Identification of the enzymatic mechanism of nitroglycerin bioactivation

Abstract

Nitroglycerin (glyceryl trinitrate, GTN), originally manufactured by Alfred Nobel, has been used to treat angina and heart failure for over 130 years. However, the molecular mechanism of GTN biotransformation has remained a mystery and it is not well understood why "tolerance" (i.e., loss of clinical efficacy) manifests over time. Here we purify a nitrate reductase that specifically catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN, leading to production of cGMP and relaxation of vascular smooth muscle both in vitro and in vivo, and we identify it as mitochondrial aldehyde dehydrogenase (mtALDH). We also show that mtALDH is inhibited in blood vessels made tolerant by GTN. These results demonstrate that the biotransformation of GTN occurs predominantly in mitochondria through a novel reductase action of mtALDH and suggest that nitrite is an obligate intermediate in generation of NO bioactivity. The data also indicate that attenuated biotransformation of GTN by mtALDH underlies the induction of nitrate tolerance. More generally, our studies provide new insights into subcellular processing of NO metabolites and suggest new approaches to generating NO bioactivity and overcoming nitrate tolerance.

Figure 1

(a) Purification of mtALDH from mouse macrophage RAW264.7 cells. The gradual enrichment of the 53-kDa band is shown on a 7% SDS polyacrylamide gel. Lane A, protein molecular mass marker (kDa); lane B, 100,000-g supernatant; lane C, after DEAE-cellulose; lane D, after Q-Sepharose; lane E, after butyl-Sepharose; lane F, after hydroxyapatite. (b) Kinetic analysis of purified GTN reductase in the absence of NAD⁺ (K_M = 12 μM; V_max = 3 nmol/mg per min).V_max in the presence of NAD⁺ = ≈30 nmol/mg per min.

Scheme 1

Figure 2

Effects of inhibitor treatment and GTN tolerance on GTN-induced (Left) and SNP-induced (Right) relaxation in rabbit aorta. The dose–response curves of control rings are shown as ●, and inhibitor-treated rings (1 mM) are shown as ○. (a) Chloral hydrate dose–response (○, 1 mM; ▴, 5 mM). (b) Cyanamide. (c) Acetaldehyde. (d) GTN tolerant. All inhibitor curves (a–c) are significantly different from GTN control (P < 0.01), but none are different from SNP control (P = not significant). The GTN tolerance curve (d) is significantly different from control to P < 0.05. Data are expressed as the means ± SD of 4–6 aortic rings.

Figure 3

Inhibition of GTN (1 μM) biotransformation by ALDH inhibitors and tolerance induction in rabbit aorta. (a) Concentration-dependent inhibition of 1,2-GDN formation by chloral hydrate (●), cyanamide (■), and acetaldehyde (▴). Activities are presented as the percentage of control rings (without inhibitor treatment). Data are the average of two experiments. (b) 1,2-GDN formation is reduced in tolerant vessels relative to control (P < 0.01). Data are the means ± SD of three experiments. (c) mtALDH activity is inhibited in tolerant vessels (P < 0.01). Data are the means ± SD of three experiments.

Figure 4

Inhibition of cGMP accumulation in rabbit aorta by ALDH inhibitors (1 mM × 20 min, followed by washout) and tolerance (Tol)-inducing amounts of GTN (0.3 mM × 30 min, followed by washout). Rings were (+) or were not (−) exposed to 1 μM GTN for 1 min. All bars are significantly less than control (Con) + GTN to P < 0.01, except cyanamide (Cya) + GTN, where P < 0.05. Data are presented as the means ± SD of three aortic rings. Chl, chloral hydrate; Ace, acetaldehyde.

Figure 5

mtALDH inhibitors block the hypotensive effects of GTN in vivo. (a) i.v. infusions of GTN in rabbits produce dose-dependent decrements in blood pressure (before treatment, ●) that are attenuated significantly (P < 0.05) by both cyanamide (17 mg/kg, ○) and chloral hydrate (66 mg/kg, ▵) (n = 5–7). (b) Cyanamide and chloral hydrate have no effect on SNP-mediated decreases in blood pressure (n = 7) (data for cyanamide treatment are shown). ●, before treatment; ○, after treatment.