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
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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).
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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.
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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.
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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.
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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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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. The remaining PGH2 will act directly or indirectly (via formation of thromboxane A2 [TXA2]) on the thromboxane receptor (TxR), leading to enhanced constriction.
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Figure 7 Effects of in vitro incubation of control and tolerant aortas with U51605 (100 µmol/l) on the nitroglycerin (NTG) concentration-response relationship. Data are expressed as the mean value ± SEM.
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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.
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