Bioactivation of Nitroglycerin

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 veins, leading to reduction of cardiac preload and oxygen consumption. At higher doses, arterial resistance and cardiac output are decreased as well, resulting in a drop of diastolic and systolic blood pressure, respectively. Despite the considerable therapeutic implications of organic nitrates, the molecular mechanisms underlying their biological effects are still unclear and highly controversial. It is known that bioactivation of GTN results in formation of nitric oxide (NO) or a related species activating soluble guanylate cyclase (sGC) which forms cyclic GMP (cGMP), an established cellular messenger of smooth muscle relaxation. Several mechanism have been suggested to explain NO release from organic nitrates, but virtually all of them have been questioned by subsequent studies. Recently, Dr. Stamler and coworkers at Duke University identified mitochondrial aldehyde dehydrogenase (mtALDH) as highly efficient GTN metabolizing enzyme [Chen et al., Proc. Natl. Acad. Sci. U.S.A. 99, 8306-11, 2002]. Based on their findings that established (but non-selective) mtALDH inhibitors diminished GTN-induced vascular relaxation and cGMP accumulation, the authors proposed that mtALDH is the key enzyme of GTN bioactivation. Although this paper led a Nobel laureate to write an Editorial entitled "After 130 years, the molecular mechanism of action of nitroglycerin is revealed" [Ignarro, Proc. Natl. Acad. Sci. U.S.A. 99, 7816-7, 2002], the study fails to explain how mtALDH-catalyzed GTN metabolism, leading to inactive nitrite as initial reaction product, could trigger sGC activation. It is the aim of the project to address this issue and to clarify the potential involvement of mtALDH and other enzymatic pathways in GTN bioactivation. To account for the uncertain identity of the active intermediate (NO or a related species), purified sGC will be used as highly sensitive sensor for GTN bioactivity. The role of mtALDH will be explored by co-incubation of the purified recombinant human liver enzyme (provided by Dr. Wing Ming Keung, Harvard University, Boston, U.S.A.) with sGC and determination of cyclic GMP formation in the presence of mtALDH cofactors and inhibitors. Studies with isolated mitochondria are aimed to reveal the possible involvement of the respiratory electron transfer chain in conversion of the inactive mtALDH product nitrite into the bioactive species. The sGC activity data will be compared with GTN metabolism (formation of 1,2- and 1,3-glyceroldinitrate), and NO release determined under identical conditions. In addition, potent and selective mtALDH inhibitors will be tested for interference with GTN-induced relaxation of vascular and non- vascular smooth muscle tissues. The proposed work is expected to provide new insights into the molecular mechanisms underlying the biological action of GTN and other organic nitrates. The results may have important implications for nitrate therapy and the development of new antianginal drugs.

Nitroglycerin (glycerol trinitrate, GTN) and other organic nitrates ("nitrovasodilators") have 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 veins, leading to reduction of cardiac preload and oxygen consumption. At higher doses, arterial resistance and cardiac output are decreased as well, resulting in a drop of diastolic and systolic blood pressure, respectively. Despite the considerable therapeutic implications of organic nitrates, the molecular mechanisms underlying their biological effects are still unclear and highly controversial. It is known that bioactivation of GTN results in formation of nitric oxide (NO) or a related species activating soluble guanylate cyclase (sGC) which forms cyclic GMP (cGMP), an established cellular messenger of smooth muscle relaxation. Several mechanisms have been suggested to explain NO release from organic nitrates, but virtually all of them have been questioned by subsequent studies. Recently, Dr. Stamler and coworkers at Duke University identified mitochondrial aldehyde dehydrogenase (ALDH2) as highly efficient GTN metabolizing enzyme, but it has remained unclear whether ALDH2 is indeed the key enzyme of GTN metabolism and how the enzymatic reaction is linked to sGC activation. In this project we used purified sGC as highly sensitive detector for NO-like bioactivity generated from GTN by isolated mitochondria. Our results indicate that ALDH2 is essential for the effect of therapeutically relevant low GTN concentrations. The main product of the ALDH2 reaction appears to be inorganic nitrite, which becomes reduced to NO by cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain. In addition, we found that ALDH2 becomes inactivated in the course of catalytic turnover by oxidation of essential SH groups. This mechanism-based enzyme inactivation by GTN and other organic nitrates may provide a molecular explanation for the development of nitrate tolerance, which is characterized by a loss of therapeutic effect upon continuous administration of organic nitrates to patients. Therefore, the results of this project should disclose new therapeutic strategies to prevent nitrate tolerance in humans and contribute to the development of improved nitrovasodilators.