After 130 years, the molecular mechanism of action of nitroglycerin is revealed.

N itroglycerin, which was originally synthesized by Ascanio Sobrero, was used by Alfred Nobel to manufacture dynamite. It was in Nobel’s dynamite factories in the late 1860s that the antianginal effect of nitroglycerin was discovered. Two interesting observations were made. First, factory workers on Monday mornings often complained of headaches that disappeared over the weekends. Second, factory workers suffering from angina pectoris or heart failure often experienced relief from chest pain during the work week, but which recurred on weekends. Both effects were attributed to the vasodilator action of nitroglycerin, which quickly became apparent to the physicians and physiologists in local communities. But what was the mechanism of this vasodilator action of the most powerful explosive chemical discovered in the nineteenth century? The answer to this question was not to come for another century. In the late 1970s and early 1980s, the vasodilator effect of nitroglycerin was discovered to be caused by nitric oxide (NO), which was apparently generated from nitroglycerin in vascular smooth muscle (1–4). These early observations on NO culminated less than 10 years later, in 1986, with the discovery that mammalian cells synthesize NO (5). In 1998, about 130 years after Alfred Nobel’s invention of dynamite and the first observed clinical benefit of nitroglycerin, the Nobel Prize in Physiology or Medicine was awarded for ‘‘Nitric Oxide as a Signaling Molecule in the Cardiovascular System’’. Despite these achievements, the precise molecular mechanism by which NO is generated from nitroglycerin remained elusive until the work of Chen et al. (6), reported in this issue of PNAS. Previous studies showed that the bioactivation of nitroglycerin somehow involved thiols or sulfhydryl-containing compounds, and that NO or NO-containing compounds constituted the biologically active species (1–5, 7). The earliest studies suggested that an interaction between nitroglycerin and sulfhydryl (-SH)containing cellular receptors was necessary for vascular smooth muscle relaxation to occur and that repeated administration of nitroglycerin caused sulfhydryl depletion (via oxidation) and consequent tolerance to further vasodilation (7–9). Subsequent studies addressing the activation of cytosolic guanylate cyclase by organic nitrate esters (nitroglycerin), organic nitrite esters (isoamyl nitrite), and nitroso compounds revealed that a chemical reaction occurred between the nitro compound and a thiol to generate an intermediate S-nitrosothiol, which then decomposed with the liberation of NO (3). Tolerance to nitroglycerin was explained simply by thiol utilization and depletion in the presence of excess nitroglycerin, thereby resulting in deficient production of S-nitrosothiol and NO. This working hypothesis was supported by animal and clinical studies showing that the administration of relatively large doses of cysteine or N-acetylcysteine could prevent or reverse the tolerance to the vasodilator action of repeated administration of nitroglycerin (see ref. 5). There were many unanswered questions associated with these earlier studies, however. The molecular mechanism of the interaction between nitroglycerin and thiol to generate S-nitrosothiol and NO remained an enigma. Moreover, the basis of the earlier hypotheses was activation of cytosolic guanylate cyclase in enzyme reaction mixtures and not vascular smooth muscle relaxation (3). Isolated enzyme reaction mixtures or broken cell preparations are very different from intact cells or tissues. The early work with cellular extracts did not address the likely possibility that the reaction between nitroglycerin and thiol might be enzymatic in nature. In fact, the evidence was in favor of a nonenzymatic chemical reaction (3). Subsequent studies suggested that one or more enzymatic mechanisms might be responsible for the bioactivation of nitroglycerin (10–16). However, none of these enzyme systems could catalyze the selective formation of 1,2-glyceryl dinitrate from nitroglycerin and no correlation could be found between tolerance to nitroglycerin action and tolerance to enzyme activities. The article by Chen et al. (6) uncovers the role of mitochondrial aldehyde dehydrogenase, which specifically generates 1,2glyceryl dinitrate from nitroglycerin, in the bioactivation of nitroglycerin to elicit vasorelaxation and in the development of tolerance to nitroglycerin. Chen et al. (6) used several ingenious approaches to elucidate the enzymatic mechanism of bioactivation of nitroglycerin: a source of large numbers of cells so that the lack of starting material would not be a limiting factor. By using mouse macrophages grown in cell culture, physiologically relevant, relatively low concentrations of nitroglycerin (0.1 M) were shown to generate 1,2-glyceryl dinitrate through the catalytic action of an enzyme that was virtually identical to mouse mitochondrial aldehyde dehydrogenase. Mitochondrial aldehyde dehydrogenase purified from bovine liver showed identical catalytic properties to the mouse enzyme. Inhibitors of aldehyde dehydrogenase, such as cyanamide and chloral hydrate, blocked the formation of 1,2-glyceryl dinitrate from nitroglycerin. Aldehyde dehydrogenase possesses esterase activity (17) in addition to the classical NAD dependent dehydrogenation activity, and the catalytic action on nitroglycerin was analogous to its esterase activity, with the important exception that nitrite (NO2 ) rather than nitrate (NO3 ) was a product of the enzymatic reaction. Thus, these observations were in agreement with the earliest biological findings that nitroglycerin is metabolized by tissues to inorganic nitrite or NO2 (3–5, 7–9). The classical sulfhydryl requirement for vascular smooth muscle relaxation by nitroglycerin (7) was explained as a chemical reaction between nitroglycerin and thiol sulfhydryl group to generate an intermediate Snitrosothiol species, which then decomposed with the liberation of NO (3). Other explanations and hypotheses were offered, but none of them could be replicated or confirmed across different tissues (18–19). Therefore, the selective conver-