An essential role for mitochondrial aldehyde dehydrogenase in nitroglycerin bioactivation

Abstract

The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALDH(-/-) mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NO-based cellular signals that may originate independently of NO synthase activity.

Fig. 1.

Generation from GTN of NO bioactivity (guanylate cyclase activation) depends on mtALDH. (A) Mitochondria isolated from mouse or rat liver generate and export GTN-derived NO bioactivity, as revealed by activation of guanylate cyclase (cGMP production) in coincubated RFL-6 cells. The activation of guanylate cyclase by aqueous NO and by S-nitrosoglutathione (GSNO) is shown for comparison. (B) Activation of guanylate cyclase by mitochondria plus GTN is eliminated in the presence of the ALDH inhibitor chloral hydrate (Chlor, 1 mM), and NO bioactivity is scavenged by (oxy)hemoglobin (Hb, 10 μM). (C) Generation of NO bioactivity from GTN (1 μM) is abrogated in mitochondria isolated from mtALDH^-/- liver. (D) In segments of intact aorta, deletion of mtALDH eliminates cGMP production from 0.1 and 1.0 μM GTN and substantially inhibits production from 10 μM GTN. In all experiments, GTN was present for 1 min before samples were harvested.

Fig. 2.

Metabolism of GTN by mitochondria and intact vascular tissue. (A) Mitochondria isolated from wild-type mouse liver generate 1,2-GDN from GTN (1 μM) in large preference over 1,3-GDN, and generation of 1,2-GDN is virtually eliminated in mitochondria isolated from liver of mtALDH^-/- mice. In contrast, a postmitochondrial fraction generates 1,3-GDN in preference to 1,2-GDN, and elimination of mtALDH has no effect. Note that, because the mitochondrial fraction contains only ≈2% of cellular protein, mtALDH will account for only a small proportion of the total GTN-metabolizing activity of liver, consistent with the well established role of hepatocytes in systemic clearance of GTN. (B) mtALDH is absent from the postmitochondrial fraction as assessed by Western blotting (Homog, unfractionated liver homogenate; Mito, isolated mitochondria; Sup, postmitochondrial supernatant; see Materials and Methods). In the example shown (representative of three separate experiments), each lane was loaded with 50 μg of protein with the exception of the lane containing 0.25 μg of purified human mtALDH standard (STD). (C) Generation of 1,2-GDN is selectively reduced vs. 1,3-GDN in aortic segments from mtALDH^-/- vs. wild-type mice. Note also that the mtALDH^-/- aorta generates 1,2-GDN and 1,3-GDN equally, and that the preference of wild-type aorta for production of 1,2-GDN vs. 1,3-GDN decreases with increasing GTN concentration. In A and C, GTN was present for 5 min before samples were harvested.

Fig. 3.

mtALDH mediates GTN-induced vasodilation in vitro and in vivo. (A) Aortic relaxation in vitro. (Upper) Polygraph tracings of strain gauge output illustrate typical dose-response relations in vitro for GTN- and SNP-induced vasorelaxation of aortic ring segments from wild-type (wt) and mtALDH^-/- mice. Initial tension was induced with prostaglandin F_2α (PGF). Dosages of GTN and of SNP are given as log molar concentrations. (Lower) Graphical summaries of dose-response curves illustrate that GTN-induced relaxation (Left) is essentially eliminated in mtALDH^-/- aorta at GTN concentrations less than ≈0.5 μM and that the attenuation of responsiveness of mtALDH^-/- vs. wild-type aorta decreases with increasing GTN concentration (n = 12; ^*, P < 0.05). (Note that the dose-response curve for wild-type aorta is biphasic, with a distinct region of reduced slope.) (Right) In contrast, vasorelaxation induced by the NO donor SNP (n = 6) or by the nitrovasodilator ISDN (n = 2-4) is indistinguishable between wild-type and mtALDH^-/- aorta. (B) Blood pressure in vivo. The decrease in mean arterial blood pressure induced by i.v. administration of GTN is significantly attenuated in mtALDH^-/- vs. wild-type mice, whereas the response to SNP is unaltered (n = 12-13; ^*, P < 0.05).

Fig. 4.

Inactivation of mtALDH underlies both mechanism-based tolerance to GTN and suppressed responsiveness to GTN induced by chloral hydrate. (A) After induction of tolerance by exposure to GTN before assay (300 μM, 0.5 h), the dose-response curve from wild-type (wt) aorta is superimposable on the curve from mtALDH^-/- aorta, reflecting a selective suppression of responsiveness at lower GTN concentrations. The dose-response curve from mtALDH^-/- aorta is unaffected by prior exposure to GTN. (B) The effects of the ALDH inhibitor chloral hydrate (chlor. hyd.) on the dose-response curve from wild-type aorta is identical to the effects of tolerance, and chloral hydrate has no effect on the responsiveness of mtALDH^-/- aorta. (C) Dose-response curves fitted to the GTN-induced vasodilatory responses of wild-type aorta (1 nM to 1 μM) and of mtALDH^-/- aorta (100 nM to 100 μM) indicate a ≈120-fold difference in the functional EC₅₀ of the mtALDH-dependent and mtALDH-independent (high K_m) mechanisms of GTN bioactivation.