Nitroglycerin (NTG) remains one of the most useful drugs in the symptomatic treatment of congestive heart failure and myocardial ischemia.1 Organic nitrates have an excellent therapeutic profile; they are generally fast acting, safe, and free of serious side effects.2 However, the beneficial effect of NTG is rapidly lost due to the development of nitrate tolerance, a phenomenon that is characterized by an impaired vasorelaxation to NTG. According to animal experiments, this tolerance is accompanied by increased oxidative stress production.3 The triggering events leading to this redox imbalance remain controversial because several cellular enzyme systems were shown to be altered by sustained in vivo exposure to NTG. NADPH oxidase has been suggested to be the major superoxide anion-generating enzyme in the vascular wall, which is stimulated by the potent vasoconstrictor peptide angiotensin II (Ang II).4 In patients who are nitrate tolerant, elevated circulating Ang II levels have been observed and seem to be associated with an increase in body weight and a decrease in hematocrit. This physiological response to NTG has been called “pseudo-tolerance.” Another enzyme source was first reported by Munzel et al,5 who suggested that in nitrate tolerance, eNOS exists in a state called “eNOS uncoupled” promoting superoxide anion rather than nitric oxide (NO) production. Accordingly, in animal experiments, long-term angiotensin-converting enzyme (ACE) inhibition with ramipril treatment, which seems to interact with both pathways (ie, eNOS, NAP(H)oxidase), protects against the development of nitrate tolerance.6,7 Nevertheless, our previous study in the rat suggests that the ramipril-induced protection against nitrate tolerance is mainly related to an upregulation of the eNOS pathway.8 Ramipril treatment increases NO availability, resulting not only in a downregulation of oxidative enzymes but also in a direct scavenging of superoxide anion. L-NAME, the nonselecitve NOS inhibitor, as well as the bradykinin BK2 antagonist Icatibant (HOE140), inhibits this protective effect of ramipril, suggesting that this protection is eNOS and BK2 dependent.8 Icatibant abolishes not only ramipril-induced protection but also that of an AT1 receptor blocker (losartan), suggesting that in the rat, protection against nitrate tolerance by both ACE inhibitor and sartan is mainly dependent on the eNOS pathway.8 Nevertheless, L-NAMEseems to have effects other than eNOS inhibition, such as an increase in ACE activity, leading to augmented levels of Ang II.9
To accurately assess the involvement of the eNOS pathway in ramipril-induced protection against nitrate tolerance, we studied the effect of long-term ACE inhibition on the development of nitrate tolerance in eNOS-deficient (eNOS−/−) mice. By comparing the mechanisms in both mouse strains, wild-type (eNOS+/+) and eNOS−/−, this study demonstrates a direct interaction between ramipril and the vascular NADPH oxidase pathway, and the contribution of NADPH oxidase to nitrate tolerance.
Male and female eNOS−/− and wild-type mice (WT, eNOS+/+, C57Bl/6J) were purchased from Jackson Laboratories (Bar Harbor, ME) and were bred at the ULB Institute of Pharmacy. Mice were maintained in a temperature- (21°C) and relative humidity (60%)-controlled room with a 12-h light and dark cycle and were allowed food and tap water ad libitum. All procedures were performed according to protocols approved by the ethical committee of the ULB Faculty of Medicine and Pharmacy.
Male mice from both strains, between 4 and 6 months old and weighing 20 to 30 g, were divided into 4 groups. Group 1 received ramipril (10 mg/kg/d, dissolved in water) added to drinking water for 5 weeks and for the last 4 days they received the ACE inhibitor + nitroglycerin (NTG; 30 mg/kg/d, dissolved in alcohol and diluted in water) in co-treatment by subcutaneous injection. Group 2 received ramipril alone for 5 weeks. Group 3 received NTG alone for 4 days, and group 4 served as control (no treatment). The ramipril and NTG doses were chosen according to previous studies10-12 and preliminary experiments in mice. As in our previous studies in mice, the group receiving the solvent solution of NTG behaved exactly like the control mice receiving no treatment. To simplify the treatment protocols, the control mice received no subcutaneous injections. In some experiments, 8 mice from both strains received co-treatment with ramipril plus the bradykinin BK2 antagonist HOE 140 (0.5 mg/kg/d)8,13 by subcutaneous injection for 10 days and another 8 mice from both strains received a co-treatment of ramipril (5 weeks) + HOE140 (10 days) + NTG (the last 4 days before their sacrifice).
In another set of experiments, some WT and eNOS−/− mice aortas were treated with the AT1 blocker candesartan at a dose of 2 mg/kg/d added to their drinking water for 10 days. Cilexetil, like candesartan, is insoluble in water, and vehicle containing ethanol (0.1% vol/vol), polyethylene glycol (0.1% vol/vol), and sodium biocarbonate (5 mmol/L) were added to achieve the water solubility of candesartan.14 Some of these mice received a co-treatment of candesartan + NTG (30 mg/kg/d by a subcutaneous injection, bid, the last 4 days before being killed; candesartan-NTG).
Systolic Blood Pressure Measurements
Blood pressure was measured noninvasively using a tail-cuff method (Apollo 179, IITC Life Science Instruments, Woodland Hills, CA) on conscious, restrained mice when the animals were between 4 and 6 months of age. The mice were accommodated to the restrainers during 1 h for 4 consecutive days without taking any blood pressure measurements. On day 5, the first blood pressure measurements were taken and all blood pressures were calculated as the average of 5 to 10 measurements per day for 2 consecutive days (ie, days 5 and 6).
Vascular Relaxation Experiments
Mice between 4 and 6 months of age were killed with CO2 and the thoracic aortas were dissected, cleaned of adherent tissue, and cut into segments. The aortic rings were mounted in microvessel organ chambers (EMKA, Paris), filled with Krebs-Henseleit solution (in millimoles per liter: NaCl, 118.4; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 5), bubbled with 95% O2 and 5% CO2, and maintained at 37°C. The rings were connected to a force transducer to measure isometric tensions and recorded on a transducer data acquisition system (EMKA). Resting tension was increased stepwise to reach a final tension of 0.80 g, and rings were allowed to equilibrate for 60 min. Krebs-Henseleit solution was changed before and twice after each concentration-response curve. Cumulative concentration-response curves for acetylcholine (Ach; 10−9 to 10−4 mol/L) and NTG (10−10 to 10−4 mol/L) were generated after precontraction of vessels with 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619). The used concentration of 0.1 μmol/L gave similar plateaus in both mice strains (in WT mice aortas, n = 6: 0.88 ± 0.09 g vs 0.81 ± 0.07 g in eNOS−/− mice aortas, n = 6), thereby allowing an accurate comparison of the vasodilator effect of a drug.
All experiments were performed in the absence of a cyclooxygenase inhibitor; indeed, according to previous studies15 and our preliminary experiments, indomethacin did not affect endothelium-dependent relaxations in the mouse aorta.
NADPH Oxidase Activity Measured by the Lucigenin-Enhanced Chemiluminescence Technique
The thoracic aortas from the different mice groups of both strains were isolated and cleaned of fat and loose connective tissue. The aorta was then homogenized on ice in a lysis buffer containing Tris-HCl (50 mmol/L), EDTA (1 mmol/L), dithiothreitol (0.5 mmol/L), and protease inhibitor cocktail (pepstatinA, E-64, bestatin, leupeptin, aprotinin, 4-[2-aminoethyl]benzenesulfonyl fluoride). The samples were centrifuged; 20 μL of the supernatant were used for protein assay and 40 μL were added to the vials containing DMEM (Dulbecco's modified eagle medium without L-glutamine and phenol red; Cambrex, Belgium) with 5 μmol/L of lucigenin at 37°C. The background signal with lucigenin alone was not modified upon addition of the homogenates. The reaction was initiated by addition of NADPH (100 μmol/L) to the vials containing aortic homogenates. NADPH did not modify the basal signal in blank vials (without homogenates). The peak count was assessed in a Lumat LB 9507 Luminometer (EG&G Berthold, Oak Ridge, TN) every 5 s for 45 min. At the end of the experiment, diphenylene iodonium (DPI), a flavin inhibitor (100 μmol/L), or L-NAME, an eNOS inhibitor (10 μmol/L) were added to the vials. A buffer blank was subtracted from each reading and the NADPH oxidase activity was expressed as relative light units (RLU)/5 s/mg of total protein content. The signals from the different mice groups were converted into the percentage of the control group, which was set at 100%.
Cyclic Guanosine Monophosphate (cGMP) Tissue-Level Analysis
Total aortas from treated and control (nontreated) WT and eNOS−/− mice were incubated without tension for 30 min in Krebs' solution containing 100 μmol/L of 3-isobutyl-1-methylxanthine (IBMX), aerated with 95% O2 and 5% CO2, and thermostated at 37°C.16,17 Thereafter, total aortas were frozen in liquid nitrogen and sonicated in 6% ice-cold trichloroacetic acid and centrifuged.18 The supernatant was used for cGMP measurements and the pellet was dissolved in NaOH for protein analysis (commercial available kit, Pierce, Rockford, IL). Briefly, cGMP was extracted with water-saturated diethylether, acetylated, and quantified via enzyme immunoassay (Amersham Biosciences, Piscataway, NJ) as previously described.18 Each assay was performed in duplicate.
RNA Extractions and Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR)
Aortas from the different mice groups of both strains were snap-frozen in liquid nitrogen, and total RNA was extracted with the SV total RNA isolation system (Promega; Brussels). The extracted RNA was used for reverse transcription (Fermentas GmBH, St. Leon-Rot, Germany) in a total volume of 20 μL. The reverse transcription products were amplified by PCR for the 3 genes of interest (p22phox, gp91phox, and eNOS) and the internal standard gene GAPDH. The PCR conditions were as follows: initial denaturation step, 94°C, 5 min; then the PCR was followed during 27, 30, 33, and 36 cycles, respectively, for each gene, with the following steps-denaturation 94°C, 1 min; annealing 62°C, 1 min; elongation 72°C, 1 min.
The primers used for p22phox amplification were: TGG CCT GAT TCT CAT CAC TGG sense and GGG ACA ACT CCA CAG AAA CTC antisense (product size 580 bp); the primers used for gp91phox amplification: GCT GGG ATC ACA GGA ATT GTC sense and GCT TAT CAC AGC CAC AAG CAT T antisense (product size 597 bp); and the primers used for eNOS amplification were: TGT GTG CAT GGA TCT GGA CAC CAG GAC AA sense and GAA TGG TTG CCT TCA CAC GCT CG CCA T antisense (product size: 427 bp). The same cDNA samples were used for GAPDH cDNA amplification to confirm that equal amounts of RNA were reverse transcribed (sense: AGG TCG GTG TGA ACG GAT TTG GCC GTA T, and antisense: CCT TCT CCA TGG TGG TGA AGA CAC CAG TA; product size 308 bp).
Equal amounts of RT-PCR products were loaded on 1.5% agarose gels and absorbance of ethidium bromide-stained DNA bands was quantified by an AIDA Image Analyzer (Raytest, Straubenhardt, Germany. The intensity of expression of the genes of interest was normalized to the intensity of expression of their respective internal standard gene GAPDH. The mRNA expression of these normalized genes from the control mice of both strains was set at 100%.
ACh chloride, 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α (U-46619), NTG, NADPH, mevalonic acid lactone, bovine superoxide dismutase (SOD), dithiothreitol, DPI, N-nitro-L-arginine methyl ester (L-NAME), and IBMX were purchased from Sigma Chemical Company (St. Louis, MO).
Results were expressed as mean ± SEM, n representing the number of different animals studied. Relaxations are expressed as the percentage inhibition of tension developed by U-46619. For each concentration-effect curve, the area under the curve (AUC) was calculated and expressed in arbitrary units; the concentration causing half-maximal relaxation expressed as negative log molar concentration (pD2) was also assessed. An unpaired Student t test or a 1-way ANOVA analysis followed by Tukey's comparison test (when variance was equal between groups) or a Kruskal-Wallis test with a Dunn's posttest (nonparametric test, when variance was not equal between groups) were performed for assessing intergroup differences. For comparison of rings from the same animals, a paired Student t test was used. Significance was accepted at P < 0.05.
Body Weights and Arterial Tension from the Different Mice Groups
Body weights were not significantly modified by the different treatments in both mice strains. The systolic blood pressure was significantly elevated in eNOS−/− mice compared with the control WT mice. Ramipril was able to reduce the blood pressure in both mice strains (Table 1).
Organ Bath Studies
In contrast to aortas from control WT mice, no relaxations to ACh were observed in control eNOS−/− mice aortas. The concentration-effect curves to ACh were not modified after the different mice treatments compared with the control mice aortas (data not shown).
The in vivo NTG treatment induced a significant rightward shift of the concentration-effect curve to NTG in WT and eNOS−/− mice aortas compared with the respective control mice aortas. In WT mice, the pD2 and the AUC values of the NTG group were significantly different from those of the control group: pD2, 6.16 ± 0.17, and AUC, 893 ± 19 (n = 6) vs 6.81 ± 0.10 and 772 ± 18 (n = 6) in control group (P < 0.05 for both parameters).
In preparations from eNOS−/− mice, the pD2 and AUC values of the NTG group were 6.89 ± 0.20 and 772 ± 29 (n = 6) vs 7.58 ± 0.08 and 634 ± 18 (n = 6) for the control group (P < 0.05 for both parameters). The concentration-response curves to NTG in preparations from control and NTG groups in eNOS−/− mice were significantly shifted to the left compared with corresponding groups of the WT mice.
Ramipril treatment was able to protect against the development of nitrate tolerance in both mice strains (Fig. 1). Ramipril treatment alone did not modify the concentration-effect curves to NTG compared with the control mice aortas in both mice strains. The pD2 and AUC values in the ramipril WT group were 6.82 ± 0.15 and 772 ± 24 (n = 6; NS vs the respective pD2 and AUC values from the control WT mice aortas), and in ramipril eNOS−/− group they were 7.49 ± 0.05 and 629 ± 16 (n = 6; NS vs the respective results from the control eNOS−/− mice aortas).
The co-treatment of ramipril-NTG-HOE140 still demonstrated protection against the development of nitrate tolerance in the WT and eNOS−/− mice aortas (Fig. 1). Similar results were observed with the AT1 receptor blocker. The co-treatment of candesartan-NTG was able to inhibit the development of nitrate tolerance in the WT and eNOS−/− mice aortas (Fig. 2, Table 2). Candesartan alone did not modify the concentration effect curve compared with the respective control mice aortas (pD2 and AUC values for the candesartan WT mice aortas: 7.07 ± 0.10 and 751 ± 19 [n = 6], respectively, and pD2 and AUC values for the candesartan eNOS−/− mice aortas: 7.57 ± 0.15 and 649 ± 32 [n = 6]).
NADPH Oxidase Activity
NADPH oxidase activity was significantly increased after 4 days of NTG treatment in both mice strains compared with the aorta homogenates from the respective control mice. The co-treatment with Ramipril-NTG in both strains abolished this enhanced activity (NS vs the respective control groups). Ramipril treatment alone did not reduce the NADPH oxidase activity compared with the aorta homogenates from the respective control mice from both strains (Fig. 3). NADPH oxidase activity was slightly but not significantly increased in the aorta homogenates from the control eNOS−/− mice compared with the control WT mice aorta homogenates. The addition of DPI completely abolished the NADPH oxidase activity, whereas L-NAME had no effect.
cGMP Tissue-level Analysis
Aortic cGMP levels were significantly increased after ramipril treatment alone in WT mice aortas compared with those of the control group. Icatibant was able to counteract this increase in cGMP levels observed after ramipril treatment. In all eNOS−/− mice groups, aortic cGMP levels were significantly decreased (Fig. 4).
The gp91phox mRNA expression was significantly increased after NTG treatment alone in the aortas of both mice strains (Fig. 5B and 6B). In contrast, the ramipril-NTG co-treated mice aortas from both strains exhibited a reduced p22phox and gp91phox mRNA expression compared with the respective control mRNA expressions of these 2 genes (Figs. 5A, B and 6A, B). Ramipril treatment alone was also able to lessen significantly the mRNA expression of p22phox and gp91phox compared with the respective control group in WT (Fig. 5) and eNOS−/− aortas (Fig. 6). In the WT mice aortas, eNOS mRNA expression was significantly enhanced by ramipril treatment alone (comparison by Student t test but not by ANOVA; Fig. 5C), whereas in eNOS−/− mice aortas, no eNOS mRNA expression could be observed.
In this study, we showed that ramipril was able to protect against the development of nitrate tolerance in both WT and in the eNOS−/− mice via a direct interaction with the NADPH oxidase pathway. Indeed, the mRNA expressions of the membrane-bound subunits, p22phox and gp91phox, of the superoxide anion-generating enzyme were decreased after ramipril treatment in both mice strains. Ramipril was, however, also able to increase NO bioavailability as demonstrated by the increased cGMP levels in WT mice aortas and this stimulation was inhibited by co-treatment with ramipril + Icatibant, the bradykinin receptor BK2 antagonist. These results suggest that eNOS activation induced by ramipril was bradykinin dependent in the WT mice aortas. Nevertheless, Icatibant did not inhibit the protection of ramipril against the development of nitrate tolerance in WT or in eNOS−/− mice aortas. Interestingly, candesartan, an AT1 receptor blocker, was also able to protect against the development of nitrate tolerance in both mice strains. This result strengthens the hypothesis that ramipril protects against the development of nitrate tolerance by counteracting the action of Ang II, entailing a decreased NADPH oxidase activity. Accordingly, in the present study we showed that eNOS is not functionally necessary either for the ramipril-induced protection against nitrate tolerance or for the development of nitrate tolerance. These results are in line with those observed by Wang et al,12 who found a similar degree of tolerance development to NTG in WT and eNOS−/− mice aortas.
The absence of alteration in superoxide anion release despite ramipril-induced decrease in p22phox and g91phox mRNA expression may be related to the assay. Indeed, as suggested by Wagner et al19 some disruption of the enzyme complex likely occurs during the homogenization so that only partial activity of the NADPH oxidase can be detected, and therefore, marginal changes induced by decreased mRNA expression of the oxidase subunits (p22phox or g91phox) or also by the enhanced mRNA expression of eNOS do not translate in significant superoxide basal release alteration.
Our results in mice aortas are in contrast to our previous study on rat aortas,8 in which the protective effect of ramipril against the development of nitrate tolerance is abolished after L-NAME or Icatibant co-treatment. As mentioned above, the eNOS inhibitor L-NAME can also stimulate the Ang II converting enzyme and therefore, at the high dose used, L-NAME may have some eNOS-independent actions. The discrepancies between rat and mouse preparations could also be related to the fact that the BK2 receptor seems to be functionally different in the 2 species. Indeed, we failed to demonstrate a relaxation to bradykinin in these mice aortas (data not shown), which was not the case in rat preparations.18,20 This is in line with previous investigations,21,22 showing that the BK2 receptor is not functionally expressed in these mice vessels. Thus, a functional difference between the bradykinin receptors could explain why Icatibant did not inhibit the protective effect of ramipril in the mice aortas. Another discrepancy between our 2 studies is that after ACE inhibitor treatment, the relaxations to ACh were potentiated in the rat but not in the mouse. Furthermore, in contrast to the present study, nitrate tolerance in the rat aortas was accompanied by impaired ACh-induced relaxation. In fact, the data on a cross-tolerance to ACh and/or various NO donors are conflicting.8,23-30 The extent of tolerance likely plays a major role in the development of this cross-tolerance to non-nitrate sources. Indeed, a 10-fold vs a 3-fold rightward shift of the concentration-response curves to NTG was observed in the rat aortas vs mouse preparations.31 This may be due to a higher NTG dose (50 mg/kg/d) used to induce tolerance in the rats, but it is probably also due to species differences.2,29,30 In line with this last hypothesis, mice vessels, in contrast to the rat aortas, seem to possess an EC-SOD enzyme in their wall that may protect effectively against a redox imbalance.32 According to Karlsson and Marklund,32 the wide diversity of EC-SOD in the vascular system of mammals with regard to total amount, division into fractions, and distribution between plasma and endothelium may account for the marked species variability in superoxide radical toxicity.
Long-term ramipril treatment in mice protects against the development of nitrate tolerance by counteracting NTG-induced increase in O2−· production. This protection involves a direct interaction with the NADPH oxidase pathway and seems to be completely independent of the eNOS pathway.
The authors gratefully acknowledge Sanofi-Aventis for the supply of ramipril and AstraZeneca for the supply of candesartan. Anne Otto is a recipient of a doctoral fellowship from the Ministère de la Culture, de l'Enseignement Supérieur et de la Recherche, BRF 02/022 (Luxembourg), and this work was supported by the Fondation pour la Chirurgie Cardiaque.
1. Abrams J. Mechanisms of action of the organic nitrates in the treatment of myocardial ischemia. Am J Cardiol
2. Fung HL. Biochemical mechanism of nitroglycerin action and tolerance: is this old mystery solved? Annu Rev Pharmacol Toxicol
3. Munzel T, Kurz S, Rajagopalan S, et al. Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase. A new action for an old drug. J Clin Invest
4. Griendling KK, Minieri CA, Ollerenshaw JD, et al. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res
5. Munzel T, Li H, Mollnau H, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res
6. Kaesemeyer WH, Ogonowski AA, Jin L, et al. Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate. Br J Pharmacol
7. Kurz S, Hink U, Nickenig G, et al. Evidence for a causal role of the renin-angiotensin system in nitrate tolerance. Circulation
8. Berkenboom G, Fontaine D, Unger P, et al. Absence of nitrate tolerance after long-term treatment with ramipril: an endothelium-dependent mechanism. J Cardiovasc Pharmacol
9. Suda O, Tsutsui M, Morishita T, et al. Long-term treatment with N(omega)-nitro-L-arginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthase-deficient mice. Circulation
10. Marin-Castano ME, Schanstra JP, Neau E, et al. Induction of functional bradykinin b(1)-receptors in normotensive rats and mice under chronic angiotensin-converting enzyme inhibitor treatment. Circulation
11. Wagner C, Pfeifer A, Ruth P, et al. Role of cGMP-kinase II in the control of renin secretion and renin expression. J Clin Invest
12. Wang EQ, Lee WI, Fung HL. Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice. Br J Pharmacol
13. Hiyoshi H, Yayama K, Takano M, et al. Angiotensin type 2 receptor-mediated phosphorylation of eNOS in the aortas of mice with 2-kidney, 1-clip hypertension. Hypertension
14. Taal MW, Chertow GM, Rennke HG, et al. Mechanisms underlying renoprotection during renin-angiotensin system blockade. Am J Physiol Renal Physiol
15. Levy JV. Prostacyclin-induced contraction of isolated aortic strips from normal and spontaneously hypertensive rats (SHR). Prostaglandins
16. Sabetkar M, Naseem KM, Tullett JM, et al. Synergism between nitric oxide and hydrogen peroxide in the inhibition of platelet function: the roles of soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein. Nitric Oxide
17. Qian X, Prabhakar S, Nandi A, et al. Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine. Endocrinology
18. Berkenboom G, Brekine D, Unger P, et al. Chronic angiotensin-converting enzyme inhibition and endothelial function of rat aorta. Hypertension
19. Wagner AH, Kohler T, Ruckschloss U, et al. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol
20. Berkenboom G, Langer I, Carpentier Y, et al. Ramipril prevents endothelial dysfunction induced by oxidized low-density lipoproteins: a bradykinin-dependent mechanism. Hypertension
21. Brandes RP, Schmitz-Winnenthal FH, Feletou M, et al. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad Sci U S A
22. Chlopicki S, Kozlovski VI, Lorkowska B, et al. Compensation of endothelium-dependent responses in coronary circulation of eNOS-deficient mice. J Cardiovasc Pharmacol
23. Laursen JB, Mulsch A, Boesgaard S, et al. In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide. Circulation
24. Munzel T, Heitzer T, Brockhoff C. Neurohormonal activation and nitrate tolerance: implications for concomitant therapy with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. Am J Cardiol
25. Mulsch A, Oelze M, Kloss S, et al. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation
26. Schulz E, Tsilimingas N, Rinze R, et al. Functional and biochemical analysis of endothelial (dys)function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment. Circulation
27. Sydow K, Daiber A, Oelze M, et al. Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance. J Clin Invest
28. Abou-Mohamed G, Kaesemeyer WH, Caldwell RB, et al. Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin. Br J Pharmacol
29. Ratz JD, McGuire JJ, Anderson DJ, et al. Effects of the flavoprotein inhibitor, diphenyleneiodonium sulfate, on ex vivo organic nitrate tolerance in the rat. J Pharmacol Exp Ther
30. Balazy M, Kaminski PM, Mao K, et al. S-Nitroglutathione, a product of the reaction between peroxynitrite and glutathione that generates nitric oxide. J Biol Chem
31. Otto A, Fontaine D, Fontaine J, et al. Rosuvastatin treatment protects against nitrate-induced oxidative stress. J Cardiovasc Pharmacol
32. Karlsson K, Marklund SL. Extracellular superoxide dismutase in the vascular system of mammals. Biochem J