A new class of organic nitrates: investigations on bioactivation, tolerance and cross-tolerance phenomena

Abstract

Background and purpose: The chronic use of organic nitrates is limited by serious side effects including oxidative stress, nitrate tolerance and/or endothelial dysfunction. The side effects and potency of nitroglycerine depend on mitochondrial aldehyde dehydrogenase (ALDH-2). We sought to determine whether this concept can be extended to a new class of organic nitrates with amino moieties (aminoalkyl nitrates).

Experimental approach: Vasodilator potency of the organic nitrates, in vitro tolerance and in vivo tolerance (after continuous infusion for 3 days) were assessed in wild-type and ALDH-2 knockout mice by isometric tension studies. Mitochondrial oxidative stress was analysed by L-012-dependent chemiluminescence and protein tyrosine nitration.

Key results: Aminoethyl nitrate (AEN) showed an almost similar potency to glyceryl trinitrate (GTN), even though it is only a mononitrate. AEN-dependent vasodilatation was mediated by cGMP and nitric oxide. In contrast to triethanolamine trinitrate (TEAN) and GTN, AEN bioactivation did not depend on ALDH-2 and caused no in vitro tolerance. In vivo treatment with TEAN and GTN, but not with AEN, induced cross-tolerance to acetylcholine (ACh)-dependent and GTN-dependent relaxation. Although all nitrates tested induced tolerance to themselves, only TEAN and GTN significantly increased mitochondrial oxidative stress in vitro and in vivo.

Conclusions and implications: The present results demonstrate that not all high potency nitrates are bioactivated by ALDH-2 and that high potency of a given nitrate is not necessarily associated with induction of oxidative stress or nitrate tolerance. Obviously, there are distinct pathways for bioactivation of organic nitrates, which for AEN may involve xanthine oxidoreductase rather than P450 enzymes.

Figure 2

Vascular function of organic nitrates in wild-type and ALDH-2^−/− aortic rings and tachyphylaxis (in vitro tolerance). Concentration–relaxation response curves for 2-nitrooxyethylammoniumnitrate (AEN) (10⁻¹⁰ to 10^−3.5 M; A and B), triethanolamine trinitrate (TEAN) (10⁻⁷ to 10⁻³ M; C and D) and methyl-3-nitrooxypropanoat (NPME) (10⁻⁷ to 10⁻³ M; E and F) were obtained by isometric tension recordings in aortic segments from wild-type (B6 WT) and ALDH-2^−/− (ALDH-2 ko) mice. (A, C and E) Potency of the nitrates was compared in aorta from wild-type and ALDH-2^−/− mice. (B, D and F) Effect of repeated concentration–relaxation response curves was tested resulting in pre-incubation of the rings with the EC₇₀ (100 µM for AEN, 1 mM for TEAN and NPME). For both strains the first concentration–relaxation response curves and the subsequent are shown. (G) Comparison of the vasodilator potency of AEN, TEAN, glyceryl trinitrate (GTN) and NPME in B6 WT mice. Data are mean ± SEM of 14–30 independent experiments with tissue from 8–17 animals per group. *P < 0.05 versus WT on the same treatment. For further statistical analysis, see Table 1.

Figure 1

Structures of the organic nitrates used in this study. 2-Nitrooxyethylammoniumnitrate (AEN), methyl-3-nitrooxypropanoat (NPME) triethanolamine trinitrate (TEAN), glyceryl trinitrate (GTN), pentaerithrityl trinitrate (PETriN) and isopropyl nitrate (IPM).

Figure 3

Effect of different inhibitors on vasodilatation evoked by 2-nitrooxyethylammoniumnitrate (AEN) in wild-type mice. AEN potency was determined after pre-incubation of isolated aortic ring segments for 30 min with (A) NS2028 (3 µM), PTIO (10 µM) or miconazol (25 µM) and (B) benomyl (10 µM), L-NAME (200 µM) or allopurinol (100 µM). Data are mean ± SEM of 6–12 independent experiments with tissue from different mice. *P < 0.05 versus control without treatment.

Figure 4

Mitochondrial reactive oxygen species (ROS) formation after acute organic nitrate treatment. Isolated cardiac mitochondria (0.1 mg protein mL⁻¹) were stimulated with succinate (5 mM), the chemiluminescence signal was detected using a single photon counter in the presence of the dye L-012 (100 µM). The organic nitrates 2-nitrooxyethylammoniumnitrate (AEN), triethanolamine trinitrate (TEAN), methyl-3-nitrooxypropanoat (NPME) and isopropyl nitrate (IPM) were tested at 10 or 1000 µM in mitochondria from ALDH-2^−/− (A) or wild-type (WT) (B) mice. ROS formation was also determined for NPME, TEAN, AEN, glyceryl trinitrate (GTN) and pentaerithrityl trinitrate (PETriN) at 500 µM in mitochondria from ALDH-2^−/− mice (C). Mitochondrial ROS formation in WT versus ALDH-2^−/− mice was also measured for AEN, PETriN and GTN at 5 mM (D). All nitrates were used from stocks in dimethyl sulphoxide and the vehicle was added to controls. Data are mean ± SEM of 8–24 (A), 6–24 (B), 8 (C) and 4–6 (D) experiments with mitochondria from three to six animals per group. *P < 0.05 versus untreated control.

Figure 5

Nitration of mitochondrial proteins in response to acute organic nitrate treatment. Protein tyrosine nitration was tested in isolated cardiac mitochondria (0.2 mg protein mL⁻¹) from control mice for glyceryl trinitrate (GTN) (5–5000 µM) (A) and 2-nitrooxyethylammoniumnitrate (AEN), methyl-3-nitrooxypropanoat (NPME), triethanolamine trinitrate (TEAN) (5 mM) (B). 3-Nitrotyrosine formation was detected by dot blot analysis using a specific antibody. Dimethyl sulphoxide (DMSO) was used as a solvent control for AEN, NPME and TEAN whereas ethanol (EtOH) was used for GTN respectively; 20 µg of protein was transferred to each well. Below the densitometric quantification the original blot is shown. Data shown in (B) are mean ± SEM of eight independent experiments. *P < 0.05 versus DMSO solvent control.

Figure 6

Potency of different vasodilators after chronic 2-nitrooxyethylammoniumnitrate (AEN) or triethanolamine trinitrate (TEAN) treatment. Relaxation was assessed by recording isometric tension in aortic segments from wild-type mice. The effect of AEN treatment in vivo (150 µg·h⁻¹ per 3 days) on relaxation to acetylcholine (ACh) (A), glyceryl trinitrate (GTN) (C) and AEN (E) was tested. Similarly, the effect of TEAN (140 µg·h⁻¹ per 3 days) on relaxation to ACh (B), glyceryl trinitrate (GTN) (D) or TEAN (F) was assessed. For both nitrates dimethyl sulphoxide (DMSO) was used as a solvent control. Data show mean ± SEM of 12–19 (AEN) and 7–12 (TEAN) independent experiments with tissue from at least five animals per group. *P < 0.05 versus DMSO solvent control.

Figure 7

Potency of different vasodilators after chronic glyceryl trinitrate (GTN) treatment. Relaxation was assessed by recording isometric tension in aortic segments from wild-type mice. The effect of GTN treatment in vivo (50 µg·h⁻¹ per 3 days) on relaxation to acetylcholine (ACh) (A), GTN (B), 2-nitrooxyethylammoniumnitrate (AEN) (C) and triethanolamine trinitrate (TEAN) (D) was assessed. Data are mean ± SEM of 12–18 (ACh, GTN, TEAN) or 27–28 (AEN) independent experiments with tissue from at least seven animals per group. *P < 0.05 versus ethanol (EtOH) solvent control.

Figure 8

Mitochondrial reactive oxygen species (ROS) formation after chronic organic nitrate treatment. Isolated cardiac mitochondria (0.1 mg protein mL⁻¹) where stimulated with succinate (5 mM), the chemiluminescence signal was detected using a single photon counter in the presence of the dye L-012 (100 µM). The organic nitrates 2-nitrooxyethylammoniumnitrate (AEN), triethanolamine trinitrate (TEAN) and glyceryl trinitrate (GTN) were infused at a dose of 150, 140 or 50 µg·h⁻¹ for 3 days respectively. Data are mean ± SEM of 24 (AEN), 20–21 (TEAN) and 13–17 (GTN) independent experiments with mitochondria from at least eight animals per group. *P < 0.05 versus solvent (sham-treated) control.