Abstract
Background and PurposeL-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation.Experimental ApproachAscorbate deficiency was induced in Gulo(-/-) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo(-/-), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined.Key ResultsLike GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo(-/-) mice with the 26S proteasome inhibitor bortezomib.Conclusions and ImplicationsOur data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.
Highlights
Organic nitrates, in particular nitroglycerin (GTN), have been used for decades to treat angina pectoris, but development of tolerance limits their usefulness and precludes continuous administration to patients
The proposed mechanisms underlying the development of vascular tolerance to glycerol trinitrate (GTN) are manifold, including impaired bioactivation to NO or a related activator of soluble guanylate cyclase (Sage et al, 2000; Chen et al, 2002), desensitization of soluble guanylate cyclase (Schröder et al, 1988; Sayed et al, 2008) and oxidative stress associated with increased generation of superoxide in blood vessels leading to limited NO bioavailability due to peroxynitrite formation (Münzel et al, 1995)
There is a large body of evidence indicating that aldehyde dehydrogenase-2 (ALDH2) is a key enzyme of vascular GTN bioactivation and that exposure of blood vessels to GTN causes mechanism-based inactivation of ALDH2, resulting in impaired GTN bioactivation (Mayer and Beretta, 2008)
Summary
In particular nitroglycerin (GTN), have been used for decades to treat angina pectoris, but development of tolerance limits their usefulness and precludes continuous administration to patients. Challenged by some studies (Hinz and Schröder, 1998; Csont et al, 2002; Hanspal et al, 2002), the oxidative stress concept is supported by several reports on beneficial effects of antioxidants like ascorbate in experimental and clinical studies of nitrate tolerance (Bassenge et al, 1998; Watanabe et al, 1998; McVeigh et al, 2002) This hypothesis is questioned, by a study showing that ascorbate did not prevent tolerance development (Milone et al, 1999). AND PURPOSE L-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined
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