Subcellular distribution of ALDH2 and nitroglycerin metabolism

The research objective of this proposal is to clarify how the subcellular localization of aldehyde dehydrogenase-2 (ALDH2) affects the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) and the development of vascular nitrate tolerance. The clinical benefit of GTN and related organic nitrates in the therapy of coronary artery disease results from dilation of coronary arteries and capacitative veins, leading to improved blood supply to cardiac muscle and reduction of preload, respectively. GTN is enzymatically bioactivated in vascular smooth muscle to nitric oxide (NO) or a related species, which triggers cGMP-mediated vasodilation through activation of soluble guanylate cyclase. ALDH2 is considered as the key enzyme catalyzing GTN bioactivation, and inactivation of ALDH2 by GTN may at least partially explain the development of nitrate tolerance, i.e. the loss of therapeutic effect of GTN upon long-term application. ALDH2 has a well established role in the detoxification of ethanol-derived acetaldehyde and other reactive aldehydes in the liver. The protein is expressed as a precursor with an N-terminal signal sequence targeting ALDH2 to the mitochondrial matrix of hepatocytes. Based on the almost exclusive mitochondrial expression of ALDH2 in liver, vascular GTN bioactivation is generally believed to occur in smooth muscle mitochondria. However, we have recently shown that ALDH2 is mainly present in cytosolic fractions of rodent and human blood vessels. Since GTN-induced vasodilation of ALDH2-deficient murine aortas was restored upon cytosolic but not mitochondrial overexpression of ALDH2, GTN bioactivity appears to be determined by the subcellular distribution of the enzyme. In the proposed project we will study the subcellular localization of ALDH2 in various types of cells and tissues and sequnce the ALDH2 gene transcripts to clarify the mechanism for differential expression of the protein with and without the mitochondrial signal sequence. Special emphasis will be placed to the effects of ALDH2 localization on GTN bioactivation in blood vessels and the development of vascular nitrate tolerance. According to our working hyothesis, cytosolic ALDH2 catalyzes the formation of NO bioactivity from GTN, whereas mitochondrial GTN metabolism results in oxidative stress that compromises relaxation and may contribute to nitrate tolerance. This hypothesis will be tested by correlating the subcellular distribution of ALDH2 in arterial and venous blood vessels from various species (including humans) with GTN potency. The involvement of mitochondrial ALDH2 in nitrate tolerance will be studied in cell culture and in vivo models of mitochondrial dysfunction as well as by vascular overexpression of cytosolic and mitochondrial ALDH2 in ALDH2 knockout mice. Besides providing new insights into the cellular mechanisms underlying GTN bioactivation and the development of vascular nitrate tolerance, the proposed studies will significantly increase the current knowledge on differential ALDH2 expression in mammalian tissues.

In this project, we investigated the cellular mechanisms underlying the therapeutic effects of nitroglycerin (glyceryl trinitrate, GTN), a drug that has been used since the late 19th century for the therapy of coronary artery disease and other cardiovascular disorders. The clinical benefit of GTN and related organic nitrates results from dilation of large blood vessels, in particular, coronary arteries and large veins, leading to improved blood supply to cardiac muscle and reduction of preload, respectively. GTN is a prodrug that becomes enzymatically bioactivated in vascular smooth muscle to eventually yield nitric oxide (NO). NO causes vasodilation by inducing accumulation of the second messenger cyclic GMP (cGMP) by activating the enzyme soluble guanylate cyclase (sGC). The key enzyme of GTN bioactivation is aldehyde dehydrogenase (ALDH2). In contrast to hepatocytes, where ALDH2 is almost exclusively found in mitochondria, vascular smooth muscle contain mainly cytosolic ALDH2. In the current project, we studied the reasons and consequences of this differential subcellular localization of ALDH2 and clarified the reaction resulting in ALDH2-catalyzed NO formation. For this purpose, we used a newly developed protein-based sensor that enabled us to monitor and quantify NO formation in vascular smooth muscle cells in real time as changes in the emission of fluorescent light. The obtained data were compared with GTN bioactivation, measured as activation of sGC, showing that formation of NO from pharmacologically relevant concentrations of GTN was strictly dependent on the presence of active ALDH2. Formation of GTN-derived NO correlated excellently with sGC activation and accumulation of cGMP in intact cells, indicating that the reaction catalyzed by ALDH2 is necessary and sufficient for GTN bioactivation. Clinical application of GTN is hampered by the loss of therapeutic efficiency in patients continuously treated with the drug for several hours. The development of this tolerance to GTN is thought to be a consequence of ALDH2 inactivation, but the precise mechanism is still controversial, so we took advantage of the new NO sensor to shed light on this issue. We overexpressed a suitable ALDH2 mutant in vascular smooth muscle cells and monitored NO formation in single cells upon GTN infusion under various conditions. Our results showed that the enzyme becomes almost completely inactivated within a single turnover, but retains low level NO formation, presumably due to slow partial reactivation by an intracellular reductant. The kinetics of residual NO formation correlated with the kinetics of aortic relaxation and may explain the relatively longlived effects of GTN in vivo. According to our data, nitrate tolerance is due to a poorly characterized reaction between ALDH2 and GTN that leads to slow irreversible inactivation of the enzyme. Our results provide new insights into the cellular and molecular mechanisms of nitroglycerin bioactivation in blood vessels and pave the way for the development of new drugs, which could be useful for the treatment of coronary artery disease without causing nitrate tolerance.