant was kept on ice for immediate assay of enzyme activities. Samples were incubated for 25 min at 37°C in the presence or absence of EGTA (1 mM) or EGTA plus NG-monomethyl-L-arginine (1 mM) to determine the level of Ca2+-dependent and Ca2+-independent NO synthase activities, respectively. NO synthase activities were expressed in picomoles per minute per milligram of protein.

Activity of xanthine oxidoreductase (xanthine oxidase and xanthine dehydrogenase), the major source of superoxide in rat hearts (11), was determined from ventricular homogenates by a fluorometric kinetic assay based on the conversion of pterine to isoxanthopterine in the presence (total xanthine oxidoreductase activity) and absence (xanthine oxidase activity) of the electron acceptor methylene blue, as described by Beckman et al. (4). Ventricular homogenates were prepared as for the measurement of NO synthase activity.

Total activity of SOD was measured by a spectrophotometric assay using a kit (Randox Laboratories). Approximately 100 mg ventricular tissue was homogenized in 10 volumes of ice-cold phosphate buffer (0.01 M, pH 7.0). SOD activity in homogenates was determined by the inhibition of formazan dye formation due to superoxide generated by xanthine and xanthine oxidase.

Measurement of markers of peroxynitrite. We measured dityrosine by spectrofluorometry in the perfusate and free nitrotyrosine by enzyme-linked immunosorbent assay (ELISA) in the perfusate and in the serum as markers of peroxynitrite generation (41, 42). ELISA (Cayman Chemical; Ann Arbor, MI) was conducted as described (13). Briefly, serum or perfusate samples were deproteinized by the addition of 4 volumes of ice-cold ethanol. After centrifugation, the supernatants were evaporated in nitrogen flow, dissolved in phosphate buffer, and incubated overnight with anti-nitrotyrosine rabbit IgG and nitrotyrosine acetylcholinesterase tracer in precoated (mouse anti-rabbit IgG) microplates, followed by development with Ellman's reagent.

Proteins containing nitrotyrosine residues were used as intracellular markers of peroxynitrite formation in ventricular sections obtained from control, nitrate tolerant, and positive control hearts. Positive controls were obtained by infusion of authentic peroxynitrite (1 mmol/l final concentration) for 10 min into the perfused hearts. The ventricles were frozen in isopentane cooled in liquid nitrogen and 6-µm-thick fresh frozen sections were made by a cryostate. The sections were fixed in acetone (5 min) and, after being washed, they were blocked for 15 min in Tris-buffered saline containing 1% bovine serum albumin, 0.1% Na-azide, and 0.5% Tween 20. Slices were incubated with a primary anti-nitrotyrosine antibody (rabbit polyclonal IgG, Upstate Biotechnology; Lake Placid, NY) for 2 h at room temperature. Bovine anti-rabbit IgG was used as a secondary antibody followed by fluorescein isothiocyanate-conjugated streptavidine labeling (DAKO; Glostrup, Denmark) (19). The sections were examined with the use of confocal laser scanning microscopy (Leica DMRE; Heidelberg, Germany).

Statistical analysis. Data were expressed as means ± SE and analyzed with unpaired t-test. P < 0.05 was accepted as indicating a statistically significant difference compared with the control group.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular nitrate tolerance and cardiac function. To verify the development of nitrate tolerance after in vivo repetitive nitrate treatment, isolated aortic rings obtained from control and nitroglycerin-treated (9 × 100 mg/kg) animals were precontracted with an EC50 concentration of norepinephrine. The nitroglycerin concentrations needed to produce half-maximal relaxation were 0.09 ± 0.01 µmol/l in rings obtained from control animals versus 1.79 ± 0.28 µmol/l in nitroglycerin-treated group (P < 0.05, n = 7 in both groups). This 20-fold decrease in sensitivity to nitroglycerin observed in the present study is consistent with previous observations (6, 17, 35).

Parameters of cardiac performance in isolated working rat hearts, such as heart rate, aortic flow, coronary flow, left ventricular developed pressure, left ventricular end-diastolic pressure, and ±dP/dtmax were not affected by in vivo repetitive nitroglycerin treatment compared with the control group (Table 1).

                              
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  		Table 1.   Basal myocardial functional parameters in isolated working rat hearts

In nontolerant rats (1, 5, or 12 h after treatment with a single dose of nitroglycerin), neither cardiac function (data not shown) nor nitroglycerin sensitivity of aortic rings (12-h group: EC50 of nitroglycerin 0.1 ± 0.02 µmol/l, n = 5, nonsignificant vs. control) were significantly changed. Nitroglycerin sensitivity of aortic rings was not further tested in the 1-h and 5-h groups.

Cardiac NO content and NO synthase. Cardiac NO content was significantly elevated in the nitroglycerin-tolerant group when measured by ESR spectroscopy after ex vivo spin trapping of NO in isolated hearts (Fig. 1A).


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  		Fig. 1.   Cardiac nitric oxide (NO) content (A) and Ca2+-dependent and Ca2+-independent NO synthase (NOS) activities (B) in ventricular tissue of control and nitroglycerin-treated rats. *P < 0.05 vs. control (n = 4-7 in each groups).

To test whether elevated NO derives from exogenous nitroglycerin or endogenous enzymatic sources, we measured cardiac activities of NO synthases. Endogenous enzymatic sources of NO, Ca2+-dependent and Ca2+-independent activities were not changed in the ventricular tissue due to development of nitrate tolerance (Fig. 1B).

Cardiac superoxide. To test whether nitrate tolerance increases cardiac superoxide generation, we performed lucigenin-enhanced chemiluminescence assay in fresh cardiac tissue. Cardiac superoxide generation remained unchanged due to repetitive nitroglycerin treatment compared with the control group (Fig. 2A). To further verify this negative finding, we measured the activity of xanthine oxidoreductase enzyme complex, a major enzymatic source of superoxide in rat hearts. There was no significant difference in activities of xanthine oxidase and xanthine dehydrogenase (Fig. 2B). We also tested the total activity of SOD in the heart, the major enzyme responsible for endogenous detoxification of superoxide. SOD activity was not changed in the nitroglycerin-tolerant hearts compared with controls (Fig. 2C).


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  		Fig. 2.   Cardiac superoxide production (A), xanthine oxidase (XO), and xanthine dehydrogenase (XDH) activities (B), and superoxide dismutase (SOD) activity (C) in ventricular tissue of control and nitroglycerin-treated rats (n = 6-8 in each group).

Cardiac peroxynitrite. To estimate the peroxynitrite-generating capacity of the heart, hearts were isolated from nitroglycerin-tolerant and control rats and were perfused with a buffer supplemented with 0.3 mmol/l L-tyrosine. Formation of dityrosine and nitrotyrosine in the coronary effluent markers of peroxynitrite generation did not differ significantly between the two groups (Fig. 3A). To further study markers of peroxynitrite, we performed nitrotyrosine immunostaining in ventricular slices. Nitrotyrosine immunostaining detected by confocal laser scanning microscopy was negligible in hearts of both control and nitrate-tolerant groups, whereas an intensive nitrotyrosine staining was observed in positive control hearts, which were treated with authentic peroxynitrite (images not shown).


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  		Fig. 3.   Dityrosine and nitrotyrosine, markers of peroxynitrite (A), and 2,5-dihydroxybenzoic acid (DHBA), marker of hydroxyl radical (B), formation in perfusate from hearts perfused with 0.3 mmol/l L-tyrosine or 1 mmol/l salicylic acid in control and in vivo nitroglycerin-treated groups, respectively (n = 5-7 in each group).

In hearts of nontolerant rats (1, 5, and 12 h after treatment with a single dose of nitroglycerin), formation of dityrosine in the coronary perfusate was not significantly different compared with controls (data not shown).

Cardiac hydroxyl radical. We also tested whether the formation of hydroxyl radical, a toxic derivative of peroxynitrite and superoxide, is changed in the heart as a result of development of nitroglycerin tolerance. Isolated hearts from control and nitroglycerin-tolerant rats were perfused in the presence of 1 mmol/l salicylic acid. Formation of 2,5-dihydroxybenzoic acid from salicylate in the perfusate due to hydroxyl radical activity was not significantly different (Fig. 3B).

Systemic peroxynitrite. Finally, we studied whether in vivo nitrate treatment increased formation of peroxynitrite anion in extracardiac tissues. Therefore, serum free nitrotyrosine concentration was measured in control and nitroglycerin tolerant animals, as well as in nontolerant rats treated with a single dose of nitroglycerin as a systemic marker for peroxynitrite. Serum free nitrotyrosine increased approximately twofold in nitroglycerin tolerant rats compared with controls (Fig. 4).


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  		Fig. 4.   Free nitrotyrosine concentration in deproteinized sera of control, nitroglycerin (GTN)-tolerant, and nontolerant nitroglycerin-treated rats. Serum samples were taken 12 h after repetitive nitroglycerin treatment in the tolerant group and 1, 5, or 12 h after single nitroglycerin treatment. *P < 0.05 vs. control, n = 5-7 in each groups.

To test whether development of nitrate tolerance or nitrate treatment itself affects systemic peroxynitrite formation, serum nitrotyrosine was measured in nontolerant rats treated with a single dose of nitroglycerin. One hour after a single nitroglycerin treatment serum nitrotyrosine increased approximately twofold. However, there was no significant increase in serum nitrotyrosine level at 5 and 12 h after treatment (Fig. 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first demonstration that repetitive nitrate treatment, which leads to the development of vascular nitrate tolerance, results in a sustained (>12 h) increase in serum nitrotyrosine. This, however, does not enhance production of reactive oxygen species including peroxynitrite in the heart. We have also shown here that nitroglycerin treatment increases the bioavailability of NO in the heart even when nitrate tolerance develops. We further observed that a single, acute treatment with nitroglycerin, which does not lead to nitrate tolerance, results in a transient (<5 h) increase in serum nitrotyrosine.

Many previous studies (9, 10, 22, 24) have suggested that nitrate treatment, especially when it results in the development of nitrate tolerance, is associated with increased free-radical generation in the vasculature. For instance, Münzel et al. (22) showed a twofold increase in total superoxide production and a significant decrease in vascular SOD activity in functionally damaged nitrate tolerant aortic rings. However, no studies have examined the influence of nitrate treatment and nitrate tolerance on cardiac generation of reactive oxygen species. In contrast to earlier studies on the vascular effects of nitrates and nitrate tolerance, here we have shown that the generation of cardiac superoxide, hydroxyl radical, and peroxynitrite, enzymatic superoxide synthesis by xanthine oxidoreductase and its detoxification by SOD, as well as the nonenzymatic breakdown of superoxide and peroxynitrite yielding hydroxyl radicals, were not changed in the heart after in vivo nitroglycerin treatment resulting in nitrate tolerance. Furthermore, myocardial mechanical function was not altered either as a result of vascular nitrate tolerance. This further supports the conclusion that superoxide and peroxynitrite generation in the myocardium and in the coronary vasculature were unaffected by nitroglycerin tolerance. Our data show that nitrate treatment does not enhance oxidative stress in the heart. It has been suggested that NO donors induce delayed preconditioning through the stimulation of free radical generation in the rabbit heart (26, 37). However, in those studies, a pharmacological approach (i.e., a single concentration of mercaptopropionyl glycine as a free radical scavenger) was used to investigate the involvement of free radicals in the cardioprotective effect of NO donors, and this may be less rigorous than biochemical measurements of several free radicals and their enzymatic production and breakdown as seen in the present study.

Although impairment of nitroglycerin bioconversion to NO and a decrease in NO bioavailability have been shown ex vivo in vessels from nitrate tolerant animals (23) and patients (31), these findings were refuted by others (20). Here we have demonstrated that cardiac NO accumulated after repetitive nitroglycerin treatment in vivo. This shows that nitrate treatment increases the bioavailability of NO in the heart even in the state of vascular nitrate tolerance. Because the cardiac activities of NO synthases did not change, the enhanced NO level must derive from nitroglycerin accumulated in the myocardium due to exogenous nitroglycerin treatment. This is in accordance with the observation by Torfgård et al. (39) showing the accumulation of nitroglycerin in cardiac tissue after long-term nitroglycerin treatment.

Increased cardiac NO due to nitrate treatment could result in enhanced formation of peroxynitrite in the present study. However, recent studies (18) suggest that peroxynitrite generation is mainly dependent on superoxide concentrations. Accordingly, the level of superoxide did not change after nitrate treatment in our experiments. Despite the increased level of cardiac NO, markers of peroxynitrite formation in the coronary perfusate (dityrosine and free nitrotyrosine) as well as in cardiac tissue (nitration of protein tyrosine residues) were not significantly affected by in vivo nitroglycerin treatment resulting in vascular nitrate tolerance. This shows that the generation of peroxynitrite either in the coronary vasculature or in the myocardium is not affected by nitrate treatment. Although enhanced vascular peroxynitrite formation has been reported in nitrate tolerance (9, 21), this was accompanied by increased superoxide generation in the vasculature. Nevertheless, in the heart (including coronary vasculature), we have not found any evidence of elevated superoxide or peroxynitrite formation. However, we did observe a significant increase in serum free nitrotyrosine concentration in nitrate-tolerant rats 12 h after the last nitroglycerin injection. This shows a sustained, increased production of peroxynitrite in extracardiac tissues, possibly in the extracardiac vasculature. This is in accordance with previous findings (9, 10, 21, 24) showing increased generation of reactive oxygen species in vascular tissue due to nitrate tolerance. To test whether increased generation of peroxynitrite is a general phenomenon of nitrate treatment or whether it is specific for nitrate tolerance, we also measured serum nitrotyrosine after a single dose of nitroglycerin, which does not lead to tolerance development. Increase in serum nitrotyrosine was found 1 h after administration of a single dose of nitroglycerin. However, this increase was not observed 5 and 12 h after treatment. This finding suggests that nitroglycerin treatment in the absence of nitrate tolerance leads to a transient increase in extracardiac peroxynitrite formation. This is in accordance with findings of Dikalov et al. (9), showing that the biotransformation of nitroglycerin is accompanied by superoxide formation in endothelial and smooth muscle cells in the absence of nitrate tolerance. The reason why nitrate tolerance extends the time frame of peroxynitrite formation is not clear; however, increased accumulation of tissue nitroglycerin is a plausible explanation (39).

Unfortunately, the endogenous formation of peroxynitrite cannot be directly detected in biological systems. Therefore, dityrosine and nitrotyrosine, products of the reaction between peroxynitrite and tyrosine, are the most often used markers of peroxynitrite, despite some criticism. Some in vitro biochemical data suggested that peroxynitrite does not cause tyrosine nitration at physiological pH (28), but this has been recently refuted (18, 30). Moreover, peroxynitrite-mediated nitration of tyrosine residues was proposed to be the most likely mechanism of nitrotyrosine formation in vivo (30). Myeloperoxidase activity in the presence of relatively high concentrations of nitrite may lead to nitrotyrosine formation (12). However, both myeloperoxidase activity and nitrite concentration were negligible because granulocyte-free, crystalloid-perfused hearts were used to test cardiac peroxynitrite formation in this study (13). In addition, concurrent measurement of nitrotyrosine and dityrosine decreases the possibility of false estimation of peroxynitrite generation (29). The possibility of enhanced myeloperoxidase activity in extracardiac tissues contributing to serum nitrotyrosine formation, however, cannot be excluded.

Clinical trials (17, 25, 38) have suggested that nitrate therapy may worsen the prognosis and survival in ischemic heart disease, although further studies are required to confirm these findings. Animal studies (9, 10, 22, 24) showed enhanced generation of oxygen free radicals and impaired vascular function associated with nitrate treatment. Moreover, vascular nitrate tolerance has been reported (36) to interfere with the cardioprotective action of classic preconditioning in rabbits in vivo. These observations may lead to a declining confidence in the use of nitrate therapy in patients. However, here we have provided several lines of evidence showing that in vivo nitroglycerin treatment leading to vascular nitrate tolerance does not result in oxidant stress and subsequent functional damage in cardiac tissue. Furthermore, we (7, 16) have shown previously that nitroglycerin exerts a direct anti-ischemic effect on the myocardium, which does not diminish even after the development of vascular nitrate tolerance. These findings may support the safety of nitrate therapy even in the state of nitrate tolerance. However, nitroglycerin treatment, especially when leading to vascular nitrate tolerance, induces an increase in serum nitrotyrosine, a marker for systemic peroxynitrite formation. This shows that nitrate treatment may enhance oxidative stress in extracardiac tissues. The pathological significance of extracardiac oxidative stress due to nitrate treatment requires further investigation. Nevertheless, our results that nitroglycerin may induce oxidative stress in extracardiac tissue but not in the heart suggest that either cardioselective nitrates, nitrate compounds with additional antioxidant activity, or combination therapy with nitrates and antioxidants may increase the safety of organic nitrates in the chronic treatment of ischemic heart disease. Combined treatment with nitroglycerin and the antioxidant vitamin C has been used successfully to attenuate the development of nitrate tolerance in animal studies and humans (3, 8). Our present study opens a new perspective for "nitrate-antioxidant" treatment in ischemic heart disease.

In conclusion, in vivo nitroglycerin treatment, which results in vascular nitrate tolerance, may enhance oxidative stress in extracardiac tissues, but does not increase formation of oxygen free radicals and peroxynitrite in the heart. Moreover, it increases the bioavailability of cardiac NO. These findings support the safety of nitrate therapy even in the state of vascular nitrate tolerance although the importance and possible risk of nitrate-induced increase in extracardiac peroxynitrite generation requires further investigation. Our results provide a rationale for the development of cardioselective nitrates and promote further clinical studies on combination therapy with nitrates and antioxidants.


    ACKNOWLEDGEMENTS

This work was supported by Hungarian Scientific Research Fund Grant T029843, Hungarian Ministries of Education and Health Grants FKFP-0340/2000, FKFP-0057/2001, and ETT-51/2000, Hungarian National R&D Program Grants NFKP-1/040 and NFKP-1/001, Hungarian North Atlantic Treaty Organization Cooperative Linkage Grant LST.CLG.976650, and by a cooperative grant from the British Council and the Hungarian Government. R. Schulz is a senior scholar of the Alberta Heritage Foundation for Medical Research.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Ferdinandy, Cardiovascular Research Group, Dept. of Biochemistry, Univ. of Szeged, Faculty of Medicine, Dóm tér 9, Szeged, Hungary H-6720 (E-mail: peter{at}bioch.szote.u-szeged.hu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 28, 2002;10.1152/ajpheart.00817.2001

Received 17 September 2001; accepted in final form 26 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ambrosio, G, Zweier JL, and Flaherty JT. The relationship between oxygen radical generation and impairment of myocardial energy metabolism following post-ischemic reperfusion. J Mol Cell Cardiol 23: 1359-1374, 1991[ISI][Medline].

2.   Banerjee, S, Tang XL, Qiu Y, Takano H, Manchikalapudi S, Dawn B, Shirk G, and Bolli R. Nitroglycerin induces late preconditioning against myocardial stunning via a PKC-dependent pathway. Am J Physiol Heart Circ Physiol 277: H2488-H2494, 1999[Abstract/Free Full Text].

3.   Bassenge, E, Fink N, Skatchkov M, and Fink B. Dietary supplement with vitamin C prevents nitrate tolerance. J Clin Invest 102: 67-71, 1998[ISI][Medline].

4.   Beckman, JS, Parks DA, Pearson JD, Marshall PA, and Freeman BA. A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues. Free Radic Biol Med 6: 607-615, 1989[ISI][Medline].

5.   Csonka, C, Szilvássy Z, Fülöp F, Páli T, Blasig IE, Tosaki A, Schulz R, and Ferdinandy P. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts. Circulation 100: 2260-2266, 1999[Abstract/Free Full Text].

6.   Csont, T, Páli T, Szilvássy Z, and Ferdinandy P. Lack of correlation between myocardial nitric oxide and cyclic guanosine monophosphate content in both nitrate-tolerant and -nontolerant rats. Biochem Pharmacol 56: 1139-1144, 1998[ISI][Medline].

7.   Csont, T, Szilvássy Z, Fülöp F, Nedeianu S, Páli T, Tosaki A, Dux L, and Ferdinandy P. Direct myocardial anti-ischaemic effect of GTN in both nitrate-tolerant and nontolerant rats: a cyclic GMP-independent activation of KATP. Br J Pharmacol 128: 1427-1434, 1999[ISI][Medline].

8.   Dikalov, S, Fink B, Skatchkov M, and Bassenge E. Comparison of glyceryl trinitrate-induced with pentaerythrityl tetranitrate-induced in vivo formation of superoxide radicals: effect of vitamin C. Free Radic Biol Med 27: 170-176, 1999[ISI][Medline].

9.   Dikalov, S, Fink B, Skatchkov M, Sommer O, and Bassenge E. Formation of reactive oxygen species in various vascular cells during glyceryltrinitrate metabolism. J Cardiovasc Pharmacol 3: 51-62, 1998.

10.   Dikalov, S, Fink B, Skatchkov M, Stalleicken D, and Bassenge E. Formation of reactive oxygen species by pentaerithrityltetranitrate and glyceryl trinitrate in vitro and development of nitrate tolerance. J Pharmacol Exp Ther 286: 938-944, 1998[Abstract/Free Full Text].

11.   Downey, JM, Hearse DJ, and Yellon DM. The role of xanthine oxidase during myocardial ischemia in several species including man. J Mol Cell Cardiol 20, Suppl2: 55-63, 1988[ISI][Medline].

12.   Eiserich, JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, and van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391: 393-397, 1998[Medline].

13.   Ferdinandy, P, Danial H, Ambrus I, Rothery RA, and Schulz R. Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure. Circ Res 87: 241-247, 2000[Abstract/Free Full Text].

14.   Ferdinandy, P, and Schulz R. Peroxynitrite: toxic or protective in the heart? Circ Res 88: E12-E13, 2001[ISI][Medline].

15.   Ferdinandy, P, Szilvássy Z, and Baxter GF. Adaptation to myocardial stress in disease states: is preconditioning a healthy heart phenomenon? Trends Pharmacol Sci 19: 223-229, 1998[Medline].

16.   Ferdinandy, P, Szilvássy Z, Csont T, Csonka C, Nagy E, Koltai M, and Dux L. Nitroglycerin-induced direct protection of the ischaemic myocardium in isolated working hearts of rats with vascular tolerance to nitroglycerin. Br J Pharmacol 115: 1129-1131, 1995[ISI][Medline].

17.   GISSI-3. Effects of lisinopril and transdermal glyceryl trinitrate singly and together on 6-week mortality and ventricular function after acute myocardial infarction. Gruppo Italiano per lo Studio della Sopravvivenza nell'infarto Miocardico. Lancet 343: 1115-1122, 1994[ISI][Medline].

18.   Goldstein, S, Czapski G, Lind J, and Merenyi G. Tyrosine nitration by simultaneous generation of ( · )NO and O-(2) under physiological conditions. How the radicals do the job. J Biol Chem 275: 3031-3036, 2000[Abstract/Free Full Text].

19.   Krenács, T, and Rosendaal M. Immunohistological detection of gap junctions in human lymphoid tissue: connexin43 in follicular dendritic and lymphoendothelial cells. J Histochem Cytochem 43: 1125-1137, 1995[Abstract].

20.   Laursen, JB, Mülsch A, Boesgaard S, Mordvintcev P, Trautner S, Gruhn N, Nielsen-Kudsk JE, Busse R, and Aldershvile J. In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide. Circulation 94: 2241-2247, 1996[Abstract/Free Full Text].

21.   Mihm, MJ, Coyle CM, Jing L, and Bauer JA. Vascular peroxynitrite formation during organic nitrate tolerance. J Pharmacol Exp Ther 291: 194-198, 1999[Abstract/Free Full Text].

22.   Münzel, T, Hink U, Yigit H, Macharzina R, Harrison DG, and Mülsch A. Role of superoxide dismutase in in vivo and in vitro nitrate tolerance. Br J Pharmacol 127: 1224-1230, 1999[ISI][Medline].

23.   Münzel, T, Li H, Mollnau H, Hink U, Matheis E, Hartmann M, Oelze M, Skatchkov M, Warnholtz A, Duncker L, Meinertz T, and Forstermann U. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res 86: E7-E12, 2000[ISI][Medline].

24.   Münzel, T, Sayegh H, Freeman BA, Tarpey MM, and Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest 95: 187-194, 1995[ISI][Medline].

25.   Nakamura, Y, Moss AJ, Brown MW, Kinoshita M, and Kawai C. Long-term nitrate use may be deleterious in ischemic heart disease: a study using the databases from two large-scale postinfarction studies. Multicenter Myocardial Ischemia Research Group. Am Heart J 138: 577-585, 1999[ISI][Medline].

26.   Nakano, A, Liu GS, Heusch G, Downey JM, and Cohen MV. Exogenous nitric oxide can trigger a preconditioned state through a free radical mechanism, but endogenous nitric oxide is not a trigger of classical ischemic preconditioning. J Mol Cell Cardiol 32: 1159-1167, 2000[ISI][Medline].

27.   Parker, JD, and Parker JO. Nitrate therapy for stable angina pectoris. N Engl J Med 338: 520-531, 1998[Free Full Text].

28.   Pfeiffer, S, and Mayer B. Lack of tyrosine nitration by peroxynitrite generated at physiological pH. J Biol Chem 273: 27280-27285, 1998[Abstract/Free Full Text].

29.   Pfeiffer, S, Schmidt K, and Mayer B. Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite. Implications for tyrosine modification by nitric oxide/superoxide in vivo. J Biol Chem 275: 6346-6352, 2000[Abstract/Free Full Text].

30.   Reiter, CD, Teng RJ, and Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 275: 32460-32466, 2000[Abstract/Free Full Text].

31.   Sage, PR, de La Lande IS, Stafford I, Bennett CL, Phillipov G, Stubberfield J, and Horowitz JD. Nitroglycerin tolerance in human vessels: evidence for impaired nitroglycerin bioconversion. Circulation 102: 2810-2815, 2000[Abstract/Free Full Text].

32.   Schulz, R, Dodge KL, Lopaschuk GD, and Clanachan AS. Peroxynitrite impairs cardiac contractile function by decreasing cardiac efficiency. Am J Physiol Heart Circ Physiol 272: H1212-H1219, 1997[Abstract/Free Full Text].

33.   Schulz, R, Nava E, and Moncada S. Induction and potential biological relevance of a Ca(2+)-independent nitric oxide synthase in the myocardium. Br J Pharmacol 105: 575-580, 1992[ISI][Medline].

34.   Shah, AM, and MacCarthy PA. Paracrine and autocrine effects of nitric oxide on myocardial function. Pharmacol Ther 86: 49-86, 2000[ISI][Medline].

35.   Silver, PJ, Pagani ED, de Garavilla L, Van Aller GS, Volberg ML, Pratt PF, and Buchholz RA. Reversal or nitroglycerin tolerance by the cGMP phosphodiesterase inhibitor zaprinast. Eur J Pharmacol 199: 141-142, 1991[ISI][Medline].

36.   Szilvássy, Z, Ferdinandy P, Bor P, Jakab I, Szilvássy J, Nagy I, Lonovics J, and Koltai M. Loss of preconditioning in rabbits with vascular tolerance to nitroglycerin. Br J Pharmacol 112: 999-1001, 1994[ISI][Medline].

37.   Takano, H, Tang XL, Qiu Y, Guo Y, French BA, and Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res 83: 73-84, 1998[Abstract/Free Full Text].

38.   Teo, KK, and Catellier DJ. Long-term nitrate use in chronic coronary artery disease: need for a randomized controlled trial. Am Heart J 138: 400-402, 1999[ISI][Medline].

39.   Torfgård, K, Ahlner J, Axelsson KL, Norlander B, and Bertler A. Tissue levels of glyceryl trinitrate and cGMP after in vivo administration in rat, and the effect of tolerance development. Can J Physiol Pharmacol 69: 1257-1261, 1991[ISI][Medline].

40.   Tosaki, A, Bagchi D, Páli T, Cordis GA, and Das DK. Comparisons of ESR and HPLC methods for the detection of OH · radicals in ischemic/reperfused hearts: a relationship between the genesis of free radicals and reperfusion arrhythmias. Biochem Pharmacol 45: 961-969, 1993[ISI][Medline].

41.   Van der Vliet, A, Eiserich JP, O'Neill CA, Halliwell B, and Cross CE. Tyrosine modification by reactive nitrogen species: a closer look. Arch Biochem Biophys 319: 341-349, 1995[ISI][Medline].

42.   Yasmin, W, Strynadka KD, and Schulz R. Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 33: 422-432, 1997[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 283(1):H69-H76
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