Role of vitamin C in nitroglycerin-induced vasodilation

Nitroglycerin (glyceroltrinitrate, GTN) has been used since more than 130 years for the therapy of coronary artery disease which is caused by insufficient oxygen supply to the heart. The clinical benefit of GTN and related organic nitrates results from dilation of blood vessels. At low doses, the predominant action of GTN is relaxation of large vessels (coronary arteries, large veins) leading to improved blood supply to cardiac muscle and reduction of preload. At higher doses, arterial resistance and cardiac output are decreased as well, resulting in a drop of diastolic and systolic blood pressure, respectively. Nitrate therapy in patients is hampered by loss of effect upon continuous application of GTN (nitrate tolerance). At the molecular level, the effect of GTN involves enzymatic and/or non- enzymatic bioactivation to yield nitric oxide (NO) which activates soluble guanylate cyclase, leading to cGMP- mediated vasodilation. The key enzyme of GTN bioactivation is thought to be mitochondrial aldehyde dehydrogenase (ALDH2). Since GTN causes oxidative inactivation of ALDH2, a reducing cofactor is required for sustained catalysis. Nitrate tolerance may be a consequence of vascular depletion of this cofactor. We have recently discovered that ascorbate deficiency leads to an about 100-fold decrease in the potency of GTN without considerably affecting general vascular function. These results suggest that ascorbate is essential for GTN bioactivation and that nitrate tolerance may result from vascular ascorbate depletion. To test this hypothesis we will study GTN metabolism/ bioactivation in blood vessels and mitochondria isolated from guinea pigs fed either standard ascorbate containing or ascorbate-free diet. Special emphasis will be placed on the modulation of ALDH2 inactivation/reactivation kinetics by ascorbate. The details of ALDH2 inactivation/reactivation, in particular the involvement of critical cysteine residues in the active site, will be studied with purified recombinant human liver ALDH2. To clarify whether continuous application of GTN causes ascorbate depletion, attempts will be made to measure the possibly very low levels of ascorbate in vascular tissue and to correlate the data with the sensitivity of the blood vessels to GTN-induced relaxation. In addition, in collaboration with a group in the USA we will measure the hemodynamic effects of GTN in L-gulonolactone oxidase knockout mice, which are unable to synthesize ascorbate. This genetic mouse model of vitamin C deficiency will be established in our laboratory for future studies. The proposed work is expected to provide new insights into the molecular mechanisms underlying GTN bioactivation and development of nitrate tolerance in blood vessels. The key role of ascorbate in GTN bioactivation may have important implications for GTN therapy of patients with cardiovascular and non-cardiovascular diseases that are associated with oxidative stress and ascorbate deficiency.

Nitroglycerin (glyceroltrinitrate, GTN) has been used since more than 130 years for the therapy of coronary artery disease which is caused by insufficient oxygen supply to the heart. The clinical benefit of GTN and related organic nitrates results from dilation of blood vessels. At low doses, the predominant action of GTN is relaxation of large vessels (coronary arteries, large veins) leading to improved blood supply to cardiac muscle and reduction of preload. At higher doses, arterial resistance and cardiac output are decreased as well, resulting in a drop of diastolic and systolic blood pressure, respectively. Nitrate therapy in patients is hampered by loss of effect upon continuous application of GTN (nitrate tolerance). At the molecular level, the effect of GTN involves enzymatic and/or non- enzymatic bioactivation to yield nitric oxide (NO) which activates soluble guanylate cyclase, leading to cGMP- mediated vasodilation. The key enzyme of GTN bioactivation is thought to be mitochondrial aldehyde dehydrogenase (ALDH2). Since GTN causes oxidative inactivation of ALDH2, a reducing cofactor is required for sustained catalysis. Nitrate tolerance may be a consequence of vascular depletion of this cofactor. We have recently discovered that ascorbate deficiency leads to an about 100-fold decrease in the potency of GTN without considerably affecting general vascular function. These results suggest that ascorbate is essential for GTN bioactivation and that nitrate tolerance may result from vascular ascorbate depletion. To test this hypothesis we will study GTN metabolism/ bioactivation in blood vessels and mitochondria isolated from guinea pigs fed either standard ascorbate containing or ascorbate-free diet. Special emphasis will be placed on the modulation of ALDH2 inactivation/reactivation kinetics by ascorbate. The details of ALDH2 inactivation/reactivation, in particular the involvement of critical cysteine residues in the active site, will be studied with purified recombinant human liver ALDH2. To clarify whether continuous application of GTN causes ascorbate depletion, attempts will be made to measure the possibly very low levels of ascorbate in vascular tissue and to correlate the data with the sensitivity of the blood vessels to GTN-induced relaxation. In addition, in collaboration with a group in the USA we will measure the hemodynamic effects of GTN in L-gulonolactone oxidase knockout mice, which are unable to synthesize ascorbate. This genetic mouse model of vitamin C deficiency will be established in our laboratory for future studies. The proposed work is expected to provide new insights into the molecular mechanisms underlying GTN bioactivation and development of nitrate tolerance in blood vessels. The key role of ascorbate in GTN bioactivation may have important implications for GTN therapy of patients with cardiovascular and non-cardiovascular diseases that are associated with oxidative stress and ascorbate deficiency.