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 À/À 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, PETriNchromophore 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 À/À 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.

Bioactivation of Pentaerythrityl Tetranitrate by Mitochondrial Aldehyde Dehydrogenase

“…PETN was approximately 25-fold less potent on mouse (EC 50 ϭ 0.8 M) than on rat (EC 50 ϭ 0.03 M) aortas. A similar difference in potency of PETN has been reported previously [EC 50 values of 8.5 nM in rat and 170 nM in mouse (Wenzel et al, 2007a) …”

“…Previous studies have shown that vasorelaxation to PETN is attenuated by pharmacological inhibition or gene deletion of ALDH2 Mollnau et al, 2006;Wenzel et al, 2007a). Although these studies provided circumstantial evidence for a role of ALDH2 in vascular bioactivation of PETN, enzymatic metabolism of the tetranitrate has not been demonstrated so far.…”

“…Although the low-affinity pathway may reflect a combination of various enzymatic and possibly nonenzymatic reactions (Fung, 2004), the high-affinity pathway seems to be catalyzed exclusively by ALDH2 (Chen et al, 2002(Chen et al, , 2005. Meanwhile, there is general agreement that ALDH2 is essentially involved in vasorelaxation induced by GTN (DiFabio et al, 2003;Kollau et al, 2005;Huellner et al, 2008) and PETN Wenzel et al, 2007a) but does not contribute to the vascular effects of the isosorbide nitrates (Chen et al, 2005).…”

Monitoring White Blood Cell Mitochondrial Aldehyde Dehydrogenase Activity: Implications for Nitrate Therapy in Humans

“…For instance, we demonstrated that the nitrate pentaerythritol tetranitrate (PETN), although still undergoing mitochondrial activation by the ALDH-2 (Wenzel et al, 2007b), does not induce tolerance and does not stimulate vascular superoxide production in a rat model of nitrate tolerance, two phenomena that probably follow the activation of the antioxidant enzyme heme oxygenase-1 (HO-1) by PETN (Wenzel et al, 2007c). Mitochondrial reactive oxygen species formation was identified as a hallmark of GTN-induced nitrate tolerance (Sydow et al, 2004;Daiber et al, 2005;Esplugues et al, 2006), leading to oxidative ALDH-2 inhibition that could be prevented or reversed in an experimental rat model by (dihydro)lipoic acid (Wenzel et al, 2007a).…”

Searching for New NO-Donor Aspirin-like Molecules: A New Class of Nitrooxy-acyl Derivatives of Salicylic Acid

“…With the length of the chain being equal, the dinitrooxy-substituted compounds are always more active than the corresponding mononitroxy analogues, in keeping with the statement that the number of nitrate groups determines reactivity and potency of organic nitrates. 46 When the vasodilator experiments were repeated in the presence of 1 µM ODQ, a decrease in the potencies was observed, in keeping with NOinduced activation of the sGS.…”