Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Matteo Beretta,Karl Gruber,Alexander Kollau,Michael Russwurm,Doris Koesling,Walter Goessler,Wing Ming Keung,Kurt Schmidt,Bernd Mayer

doi:10.1074/jbc.m801182200

Matteo Beretta, Karl Gruber + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m801182200

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Abstract

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Highlights

Mulation in vascular smooth muscle [1]
ALDH-catalyzed GTN Metabolism—Determination of 1,2and 1,3-glycerol dinitrate (GDN) formation by radio thin layer chromatography revealed that both ALDH isoforms catalyze GTN metabolism (Fig. 1)
The present study demonstrates that GTN metabolism catalyzed by purified ALDH isoforms is associated with formation of free nitric oxide (NO) radical, resulting in pronounced soluble guanylate cyclase (sGC) activation

Summary

Introduction

Mulation in vascular smooth muscle [1]. In virtually all cells and tissues, the slow reaction of GTN with thiols, in particular GSH, yields 1,2- and 1,3-GDN together with stoichiometric amounts of inorganic nitrite [2], but thiol-triggered GTN metabolism is not associated with GTN bioactivation except the still poorly understood reaction of GTN with L-cysteine that leads to formation of a bioactive species with NO-like properties [3]. In the presence of this low GTN concentration, ALDH2 (Fig. 1B) exhibited ϳ3-fold higher activity with respect to 1,2-GDN formation

Results

Conclusion