Author + information
- Received January 17, 2003
- Revision received May 13, 2003
- Accepted July 1, 2003
- Published online November 19, 2003.
- Ulrich Hink, MD*,
- Matthias Oelze, PhD*,
- Philip Kolb, MD*,
- Markus Bachschmid, PhD‡,
- Ming-Hui Zou, PhD‡,
- Andreas Daiber, PhD*,
- Hanke Mollnau, MD*,
- Michael August, PhD*,
- Stefan Baldus, MD*,
- Nikos Tsilimingas, MD*,
- Ulrich Walter, MD†,
- Volker Ullrich, PhD‡ and
- Thomas Münzel, MD*,* ()
- ↵*Reprint request and correspondence:
Dr. Thomas Münzel, Abteilung für Kardiologie, Universitäts-Krankenhaus Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.
Objectives We tested whether in vivo nitroglycerin (NTG) treatment causes tyrosine nitration of prostacyclin synthase (PGI2-S), one of the nitration targets of peroxynitrite, and whether this may contribute to nitrate tolerance.
Background Long-term NTG therapy causes tolerance secondary to increased vasoconstrictor sensitivity and increased vascular formation of reactive oxygen species. Because NTG releases nitric oxide (NO), NTG-induced stimulation of superoxide production should increase vascular nitrotyrosine levels, compatible with increased formation of peroxynitrite, the reaction product from NO and superoxide.
Methods New Zealand White rabbits and Wistar rats were treated with NTG (0.4 mg/h for 3 days). Tolerance was assessed with isometric tension studies. Vascular peroxynitrite levels were quantified with luminol-derived chemiluminescence (LDCL) and peroxynitrite scavengers, such as uric acid and ebselen. As a surrogate parameter for the assessment of the activity of cyclic guanosine monophosphate-dependent kinase-I (cGK-I; the final signaling pathway for NO), the phosphorylation of the vasodilator-stimulated phosphoprotein (P-VASP) at serine 239 was analyzed.
Results Nitroglycerin treatment increased LDCL, and the inhibitory effect of uric acid and ebselen on LDCL was augmented in tolerant rings. Immunoprecipitation of 3-nitrotyrosine–containing proteins and immunohistochemistry analysis identified PGI2-S as a tyrosine-nitrated protein. Accordingly, conversion of (14C)-PGH2into 6-keto-PGF1α(=PGI2-S activity) was strongly inhibited. In vitro incubation of tolerant rings with ebselen and uric acid markedly increased the depressed P-VASP levels and improved NTG sensitivity of the tolerant vasculature.
Conclusions Nitroglycerin-induced vascular peroxynitrite formation inhibits the activity of PGI2-S as well as NO, cGMP, and cGK-I signaling, which may contribute to vascular dysfunction in the setting of tolerance.
Nitrates are still widely used in the management of coronary artery disease, including patients with stable and unstable angina, acute myocardial infarction, and congestive heart failure (1). The therapeutic efficacy of these nitrates is due to peripheral venous and arterial dilation that results in de- creased myocardial oxygen consumption. Nitrates also dilate large coronary arteries and collateral channels (1), while having minimal or no effect on arteriolar tone (2). It is assumed that nitroglycerin (NTG) induces vasorelaxation by releasing the vasoactive principle nitric oxide (NO) via an enzymatic biotransformation step. This may include glutathione S transferases (3), the cytochrome p450 system (4), xanthine oxidoreductases (5), or the mitochondrial aldehyde dehydrogenase (6). The NTG metabolite NO activates the target enzyme soluble guanylyl cyclase (sGC) and increases tissue levels of the second messenger cyclic guanosine monophosphate (cGMP). Cyclic guanosine monophosphate, in turn, activates a cGMP-dependent protein kinase (cGK-I), which has been shown to mediate vasorelaxation via phosphorylation of proteins that regulate intracellular Ca2+levels (7).
Two major drawbacks of nitrate therapy have been identified as important: the rapid development of tolerance within 24 to 48 h of continuous NTG treatment (8,9)and the development of endothelial dysfunction during prolonged NTG treatment (so called cross-tolerance) (10–12). Recently, we and others have shown that NTG therapy increases vascular superoxide, which reduces the bioavailability of NO and thus may interfere with activation of sGC (13)and cGK-I (13,14). Because NTG releases NO in the vascular wall, NTG-induced superoxide production should ultimately lead to increased formation of the NO/superoxide reaction product peroxynitrite. Indeed, several groups have shown that in vitro or in vivo exposure to NTG leads to increased urinary (15)and vascular nitrotyrosine content (16,17), compatible with increased peroxynitrite formation. This peroxide is unstable and rapidly decomposes to nitrite and nitrate with a half-life of <1 s (18). Recent studies have also shown that peroxynitrite preferentially inactivates prostacyclin synthase (PGI2-S) by a mechanism dependent on tyrosine nitration (19,20). Inactivation of PGI2-S by peroxynitrite could have confounding effects on NTG vasodilator potency because several groups have shown that NTG and/or the active metabolite NO has potent stimulatory effects on PGI2-S and that a considerable part of NTG-induced vasodilation is due to the release of PGI2(21,22).
The aim of the present study was therefore to test whether NTG treatment results in increased vascular peroxynitrite formation and whether this may lead to nitration and inhibition of PGI2-S and inhibition of cGK-I activity as a cause of nitrate tolerance.
Animal model: in vivo nitrate tolerance
Luminol-derived chemiluminescence (LDCL)
Peroxynitrite production in intact vessels was measured with LDCL (100 μmol/l) as described (24). Because luminol detects superoxide as well as hydrogen peroxide and peroxynitrite, we used peroxynitrite trapping agents such as uric acid and ebselen to quantify vascular peroxynitrite formation, as described (24).
Detection of cGK-I expression, cGK-I activity, and phosphorylation of vasodilator-stimulated phosphoprotein (P-VASP) at serine 239
Aortic tissue was frozen and homogenized in liquid nitrogen. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting were performed as described (25). All data reported here indicate P-VASP at serine 239.
In separate experiments, the aortas from control and NTG-treated rabbits were incubated with ebselen and uric acid (10 μmol/l), scavengers for peroxynitrite or peroxynitrite-derived radicals, to test whether the cGMP signaling pathway downstream of sGC is impaired by increased vascular peroxynitrite levels.
Organ chamber experiments
Vasodilator responses to NTG were assessed in organ chambers, as described recently (10). To determine the effects of PGI2-S on vascular sensitivity to NTG, vessels were incubated for 30 min with the PGI2-S/thromboxane A2(TXA2) inhibitor U51605 (100 μmol/l) (26). These experiments were performed in the absence of indomethacin.
Immunoprecipitation and Western blots
Immunoprecipitation of nitrotyrosine-containing proteins was done as described recently (19). For Western blots, protein precipitates were separated by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane (19). After blocking, the membranes were incubated with a polyclonal antibody directed against PGI2-S, following immunostaining and documentation of positive bands by enhanced chemiluminescence.
Aortic tissue from Wistar rats (sham or NTG-treated) was fixed in 4% formalin buffer for 1 h at 4°C. Then, the sucrose concentration was increased in three steps up to 30% (wt/vol) with 0.05% (wt/vol) of cacodylic acid. The tissue was then embedded in tissue tek (Sakura Finetek/Science Services, Munich, Germany), and cryosections of 10 μm were prepared and mounted on poly-l-lysine–coated slides. After air drying, the slides were blocked with 10% bovine serum albumin (wt/vol) and 3% (vol/vol) Triton-X100 in phosphate-buffered saline (PBS) for 1 h at room temperature. The slides were incubated with either the primary antibodies against PGI2-S (dilution 1:100; gift of Dr. T. Klein from Altana Pharma, Konstanz, Germany) or monoclonal antibody against nitrotyrosine (clone 1A6; dilution 1:40; Upstate, Hamburg, Germany), or both, at 4°C overnight. The specificity of nitrotyrosine staining was confirmed by blocking the antibody with 10 mmol/l 3-nitrotyrosine in PBS or by a reduction of nitrotyrosine to aminotyrosine with sodium dithionite. For visualization, the secondary antibodies (Alexa 568 anti-rabbit immunoglobulin G [IgG] or Alexa 488 anti-mouse IgG; dilution 1:300; Molecular Probes/MoBiTec, Göttingen, Germany) were incubated for 1 h at room temperature. Sections were then rinsed, embedded in glycerin-PBS, and examined under a fluorescent microscope (DM-IRB; Leica) connected with a digital imaging system (Spot-RT; Diagnostic Instruments/Visitron Systems, Puchheim, Germany). Negative controls were performed by eliminating the primary antibodies.
Activity assay of PGI2-S
Activity of PGI2-S was assessed as described recently (19). Homogenates from control or NTG-treated aortas (3 mg protein/ml) were incubated with 100 μmol/l (14C)-PGH2for 3 min. The reaction was stopped by acidification with 1 N HCl to pH 3.5. The incubation media were extracted with 3 volumes of ethyl acetate, and after centrifugation, the organic phases were evaporated to dryness under nitrogen. Samples were then resuspended in 60 μl of ethyl acetate and subsequently separated by thin-layer chromatography (ethyl acetate/water/isooctane/acetic acid ratio 90:100:50:20). Prostanoids were quantified as previously described (19).
Results are expressed as the mean value ± SEM. The median effective dose (ED50) value for each experiment was obtained by logit transformation. To compare LDCL values, PGI2-S, tyrosine-nitrated PGI2-S, cGK-I expression, and P-VASP in vessels from sham and NTG-treated animals, one-way analysis of variance (ANOVA) was employed. The ED50values (potency) for each experiment were obtained by logarithmic transformation. Vascular responses were compared by using ANOVA with ED50and maximal relaxations (efficacy) as dependent variables. The Scheffé post hoc test was used to examine differences between groups when significance was indicated. A p value <0.05 was considered statistically significant.
Nitroglycerin treatment, vascular production of reactive oxygen species, and cGK-I signaling
Treatment of New Zealand White rabbits for three days with NTG patches significantly increased LDCL (p < 0.05) (Fig. 1). To specify the nature of reactive oxygen species detected by LDCL, ebselen and uric acid were employed as selective trapping agents for peroxynitrite and derived radicals. The marked inhibitory effects in both groups indicate that peroxynitrite contributes to LDCL with the absolute rate of peroxynitrite formation, in particular, in vessels from NTG-treated animals. Because increased vascular peroxynitrite formation may indicate a reduced vascular NO content, we used P-VASP as an indirect readout for vascular NO bioavailability (Fig. 2), as done previously. In the setting of tolerance, P-VASP was reduced. The addition of uric acid or ebselen slightly but significantly enhanced the formation of P-VASP in vessels from sham-treated animals, and this was strikingly enhanced in tissue from NTG-treated animals, in agreement with the data presented in Figure 1. Accordingly, in vitro incubation of tolerant tissue with ebselen led to an improvement of the NTG dose-response relationship (Fig. 3) (ED50—control: 7.75 ± 0.04; control + ebselen: 7.76 ± 0.04; NTG: 6.97 ± 0.06; NTG + ebselen: 7.46 ± 0.07; Scheffé post hoc test—control vs. NTG: p < 0.001; NTG vs. NTG + ebselen: p = 0.0023).
Effects of NTG treatment on PGI2-S expression, tyrosine nitration, and activity
Activity of PGI2-S is usually already inhibited at micromolar concentrations of peroxynitrite. This would predict an inhibition of PGI2-S under prolonged exposure to NTG if simultaneous formation of NO and superoxide occurred (Figs. 1 and 2). Figure 4summarizes the effects of in vivo NTG treatment on the expression and tyrosine nitration of PGI2-S. Treatment with NTG did not modify the expression of PGI2-S, whereas the amount of tyrosine-nitrated PGI2-S was strongly increased. Using a different model of nitrate tolerance (NTG infusion in rats) and immunohistochemical detection of nitrated PGI2-S, the results obtained from rabbit aortas were confirmed (Fig. 5). In slices of aortic rings from rats treated for three days with NTG, there was an increased nitrotyrosine content, without altering PGI2-S expression. The specificity of the 3-nitrotyrosine antibody in tolerant tissue was confirmed by three control experiments. The antibody was blocked by co-incubation with authentic 3-nitrotyrosine (5G); protein-bound 3-nitrotyrosine was reduced by sodium dithionite before antibody incubation (5H); and only the secondary antibody was used (5I). These results therefore support the findings obtained with immunoprecipitation.
The functional consequences for PGI2-S activity are depicted in Figure 6. Treatment with NTG selectively inhibited PGI2-S activity, as evidenced by its inhibitory effect on the conversion of PGH2to 6-keto-PGF1α, a stable metabolite of PGI2. A simultaneous shift of 6-keto-PGF1αto PGE2was observed, suggesting the conversion of PGH2to PGE2by enzymatic or nonenzymatic pathways.
Involvement of PGI2in NTG-induced relaxation of control and tolerant aortas
In vitro incubation of control tissue with U51605, the dual blocker of PGI2-S and TXA2S, which has been previously shown to mimic the effects of authentic peroxynitrite in organ chamber experiments (19), significantly shifted NTG's dose-response relationship to the right (Fig. 7). In contrast, it failed to modify vascular NTG responses in vessels from NTG-treated rabbits, compatible with a substantially decreased activity of PGI2-S. (ED50—control: 7.91 ± 0.07; control + U51605: 7.54 ± 0.07; NTG: 7.28 ± 0.06; NTG + U51605: 7.37 ± 0.04; Scheffé post-hoc test—control vs. C + U51605: p = 0.0019).
The present studies demonstrate that NTG treatment increases vascular peroxynitrite formation. Immunoprecipitation experiments and immunohistochemical analysis identified PGI2-S as a nitrated protein in rabbit and rat aortas in response to in vivo NTG treatment, leading to a marked inhibition of PGI2-S activity. Likewise, a strong inhibition of cGK-I activity was observed. In vitro trapping of peroxynitrite with ebselen and of peroxynitrite-derived radicals with uric acid improved cGK-I activity and partially increased vascular NTG sensitivity, pointing to a decisive role of peroxynitrite in NTG-induced vascular dysfunction.
Nitroglycerin induces vasorelaxation by releasing the vasoactive principal NO via an enzymatic biotransformation step within mitochondria (6). The long-term use of NTG is limited due to the rapid development of tolerance (9), which may be at least in part due to increased production of reactive oxygen species such as superoxide (10), favoring the formation of the NO/superoxide reaction product peroxynitrite. Peroxynitrite is unstable and decomposes rapidly to nitrite and nitrate, with a half-life of <1 s at physiologic pH (18), but during this process, it also forms hydroxyl and nitrogen dioxide radicals. Once formed, peroxynitrite nitrates tyrosine residues in proteins, which can be taken as a footprint for peroxynitrite in tissues (18). Recent studies demonstrated that in vitro treatment of vascular tissue or in vivo treatment of animals and patients with NTG increased the vascular and urinary nitrotyrosine content (15–17), which can be considered as a marker of peroxynitrite-dependent oxidative damage.
The data presented with the present studies go along with this concept. Prolonged treatment with NTG led to about a threefold increase in the luminol assay signal compared with aortic segments from sham-treated controls. However, luminol-enhanced chemiluminescence lacks specificity for peroxynitrite, as it has been shown to increase also in response to hydrogen peroxide, superoxide, and hydroxyl radicals. To address this issue, we used ebselen and uric acid (Fig. 1), which have been shown to possess a relatively potent peroxynitrite trapping efficacy and have been recently used to quantify vascular peroxynitrite formation in an animal model of atherosclerosis (24). Because both compounds markedly reduced the luminol signal, in particular, in tolerant tissue (Fig. 1), it is conceivable to conclude that a major part of the chemiluminescence signal is due to increased vascular peroxynitrite formation.
As shown before, in vivo treatment with NTG resulted in quite a substantial decrease in the activity of the cGK-I, as assessed by P-VASP formation (13,14). To address the role of peroxynitrite in inhibiting cGK-I activity, tolerant tissue was incubated with ebselen and uric acid. Both compounds not only corrected the NTG-induced decreases in P-VASP but also increased P-VASP levels to 190% and 180% of control levels, respectively (Fig. 2). Accordingly, we found that NTG sensitivity of tolerant vessels was improved by ebselen (Fig. 3). Based on the strong stimulatory effect of ebselen and uric acid on the cGK-I activity of tolerant tissue, we speculate these compounds may restore vascular sensitivity to nitrates by inhibiting peroxynitrite-mediated interference with endothelial nitric oxide synthase (NOSIII) (27)and/or sGC (28).
So far, no study has addressed the consequences of NTG-induced oxidative stress on the activity of vasodilating PGI2-S. Several groups have demonstrated that NTG or its vasoactive metabolite NO causes relaxation, in part via activation of cyclooxygenase-1 (COX-1) (29), leading to enhanced formation of PGI2in cultured endothelial cells and vascular tissue (21,22). The presented data go along with this concept, as the blocker of PGI2-S (i.e., U51605) significantly shifted the NTG dose-response relationship to higher concentrations (Fig. 7). More recent in vitro studies have shown that PGI2-S is a preferential nitration target of peroxynitrite (30). More importantly, these studies also revealed that tyrosine nitration of PGI2-S resulted in an almost complete inhibition of the activity of the enzyme leading to decreased PGI2formation (19,30). Because nonmetabolized PGH2can activate the TXA2/PGH2receptor of vascular smooth muscle cells with negative effects on NO-mediated vasodilation, increased vascular peroxynitrite concentrations can be theoretically considered as a marker for—but also as a mediator of—endothelial dysfunction as well as “nitrate tolerance.”
To address the consequences of NTG-induced stimulation of vascular peroxynitrite formation, Western blots of 3-nitrotyrosine immunoprecipitates exposed to a polyclonal antibody directed against PGI2-S in vessels from animals with and without NTG treatment were obtained. As depicted in Figure 4, we were able to detect a 52-kd protein band demonstrating a marked increase in the nitrated PGI2-S signal in the immunoprecipitate in tissue from tolerant aortas compared with aortas from sham-treated animals. This cannot be attributed to increased expression of the enzyme, because immunoblots of vascular homogenates from tolerant and nontolerant vessels showed comparable signal intensity for PGI2-S. To show that this phenomenon holds true for different animal models of tolerance, immunohistochemistry studies were performed in aortic tissue from NTG-treated rats (Fig. 5). As depicted in Figure 5, NTG treatment increased nitration mainly in the endothelium, subendothelial space, and adventitia. No change was observed with respect to PGI2-S expression, and co-localization of PGI2-S with nitrated protein was observed in the endothelium and adventitia and to some extent in the media.
As a functional consequence of tyrosine nitration of PGI2-S, the conversion of (14C)-PGH2into 6-keto PGF1αwas markedly inhibited in rabbit aortas (Fig. 6). We also observed a shift toward increased PGE2formation in tolerant tissue. Because the expression of PGI2-S was not affected by NTG tolerance, it is conceivable that increased tyrosine nitration primarily accounts for the observed inhibition of the activity of the enzyme in the setting of tolerance. The functional inhibition of PGI2-S activity in tolerant tissue was indirectly confirmed by experiments using the dual blocker of PGI2-S and TXA2-S—U51605, which has previously been shown to mimic the effects of authentic peroxynitrite in organ chamber experiments (26). Accordingly, in tolerant tissue, U51605 failed to further shift NTG's dose-response relationship significantly (Fig. 7), providing further evidence that the activity of this enzyme is inhibited in response to in vivo NTG treatment. Taken together, the presented data provide evidence that increased peroxynitrite formation in response to long-term NTG treatment inhibits the formation of PGI2, which may contribute to endothelial dysfunction and an enhanced propensity of the vasculature to vasoconstrict.
The demonstration of a crucial role for peroxynitrite in mediating tolerance raises the question of whether the oxidative stress concept of tolerance contradicts recent findings by Chen et al. (6), who identified an inhibition of the mitochondrial aldehyde dehydrogenase as the crucial step for attenuation of vascular NTG sensitivity in response to long-term treatment. Although this concept may hold true for the previously observed decrease in NTG biotransformation in response to “in vitro” incubation of vascular tissue with NTG, it cannot explain why “in vivo” tolerance is associated with endothelial dysfunction (11,12)and why NTG treatment strongly inhibits the activity of cGK-I in vessels from experimental animals (13)and patients (14). The presented data may indicate that both phenomena do not necessarily exclude each other. Nitric oxide release from NTG mediated by the mitochondrial aldehyde dehydrogenase is based on a restoration of dithiol groups at the active site of the enzyme, which is dependent on glutathione or other free thiols (6). In the presence of increased oxidative stress induced, for example, by continuous NTG treatment, mitochondrial thiols will be oxidized and, as a consequence of the lack of restoration of mitochondrial aldehyde dehydrogenase, the NO release by this enzyme will cease (6). Thus, NTG-induced formation of reactive oxygen species may explain phenomena such as endothelial dysfunction and inhibition of cGK-I activity, but may also explain the observed impairment of NTG biotransformation.
Clinical implications and conclusions
Tolerance has been characterized not only by decreased sensitivity to NTG but also by endothelial dysfunction and an increase in the propensity of the vasculature to vasoconstrict. This scenario, as a consequence of NTG therapy, strikingly resembles rebound pulmonary hypertension in response to long-term inhalation therapy with NO (31). Both models are characterized by increased vasoconstrictor sensitivity, increased oxidative stress, and increases in vascular peroxynitrite levels (31). Recently, we have shown that oxidative stress markedly increases the expression of endothelin-1 in endothelial (32)and smooth muscle cells (33). Endothelin-1, in turn, stimulates vascular superoxide production, which results not only in increased endothelin-1–mediated vasoconstriction but also in increased formation of peroxynitrite (31). The latter, in turn, may cause tyrosine nitration of PGI2-S and oxidation of NOSIII, leading to an inhibition of the production of the two most important vasodilators—antithrombotic and antiproliferative signaling pathways—in vascular tissue. Therefore, it is conceivable that these specific side effects of NTG therapy not only contribute to nitrate tolerance but may also be effective in other pathologic conditions exhibiting increased vascular peroxynitrite formation. Inhibition of PGI2-S may also lead to an increased release of PGH2and activation of the TXA2/PGH2receptor, thereby enforcing these adverse effects.
The question may arise whether an inhibition of PGI2-S via tyrosine nitration has some clinical relevance, particularly when most patients with coronary artery disease are already receiving treatment with the COX inhibitor aspirin (see schematic diagrams in Figs. 6 and 8). ⇓It is important to note that anti-inflammatory drugs inhibit COX, whereas the enzyme PGI2-S uses the product of the COX reaction. Aspirin, via inhibition of COX, therefore reduces the synthesis of many prostaglandins, including PGI2and TXA2. Nitroglycerin-induced inhibition of PGI2-S will come into play, particularly when low-dose aspirin, which inhibits platelets but not vascular COX, is applied. In contrast, impairment of PGI2-S selectively reduces the concentrations of vasoprotective PGI2and may (due to increased PGH2levels) increase TXA2levels. Both effects will have adverse vascular effects.
We appreciate the technical assistance of Elisabeth Müβig (Konstanz), Hartwig Wieboldt, and Claudia Kupper (Hamburg).
☆ The present study was supported in part by the DFG Mu 4-1.
- cyclic guanosine monophosphate-dependent kinase-I
- cyclic guanosine monophosphate
- luminol-derived chemiluminescence
- nitric oxide
- endothelial nitric oxide synthase
- prostacyclin (synthase)
- phosphorylated vasodilator-stimulated phosphoprotein
- soluble guanylyl cyclase
- thromboxane A2
- Received January 17, 2003.
- Revision received May 13, 2003.
- Accepted July 1, 2003.
- American College of Cardiology Foundation
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