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Am J Physiol Heart Circ Physiol 293: H790-H797, 2007. First published April 20, 2007; doi:10.1152/ajpheart.01141.2006
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Possible usefulness of apocynin, an NADPH oxidase inhibitor, for nitrate tolerance: prevention of NO donor-induced endothelial cell abnormalities

Akiko Fukatsu,1 Toshio Hayashi,1 Asaka Miyazaki-Akita,1 Hisako Matsui-Hirai,1 Yukie Furutate,1 Asako Ishitsuka,1 Yuichi Hattori,2 and Akihisa Iguchi1

1Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya; and 2Department of Pharmacology, School of Medicine, University of Toyama, Toyama, Japan

Submitted 17 October 2006 ; accepted in final form 19 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The long-term benefits of nitroglycerin therapy are limited by tolerance development. Understanding the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent tolerance development. In this line, we tested interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2–3 days, basal production of nitrite and nitrate (NOx) was diminished. The diminished basal NOx levels were mitigated by intermittent treatment allowing an 8-h daily nitrate-free interval during the 2- to 3-day treatment period. Addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NOx that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) after continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N{omega}-nitro- L-arginine methyl ester caused a recovery effect on basal NOx and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from nitrate tolerance development.

bovine aortic endothelial cells; endothelial nitric oxide synthase; nitrate-free interval; nitroglycerin; reactive oxygen species

NITROGLYCERIN (NTG) is a nitric oxide (NO) donor that has been widely used for over a century to alleviate myocardial ischemia and anginal pain. The therapeutic benefit of NTG, however, is limited in time by the development of nitrate tolerance (15, 28, 31, 32). A number of experimental and clinical approaches to avoid the development of nitrate tolerance have been tested. It was advanced that the angiotensin-converting enzyme inhibitors N-acetyl-L-cysteine, L-arginine, and tetrahydrobiopterin (BH4) may restore sensitivity to NTG in the setting of tolerance (15, 18, 23, 28, 32), but their therapeutic significance is still a matter of considerable debate. Intermittent transdermal NTG therapy has been recommended to allow for an 8- to 12-h nitrate-free interval (1, 5, 11, 13). The disadvantage of this regime is the lack of protection during the nitrate-free period. Another potential problem of the nitrate-free period is a decrease in anginal threshold after NTG removal (9, 10), which has been shown as the development of rebound ischemia (12).

The mechanisms underlying nitrate tolerance have also been the subject of intensive investigation, and several mechanisms are currently considered important in the development of tolerance: mechanism-based processes involving impaired bioconversion of NTG into its active metabolite(s) (5, 39), neurohormonal responses (pseudotolerance) counteracting the primary vasodilator effects of NTG (7, 9), and production of O2–-derived free radicals scavenging NO, presumably the key intermediate of NTG effects (19, 21, 25). This increased oxidative stress could also limit endogenous NO bioavailability and contribute to endothelial dysfunction displayed as impaired endothelium-dependent vascular relaxation (26, 36). However, the precise nature of the mechanisms underlying NTG-induced endothelial dysfunction is still poorly defined.

In the present study, we investigated alterations in the features and functions of endothelial cells in the setting of nitrate tolerance. Changes in basal nitrite and nitrate (NOx) production, reactive oxygen species (ROS) release, and endothelial nitric oxide synthase (eNOS) expression were assessed in bovine aortic endothelial cells (BAECs) after long-term treatment with NTG, isosorbide dinitrate (ISDN), or sodium nitroprusside (SNP).

Previous reports have shown that NTG tolerance-derived ROS not only decrease NO bioavailability but also may cause inhibition of bioactivation process of NTG. Aldehyde dehydrogenase (ALDH)-2 inhibition as well as soluble guanylate cyclase desensitization resulting in vascular smooth muscle dysfunction have been suggested as possible mechanisms (4, 39). Chen et al. (4) purified a nitrate reductase that specifically catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from NTG and identified it as mitochondrial ALDH. Moreover, Sydow et al. (39) demonstrated that antioxidants/reductants decrease mitochondrial ROS production and restore ALDH-2 activity, suggesting its significant role in NTG tolerance. The sources for ROS are proposed to include activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and xanthine/xanthine oxidase (4, 24, 26, 36, 39).

The final goal of this study was to find pharmacological interventions that may protect against endothelial cell abnormalities in the setting of nitrate tolerance.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. ISDN, SNP, apocynin, allopurinol, perindoprilate, N-acetyl-L-cysteine, L-arginine, BH4, N{omega}-nitro-L-arginine methyl ester (L-NAME), and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (tiron) were purchased from Sigma-Aldrich Chemical (St. Louis, MO). Diphenyleneiodonium chloride (DPI) was obtained from Molecular Probes (Eugene, OR). NTG was a gift from Nihon Kayaku (Tokyo, Japan). Antibodies specific for eNOS (anti-ecNOS monoclonal antibody, no. 610297) and actin (mouse monoclonal antibody to beta-actin loading control, no. ab8226) were purchased from Transduction Laboratories (San Jose, CA) and Abcam (Cambridge, MA), respectively. Anti-mouse IgG (horseradish peroxidase-linked antibody, no. 7076) was obtained from Cell Signaling Technology (Beverly, MA). All other chemicals were of the highest purity commercially available.

Cell culture. BAECs (repository no. BW6001) were obtained from Bio Whittaker (Rockland, ME) and cultured as previously described (16). These cells exhibited typical cobblestone appearance and were positive for endothelial cell-specific von Willebrand factor and angiotensin I-converting enzyme activity. They tested negative for {alpha}-smooth muscle actin. BAECs were harvested in low-glucose DMEM supplemented with 10% calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air. Cells were seeded into 12-well plates and used at <12 passages. Before the experimental procedure started, medium was removed and replaced with phenol red-free low-glucose DMEM supplemented with 1% calf serum, 0.06% glutamine, and 1% penicillin-streptomycin.

NO donor treatment study design. In the first set of experiments, BAECs were treated with the NO donors NTG, ISDN, and SNP at different concentrations. These NO donors were given fresh on a daily basis. BAECs underwent the two treatment protocols detailed below. Thus cells were continuously treated with NO donors over 48 h or were given an 8-h daily nitrate-free interval after 16-h continuous treatment with NO donors over 2 days. All cells were kept in normal medium for 24 h after NO donors were removed, and then basal NOx production and eNOS protein expression levels were determined.

In another set of experiments, continuous treatment of BAECs with NO donors over 3 days was performed. On the other hand, three 6-h nitrate-free intervals subsequent to an 18-h treatment period with NO donors over 3 days were set up as intermittent treatment. After 24 h of incubation in normal medium after removal of NO donors, basal NOx production, eNOS protein and mRNA expression, and ROS levels were assessed. When the effects of apocynin, allopurinol, perindoprilate, N-acetyl-L-cysteine, L-arginine, BH4, and L-NAME were examined, they were added to the medium with application of NO donors.

Measurement of NOx production. NOx production by BAECs was assessed by the method that has been used routinely in our laboratory (8). In brief, NOx content of the medium was measured with an automated NO detector high-performance liquid chromatography (HPLC) system (ENO10; Eicom, Kyoto, Japan). Nitrate was converted to nitrite in an in-line copper-coated cadmium-reduction column (NO-RED), and nitrite content was detected with the Griess reaction. The incubated medium was not completely free from nitrite; therefore, an aliquot of medium underwent the same process as medium obtained from cultured cells. We usually used the nitrite value obtained with the medium alone as a blank, and it was subtracted from all the samples.

Analysis of intracellular ROS levels in BAECs. Intracellular ROS levels in BAECs were determined according to the dye incorporation studies of Daiber et al. (7) using the carboxylated form of dihydroethidium (DHE; Molecular Probes; Eugene, OR). DHE freely diffuses across cell membrane, is diacylated, and incorporates into hydrophobic lipid regions of the cell (2). After the appropriate amount of time following treatment with NO donors in the presence of NADPH, BAECs were incubated at 37°C for 30 min in phosphate-buffered saline (PBS) in which DHE was added at a final concentration of 10 µM. After incubation for 30 min at 37°C, the dye was aspirated and cells were trypsinized and washed once by centrifugation at 3,000 rpm for 5 min (4°C) to remove trypsin and extracellular DHE. BAECs were resuspended in PBS, transferred into 5-ml polystyrene round-bottom tubes with cell-strainer caps (Becton Dickinson, Franklin Lakes, NJ), protected from light, and kept cold until ready for analysis on a fluorescence-activated cell sorting (FACS) FACSCalibur flow cytometer (Becton Dickinson) set at 520-nm excitation. Emission filters were 610-nm band pass.

Western blot analysis. Immunoblotting was performed as demonstrated in our previous reports (16, 38). Briefly, the protein concentration was determined with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Samples of cell homogenate (5–10 µg) were subjected to electrophoresis on polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride filter membrane. To reduce nonspecific binding, the membrane was preincubated for 30 min at room temperature in Tris-buffered saline with Tween 20 [TTBS; 150 mM NaCl, 10 mM Tris (pH 8.0), and 0.05% Tween 20] containing 5% skim milk powder. The membrane was then incubated overnight with the primary antibody at 1:2,500 (for eNOS antibody) or 1:1,000 (for actin antibody) dilution in PBS (0.075 µg/ml). After an extensive washing with TTBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution for eNOS, 1:5,000 dilution for actin) for 60 min at room temperature. Blots were washed twice in TTBS and subsequently visualized with a SuperSignal West Dura Trial Kit (Pierce Biotechnology, Rockford, IL), exposed to X-ray film, and analyzed by NIH Image software produced by Wayne Rasband [National Institutes of Health (NIH), Bethesda, MD]. Equal protein loading was confirmed by Coomassie brilliant blue and amido black staining of protein in each lane of the same blot.

RNA extraction and RT-PCR. To isolate total RNA from cells, we used TRIzol reagent from Invitrogen Life Technologies (Carlsbad, CA), which is routinely used in our laboratory (38). RNA purity was determined by the ratio of optical density (OD) measured at 260 and 280 nm (OD260/OD280), and RNA quality was estimated at OD260. The concentration of total RNA was adjusted to 1 µg/ml with RNase-free distilled water.

The mRNA of eNOS was quantitatively determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) method with the use of an RT-PCR kit (Takara Shuzo; Ohtsu, Japan). The oligonucleotide sequence pair used for gene amplification in this study generated PCR products of expected size that have been sequenced to verify eNOS identity: sense primer 5'-CCGTGTCCAACATGCTGCTGGAAATCG-3', antisense primer, 5'-TAAAGGTCTTCTTCCTGGTGATGCC-3' (486 bp). cDNA was reverse transcribed from 1 µg of total RNA according to the manufacturer's instructions. The PCR conditions were 17 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s. After amplification, the resulting PCR products were subjected to agarose gel electrophoresis and ethidium bromide staining. The products were quantified with image analysis software (NIH) to standardize the amount of the target molecule; the amount of beta-actin mRNA, a ubiquitously expressed housekeeping gene, was determined with the following primer pair: sense 5'-CGAGCATTCCCAAAGTTCTACAGTG-3', antisense 5'-GGGGACCAAAAGCCTTCATACATC-3'.

Statistics. Data are presented as means ± SE of three separate experiments done in duplicate determinations. We thus performed one of three experiments on the same day with the same batch of cells. Statistical significance was estimated with the nonparametric Mann-Whitney U-test for comparison of two groups. A probability value of P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Continuous vs. intermittent treatment of BAECs with NO donors. The effects of continuous versus intermittent treatment with NO donors on basal NOx levels in BAECs were determined after cells were continuously exposed to NTG, ISDN, or SNP at two different concentrations (10–6 and 10–4 M) over 2 days or were given an 8-h daily nitrate-free interval after 16-h continuous treatment with the three NO donors over 2 days. Continuous treatment for 2 days with NO donors impaired basal NOx production in BAECs, although statistical significance was observed with NTG at the two concentrations and with SNP at the high concentration (Fig. 1). This impairment was evidently less pronounced in BAECs treated with a nitrate-free interval. Thus the basal NOx concentration in BAECs was decreased from 10.2 ± 0.3 to 7.0 ± 0.4 µM by continuous treatment with 10–4 M NTG for 2 days. However, an 8-h daily nitrate-free interval partially but significantly restored this decrease in basal NOx production (8.3 ± 0.3 µM). Paradoxically, continuous treatment with NTG and SNP at 10–4 M for 2 days increased the expression level of eNOS protein in BAECs (Fig. 2). In cells treated with a nitrate-free interval, the increase in eNOS protein expression was substantially reduced. We further tested the extent to which ROS can be generated when BAECs are continuously or intermittently treated with NO donors. Oxidation of DHE by ROS can be measured by FACS analysis. Figure 3 shows representative histograms comparing the fluorescence of oxidized DHE in cells treated with three different NO donors continuously and intermittently for 3 days. Figure 3A is a histogram comparing the fluorescence of oxidized DHE in the cells when NTG, ISDN, and SNP were given continuously for 3 days at a concentration of 10–4 M. Continuous treatment with these three NO donors produced similar degrees of increase in mean fluorescence. An 8-h daily nitrate-free interval markedly reduced the fluorescence in cells treated with NTG (Fig. 3B).


Figure 1
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  		Fig. 1. Effects of continuous or intermittent treatment with 3 nitric oxide (NO) donors, nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP), at concentrations of 10–6 and 10–4 M on basal nitrite and nitrate (NOx) production in bovine aortic endothelial cells (BAECs). Cells were continuously treated with NO donors over 2 days (filled bars) or were given an 8-h daily nitrate-free interval after 16-h continuous treatment with NO donors over 2 days (hatched bars). Control cells (C) were incubated for 2 days without NO donors (open bar). After 24 h of incubation in normal medium following removal of NO donors, basal NOx levels were measured. Data are means ± SE; n = 3. *P < 0.05.

 

Figure 2
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  		Fig. 2. Effects of continuous or intermittent treatment with NO donors NTG, ISDN, and SNP at a concentration of 10–4 M on endothelial nitric oxide synthase (eNOS) protein expression in BAECs. Cells were continuously treated with NO donors over 2 days (filled bars) or were given an 8-h daily nitrate-free interval after 16-h continuous treatment with NO donors over 2 days (hatched bars). Control cells were incubated for 2 days without NO donors (open bar). After 24 h of incubation in normal medium following removal of NO donors, eNOS protein expression was determined in BAECs by Western blot. Representative immunoblots are shown at top. Actin served as loading control. To standardize between experiments, an arbitrary density of 1 was assigned to the band obtained from the control sample. Data are means ± SE (n = 3) of eNOS/actin, expressed relative to the respective control. *P < 0.05.

 

Figure 3
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  		Fig. 3. Representative FACSCalibur fluorescence histograms constructed from BAECs loaded with dihydroethidium (DHE). A: cells were continuously treated for 3 days with NTG, ISDN, or SNP at 10–4 M. Control cells were incubated for 3 days without NO donors. After 24 h of incubation in normal medium following removal of NO donors, cells were incubated with DHE at 37°C for 30 min and then analyzed on a FACSCalibur flow cytometer as detailed in MATERIALS AND METHODS. B: histograms comparing the fluorescence of the cells when NTG at 10–4 M was continuously or intermittently administered for 3 days. Intermittently treated cells were given an 8-h daily nitrate-free interval after 16-h continuous treatment with NTG over 3 days. A and B are composites of separate runs, and the histogram represents the DHE fluorescence of ~20,000 cells.

 
Effects of pharmacological interventions on continuous NTG treatment-induced abnormalities in BAECs. The next set of experiments was designed to find pharmacological interventions that have a beneficial effect on the abnormalities observed in BAECs treated continuously with NTG. As presented in Fig. 4, L-NAME (10–3 M) showed a tendency to inhibit the reduction in basal NOx production when BAECs were continuously treated with 10–4 M NTG over 3 days, but this effect was not statistically significant. The NADPH oxidase inhibitor apocynin (10–3 M) significantly prevented the continuous NTG treatment-induced reduction in basal NOx production. Another NADPH oxidase inhibitor, DPI (10–7 M), showed the same effect on NTG-treated basal NOx as apocynin (n = 2, data not shown). Allopurinol (10–4 M), N-acetyl-L-cysteine (10–3 M), L-arginine (10–3 M), and BH4 (10–4 M) were without effect on the reduced basal NOx level in continuously NTG-treated cells. Further reduction in basal NOx concentration was found when the angiotensin-converting enzyme inhibitor perindoprilate (10–4 M) was given. We also tested tiron (10–3 M), known as a superoxide dismutase (SOD) mimetic, which showed a 1.3-fold increase in basal NOx production in BAECs treated continuously with NTG (n = 3, data not shown).


Figure 4
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  		Fig. 4. Effects of N{omega}-nitro-L-arginine methyl ester (L-NAME; LN, 10–3 M), apocynin (AP, 10–3 M), allopurinol (AL, 10–4 M), perindoprilate (PE, 10–4 M), N-acetyl-L-cysteine (NA, 10–3 M), L-arginine (LA, 10–3 M), and tetrahydrobiopterin (BH4; BH, 10–5 M) on continuous NTG treatment-induced reduction in basal NOx production in BAECs. Cells were continuously treated with 10–4 M NTG over 3 days. Control cells were incubated for 3 days without NTG. Each pharmacological agent was added to the medium with application of NTG. After 24 h of incubation in normal medium following removal of NTG, basal NOx levels were measured. Data are means ± SE; n = 3. *P < 0.05 vs. continuous treatment with NTG alone.

 
As seen for 2-day NO donor treatment, continuous treatment with NTG for 3 days resulted in a significant increase in eNOS protein expression in BAECs (Fig. 5). The increased eNOS protein expression was apparently normalized by L-NAME and apocynin. DPI and tiron appeared similar to apocynin in reversing the increased level of eNOS expression in continuously NTG-treated cells (n = 3, data not shown). Allopurinol, perindoprilate, N-acetyl-L-cysteine, L-arginine, and BH4 produced only marginal changes in eNOS protein expression in BAECs continuously treated with NTG over 3 days. There was an evident correlation between increases in protein and mRNA levels of eNOS resulting from continuous NTG treatment, as determined by RT-PCR analysis (Fig. 6). The mRNA level of eNOS in BAECs was highly upregulated by continuous treatment for 3 days with NTG. Both L-NAME and apocynin suppressed its increased expression to the level observed in control NTG-untreated BAECs. The same effect on eNOS mRNA expression level was obtained by an 8-h daily NTG-free interval.


Figure 5
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  		Fig. 5. Effects of L-NAME (10–3 M), apocynin (10–3 M), allopurinol (10–4 M), perindoprilate (10–4 M), L-arginine (10–3 M), and BH4 (10–5 M) on continuous NTG treatment-induced increase in eNOS protein expression in BAECs. Cells were continuously treated with 10–4 M NTG over 3 days. Control cells were incubated for 3 days without NTG. Each pharmacological agent was added to the medium with application of NTG. After 24 h of incubation in normal medium following removal of NTG, eNOS protein expression was determined in BAECs by Western blot. Representative immunoblots are shown at top. Actin served as loading control. To standardize between experiments, an arbitrary density of 1 was assigned to the band obtained from the control sample. Data are means ± SE (n = 3) of eNOS/actin, expressed relative to the respective control. *P < 0.05 vs. continuous treatment with NTG alone.

 

Figure 6
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  		Fig. 6. RT-PCR analysis showing the effects of a nitrate-free interval (On/Off), L-NAME (10–3 M), and apocynin (10–3 M) on the continuous NTG treatment-induced increase in gene expression for eNOS in BAECs. Cells were continuously treated with 10–4 M NTG over 3 days. Intermittent treatment was performed with an 8-h daily nitrate-free period after 16-h continuous NTG treatment over 3 days. Control cells were incubated for 3 days without NTG. L-NAME and apocynin were added to the medium with application of NTG. After 24 h of incubation in the normal medium following removal of NTG, mRNA expression levels of eNOS were analyzed by the RT-PCR method. eNOS mRNA was normalized as the ratio of its mRNA level relative to beta-actin mRNA used as an internal control. Data are means ± SE; n = 3. *P < 0.05 vs. continuous treatment with NTG alone.

 
As depicted in Fig. 7, apocynin displayed a substantial reduction in fluorescence of oxidized DHE in cells continuously treated with NTG over 3 days. Similar results were obtained with another NADPH inhibitor, DPI, and with the SOD mimetic tiron. L-NAME, perindoprilate, N-acetyl-L-cysteine, L-arginine, BH4, and allopurinol did not result in apparent change in fluorescence in cells treated with NTG (data not shown).


Figure 7
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  		Fig. 7. FACSCalibur fluorescence histograms constructed from BAECs loaded with DHE showing the effects of apocynin (10–3 M; A) and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (tiron, 10–3 M; B) on continuous NTG treatment-induced reactive oxygen species generation in the cells. Cells were continuously treated with 10–4 M NTG over 3 days. Each pharmacological agent was added to the medium with application of NTG. After 24 h of incubation in the normal medium following removal of NTG, cells were incubated with DHE at 37°C for 30 min and then analyzed on a FACSCalibur flow cytometer as detailed in MATERIALS AND METHODS.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study provides new evidence that apocynin prevents abnormal changes seen in endothelial cells after long-term continuous treatment with NTG. Apocynin is reported to be a potent inhibitor of NADPH oxidase in stimulated human neutrophils (33). Vascular NADPH oxidase, which can generate superoxide that may quench NO produced during NTG biotransformation, has been proposed to contribute to the development of nitrate tolerance due to continuous organic nitrate administration (20, 25, 35). Quite recently, Münzel et al. (23) reviewed the mechanisms, including NADPH oxidase, that may contribute to nitrate tolerance and endothelial cell dysfunctions. Furthermore, Otto et al. (29) have shown that apocynin produces protection against nitrate tolerance in vivo. Nevertheless, there is little information available to date in the literature to investigate the effect of apocynin on nitrate tolerance development in isolated blood vessels. Our present study represents the first published evidence from cultured endothelial cells for the important role of NADPH oxidase in the development of endothelial cell dysfunctions in the setting of nitrate tolerance.

Previous animal studies have revealed that long-term treatment with NTG patches or infusions leads to endothelial dysfunction (24, 25). In the present study, basal NO release from BAECs was markedly diminished after long-term continuous treatment with NTG. When BAECs were continuously treated with the three different NO-generating agents, however, the effects to diminish basal NO release from BAECs were somewhat different from each other. Continuous treatment with NTG at two different concentrations (10–6 and 10–4 M) and with SNP only at a high concentration significantly reduced basal NO release from BAECs, whereas the decrease in basal NO release by ISDN was not statistically significant even when a high concentration was used. It thus seems likely from these data that not all NO donors are equal in leading to the impairment of endothelial cell function with continuous treatment. It should be kept in mind that individual classes of NO donors may be endowed with distinct compound-specific biological activities. For instance, two enzyme systems, an NADPH-dependent cytochrome P-450 pathway and certain isoenzymes of the glutathione S-transferase family, have been proposed to account for the bioactivation of NTG (10). On the other hand, the mechanism of NO release from SNP remains incompletely understood, although both nonenzymatic and enzymatic NO release may occur in the biological system.

While decreasing basal NO production, long-term continuous treatment with NTG and SNP resulted in a significant increase in eNOS protein expression. In contrast with our findings, no change in eNOS mRNA and protein levels was shown in ovine pulmonary arterial endothelial cells exposed to SNP for 8–24 h (4, 37). Furthermore, it is believed that enzymatically generated NO may play an important negative-feedback regulatory role on eNOS protein expression (14). However, Münzel and colleagues (26) also showed in a preliminary study that NTG infusion in rats increases eNOS expression. Interestingly, continuous treatment with ISDN, which marginally affected basal NO release, had no significant effect on eNOS expression. Thus the apparent positive-feedback regulation of eNOS protein expression was accompanied by lowered eNOS enzymatic activity. Therefore, long-term continuous nitrate treatment may cause an uncoupling of eNOS by thiol depletion. The clinical implication of the uncoupling of increased eNOS expression by long-term nitrate therapy is uncertain. However, the increase in the amount of eNOS and, hence, paradoxically diminished NO production capacity may theoretically result in significant NO deficiency, leading to episodic vasospasm and hypertension despite lasting continuation of the drug in patients maintained on long-term nitrovasodilator therapy. This phenomenon may play a contributory role in the pathogenesis of nitrate tolerance.

Vascular endothelial cells have been shown to generate superoxide under both basal and stimulated conditions (34–36), although the sources of this superoxide remain unclear. Utilizing the probe DHE and FACS, we determined intracellular oxidant activity in BAECs receiving long-term continuous treatment with NTG, SNP, or ISDN. We found a substantial increase in DHE fluorescence even when any of the three NO donors was used for long-term continuous treatment. DHE is capable of being oxidized by hydrogen peroxide and lipid peroxides (3). It can be postulated, although not proven, that these peroxides are the result of generated cytoplasmic superoxide. A study using an experimental model of nitrate tolerance has suggested that endothelial dysfunction resulting from continuous NTG treatment is associated with NADPH oxidase-mediated superoxide production (24). In the endothelium, xanthine oxidase has also been identified as a significant producer of superoxide (22). However, the NADPH oxidase inhibitor apocynin but not the xanthine oxidase inhibitor allopurinol reduced oxidation of DHE in BAECs receiving long-term continuous NTG treatment. The increase in DHE fluorescence by continuous NTG treatment was also suppressed by another NADPH oxidase inhibitor, DPI. Furthermore, these NADPH oxidase inhibitors prevented the reduction in basal NO production and the increase in eNOS protein expression in continuously NTG-treated cells. In contrast, such effects were undetectable with allopurinol. Thus the xanthine/xanthine oxidase system may be one of the potential sources for endothelial superoxide but does not appear to play a significant role in the increase in superoxide production in BAECs continuously treated with nitrates. Alternatively, the contribution of superoxide formed by the action of the xanthine/xanthine oxidase pathway to the uncoupling of eNOS under long-term continuous nitrate treatment is minimal. We thus suggest that inhibition of NADPH oxidase-mediated superoxide production by apocynin may be important in preventing the uncoupling of eNOS and therefore correcting endothelial cell functions. This could be supported by the finding that tiron, an SOD mimetic, acted to protect basal NO.

Incubation of BAECs with the NOS inhibitor L-NAME blocked the reduction in basal NO production and the increase in eNOS protein expression due to long-term continuous nitrate treatment without preventing the increased superoxide production. The superoxide anion can be directly toxic, but its oxidant reactivity appears to be limited compared with other free radicals (40). However, NO contains an unpaired electron, is paramagnetic, and can react rapidly with superoxide to form peroxynitrate. Peroxynitrite is a strong oxidizing agent and can react readily with biological molecules, including protein and nonprotein sulfhydryls, DNA, and membrane phospholipids (17). This increase in peroxynitrite would then result in the oxidation of amino acids within the eNOS protein that are critical for enzyme activity, leading to dysfunction of eNOS. We thus assume that L-NAME may protect eNOS protein from the harmful action of peroxynitrite formed by the reaction of NO with superoxide under long-term continuous nitrate treatment conditions. However, L-NAME failed to lead to the recovery of basal NO production, possibly because of unmasking of its original action, that is, eNOS inhibition.

Many strategies have been proposed to prevent the phenomenon of nitrate tolerance, but the only approach that has gained clinical acceptance is a nitrate-free interval (1, 5, 6, 11, 13, 30). When BAECs were intermittently treated with nitrates, the diminished basal NO production was substantially improved, the increased expression levels of eNOS mRNA and protein were nearly completely normalized, and superoxide generation was evidently reduced. The present study thus demonstrated that a nitrate-free interval resulted in effects on nitrate-induced endothelial abnormalities similar to those of administration of the NADPH oxidase inhibitor apocynin. Our results are in accord with previous animal studies that have shown that increased vascular NADPH oxidase-mediated superoxide production seen during long-term NTG treatment can be reduced by a nitrate-free interval (27). We also tested the effects of perindoprilate, N-acetyl-L-cysteine, L-arginine, and BH4 on basal NO production, eNOS expression, and superoxide generation in continuously NTG-treated cells, because they may display significant benefits in the setting of nitrate tolerance (15, 28, 31, 32). However, none of these prevented these abnormalities in endothelial cells due to continuous NTG treatment.

In conclusion, the present results of the use of apocynin represent the potential importance of NADPH oxidase-mediated superoxide production in the uncoupling of eNOS and subsequent endothelial cell dysfunction. When nitrates were continuously administered for the long term, a nitrate-free interval also prevented the uncoupling of eNOS and therefore corrected endothelial dysfunction. This may partly account for the mechanisms by which the phenomenon of nitrate tolerance is avoided by a nitrate-free interval. However, as potential problems of nitrate-free periods of intermittent NTG therapy, the lack of protection during this nitrate-free period and the development of rebound ischemia have been pointed out (12, 30, 34). Therefore, therapeutic application of pharmacological agents preventing increases in NADPH oxidase-mediated superoxide, such as apocynin, may be promising for protection against nitrate tolerance.


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This study is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Hayashi, Dept. of Geriatrics, Nagoya Univ. Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan (e-mail: hayashi{at}med.nagoya-u.ac.jp)

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.


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