Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2-/- mice

Abstract

Background and purpose: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator.

Experimental approach: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry.

Key results: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN.

Conclusions and implications: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways.

Figure 1

Structures of PETN, PETriN, PEDN, PEMN, GTN, 1,3-GDN and spermine NONOate.

Figure 2

Concentration–relaxation curves of isolated aortic rings from C57B6 (wild type, WT) and ALDH-2^−/− mice using different vasodilators after preconstriction with prostaglandin F_2α (3 μM): (a) Effects of inhibition of sGC with ODQ on responses to PETN (b) Comparison between PETriN, PEDN and PEMN (dotted line); (c) Comparison between GTN and 1,3-GDN (d) Effects of ACh. Results are statistically summarized in Table 1.

Figure 3

Activity of cGK-1 as reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein at serine 239 (P-VASP) in response to acute challenges with organic nitrates. Aortic segments of C57B6 (WT) and ALDH-2^−/− (ALDH-2 ko) were incubated with PEDN, PETN or solvent (DMSO). Bars reflect densitometric analysis. The Western blot is representative of five independent experiments. ^$P<0.05 vs WT DMSO; ^#P<0.05 vs WT DMSO, ALDH-2 ko DMSO and ALDH-2 ko PETN, NS vs WT PETN and WT PEDN.

Figure 4

(a) Chemiluminescence nitrogen detection (CLND) of PETN and PEDN in suspensions of isolated mitochondria. Bars reflect the amount of recovered organic nitrate by CLND analysis after incubation of isolated mouse liver mitochondria with the concentration shown on the x-axis. ^*P<0.05 vs ALDH-2 ko; n.d.: not detected, e.g. peaks were below the level of quantification and therefore not elegible for analysis. (b) Activity of ALDH-2 was assessed by measuring the conversion of 2-hydroxy-3-nitro-benzaldehyde to the benzoic acid product (2H3N-BA) in isolated mitochondria. Insert shows a Western blot of ALDH-2 in extracts of aortic tissue. ^*P<0.05 vs WT.

Figure 5

Hypothetical mechanism for the reaction of a cysteine-thiol group at the active-site of ALDH-2 with a nitrate group of PETN explaining the role of the number of nitrate (–ONO₂) groups in the organic nitrate for its bioactivation. Three or more nitrate groups create a positively charged (electrophilic) environment at the nitrogens which facilitates the nucleophilic attack of an ALDH-2 thiol group with subsequent formation of a thionitrate intermediate.