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BASIC RESEARCH

Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance

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^*,*

^* University Hospital Eppendorf, Division of Cardiology, Hamburg, Germany
Department of Clinical Biochemistry, Würzburg, Germany
Department of Biology, University Konstanz, Konstanz, Germany

Manuscript received January 17, 2003; revised manuscript received May 13, 2003, accepted July 1, 2003.

^* Reprint request and correspondence: Dr. Thomas Münzel, Abteilung für Kardiologie, Universitäts-Krankenhaus Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.
muenzel{at}uke.uni-hamburg.de

	Abstract

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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)-PGH₂ into 6-keto-PGF₁ (=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.

Abbreviations and Acronyms   COX = cyclooxygenase   cGK-I = cyclic guanosine monophosphate-dependent kinase-I   cGMP = cyclic guanosine monophosphate   LDCL = luminol-derived chemiluminescence   NO = nitric oxide   NOSIII = endothelial nitric oxide synthase   NTG = nitroglycerin   PGI2(-S) = prostacyclin (synthase)   P-VASP = phosphorylated vasodilator-stimulated phosphoprotein   sGC = soluble guanylyl cyclase   TXA2 = thromboxane A2

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.

	Methods

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Animal model: in vivo nitrate tolerance.   New Zealand White rabbits and Wistar rats were studied. Tolerance was induced with NTG patches (10) or ALZET (Cupertino, California) osmotic minipumps, as described recently (23).

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 [GenBank] (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.

Immunohistochemistry.   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)-PGH₂ for 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).

Statistical analysis.   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 ED₅₀ values (potency) for each experiment were obtained by logarithmic transformation. Vascular responses were compared by using ANOVA with ED₅₀ and 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.

	Results

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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).

View larger version (15K): [in this window] [in a new window]   Figure 1 Effects of nitroglycerin (NTG) treatment on luminol-derived chemiluminescence in rabbit aortic rings. Data are expressed as the mean value ± SEM. *p < 0.05 vs. control (C) by analysis of variance (ANOVA). +p < 0.05 vs. C and NTG by ANOVA. E = ebselen; UA = uric acid (100 µmol/l each).

View larger version (29K): [in this window] [in a new window]   Figure 2 Effects of in vitro incubation of control (C) and tolerant aortic rings with ebselen (E) and uric acid (UA) (100 µmol/l each) on the activity of cyclic guanosine monophosphate-dependent kinase-I (cGK-I), as assessed by immunoblotting of phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). *p < 0.05 vs. C by analysis of variance.

View larger version (20K): [in this window] [in a new window]   Figure 3 Effects of in vitro incubation of control (C) and tolerant rabbit aortas with ebselen (100 µmol/l) on the nitroglycerin (NTG) concentration-response relationship. Data are expressed as the mean value ± SEM.

View larger version (27K): [in this window] [in a new window]   Figure 4 (A) Western blot analysis of aortas from control and nitroglycerin (NTG)-treated rabbits. (B) The left side shows the effects of NTG treatment on the expression of prostacyclin-synthase (PGI2-S). The right side shows the amount of tyrosine-nitrated PGI2-S present in 3-nitrotyrosine-immunoprecipitates. Results are representative of four to six separate experiments. p < 0.05 vs. control (C) by analysis of variance.

View larger version (95K): [in this window] [in a new window]   Figure 5 Immunohistochemical detection of nitrated prostacyclin-synthase (PGI2-S) in slices of aortic rings from rats with and without nitroglycerin treatment. In tissue from sham-treated rats, nitration was virtually absent (A), PGI2-S expression was not significantly modified (B), and hence, the overlay of both only yielded marginal background staining (C). In tolerant tissue, nitrated protein gave a clear positive signal (D), the amount of PGI2-S was comparable to control tissue (E), and the overlay resulted in a deep yellow staining for co-localization of PGI2-S and nitration (F). The specificity of the 3-nitrotyrosine antibody in tolerant tissue was confirmed by three control experiments: 1) the antibody was blocked by co-incubation with authentic 3-nitrotyrosine (G); 2) protein-bound 3-nitrotyrosine was reduced by sodium dithionite before antibody incubation (H); and 3) only the secondary antibody was used (I). Green fluorescence (Alexa 488-labeled secondary antibody) corresponds to nitrated protein; red fluorescence (Alexa 568-labeled secondary antibody) to PGI2-S; and yellow to the computer-generated overlay of both stainings and, accordingly, to nitrated PGI2-S.

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View larger version (21K): [in this window] [in a new window]   Figure 6 (Left) Effects of nitroglycerin (NTG) treatment on (14C)-PGH2–dependent prostanoid production in rabbit aortas. Results are expressed as the mean value ± SEM from five experiments. *p < 0.05 vs. control by analysis of variance. Solid bars = without NTG treatment; open bars = with NTG treatment. (Right) Schematic diagram depicting the effects of NTG-induced formation of peroxynitrite (ONOO–) on arachidonic acid metabolism. ONOO– inhibits the conversion of the cyclooxygenase product PGH2 (prostacyclin endoperoxide) by the prostacyclin synthase via tyrosine nitration. This effect can be mimicked in vitro by using the prostacyclin synthase inhibitor U51605 [GenBank] . The remaining PGH2 will act directly or indirectly (via formation of thromboxane A2 [TXA2]) on the thromboxane receptor (TxR), leading to enhanced constriction.

View larger version (21K): [in this window] [in a new window]   Figure 7 Effects of in vitro incubation of control and tolerant aortas with U51605 [GenBank] (100 µmol/l) on the nitroglycerin (NTG) concentration-response relationship. Data are expressed as the mean value ± SEM.

	Discussion

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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 PGI₂ in 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 [GenBank] ) 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 PGI₂ formation (19,30). Because nonmetabolized PGH₂ can activate the TXA₂/PGH₂ receptor 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)-PGH₂ into 6-keto PGF₁ was markedly inhibited in rabbit aortas (Fig. 6). We also observed a shift toward increased PGE₂ formation 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 [GenBank] 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 PGH₂ and activation of the TXA₂/PGH₂ receptor, 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 PGI₂ and 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 PGI₂ and may (due to increased PGH₂ levels) increase TXA₂ levels. Both effects will have adverse vascular effects.

View larger version (22K): [in this window] [in a new window]   Figure 8 Schematic diagram depicting the mechanisms underlying nitroglycerin (NTG)-induced relaxation (A) and the vascular consequences of NTG-induced vascular peroxynitrite formation (B). (A) Short-term NTG treatment causes vasorelaxation by releasing the vasoactive metabolite nitric oxide (NO), which, in turn, stimulates both soluble guanylyl cyclase (sGC) and release of prostacyclin (PGI2). Activation of sGC and adenylyl cyclase (AC) increases the formation of the second messengers cyclic guanosine monophosphate (cGMP) and cAMP. Signaling pathways activated by cAMP and cGMP may interact at different levels. Subsequent activation of cGMP- and cAMP-dependent kinase (cGK-I and cAK) will induce vasorelaxation. Activation of cyclic guanosine monophosphate-dependent kinase-I (cGK-I) and, to some extent, cAK will cause phosphorylated vasodilator-stimulated phosphoprotein (P-VASP) at serine 239. VASP is also phosphorylated at serine 157, which is primarily mediated by cAK and which was not analyzed in this study. (B) Long-term treatment with NTG stimulates the production of reactive oxygen species such as peroxynitrite (ONOO–). Peroxynitrite may, in turn, induce tolerance via inhibiting the activity of the NTG-metabolizing enzyme (mitochondrial aldehyde dehydrogenase). Peroxynitrite may also cause endothelial dysfunction via oxidization of the NOSIII co-factor tetrahydrobiopterin and by tyrosine nitration of PGI2-S associated with decreased P-VASP.

	Acknowledgments

 
We appreciate the technical assistance of Elisabeth Müßig (Konstanz), Hartwig Wieboldt, and Claudia Kupper (Hamburg).

	Footnotes

 
The present study was supported in part by the DFG Mu 4-1.

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