After 130 Years, the Molecular Mechanism of Action of Nitroglycerin Is Revealed

Commentary

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

Louis J. Ignarro*

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095

itroglycerin, which was originally syn- to occur and that repeated administration of mitochondrial aldehyde dehydroge- Nthesized by Ascanio Sobrero, was of nitroglycerin caused sulfhydryl deple- nase, which specifically generates 1,2- used by Alfred Nobel to manufacture dy- tion (via oxidation) and consequent tol- glyceryl dinitrate from nitroglycerin, in namite. It was in Nobel’s dynamite facto- erance to further vasodilation (7–9). Sub- the bioactivation of nitroglycerin to elicit ries in the late 1860s that the antianginal sequent studies addressing the activation vasorelaxation and in the development of effect of nitroglycerin was discovered. of cytosolic guanylate cyclase by organic tolerance to nitroglycerin. Two interesting observations were made. nitrate esters (nitroglycerin), organic ni- Chen et al. (6) used several ingenious First, factory workers on Monday morn- trite esters (isoamyl nitrite), and nitroso approaches to elucidate the enzymatic ings often complained of headaches that compounds revealed that a chemical re- mechanism of bioactivation of nitroglyc- disappeared over the weekends. Second, action occurred between the nitro com- erin: a source of large numbers of cells so factory workers suffering from angina pound and a thiol to generate an inter- that the lack of starting material would not pectoris or heart failure often experienced mediate S-nitrosothiol, which then be a limiting factor. By using mouse mac- relief from chest pain during the work decomposed with the liberation of NO (3). rophages grown in cell culture, physiolog- week, but which recurred on weekends. Tolerance to nitroglycerin was explained ically relevant, relatively low concentra- Both effects were attributed to the vaso- simply by thiol utilization and depletion in tions of nitroglycerin (0.1 ␮M) were dilator action of nitroglycerin, which the presence of excess nitroglycerin, shown to generate 1,2-glyceryl dinitrate quickly became apparent to the physicians thereby resulting in deficient production through the catalytic action of an enzyme and physiologists in local communities. of S-nitrosothiol and NO. This working that was virtually identical to mouse mi- But what was the mechanism of this va- hypothesis was supported by animal and tochondrial aldehyde dehydrogenase. Mi- sodilator action of the most powerful ex- clinical studies showing that the adminis- tochondrial aldehyde dehydrogenase pu- plosive chemical discovered in the nine- tration of relatively large doses of cysteine rified from bovine liver showed identical teenth century? The answer to this or N-acetylcysteine could prevent or re- catalytic properties to the mouse enzyme. question was not to come for another verse the tolerance to the vasodilator ac- Inhibitors of aldehyde dehydrogenase, century. In the late 1970s and early 1980s, tion of repeated administration of nitro- such as cyanamide and chloral hydrate, the vasodilator effect of nitroglycerin was glycerin (see ref. 5). There were many blocked the formation of 1,2-glyceryl di- discovered to be caused by nitric oxide unanswered questions associated with nitrate from nitroglycerin. Aldehyde de- (NO), which was apparently generated these earlier studies, however. The molec- hydrogenase possesses esterase activity from nitroglycerin in vascular smooth ular mechanism of the interaction be- (17) in addition to the classical NADϩ- muscle (1–4). These early observations on tween nitroglycerin and thiol to generate dependent dehydrogenation activity, and NO culminated less than 10 years later, in S-nitrosothiol and NO remained an the catalytic action on nitroglycerin was 1986, with the discovery that mammalian enigma. Moreover, the basis of the earlier analogous to its esterase activity, with the Ϫ cells synthesize NO (5). In 1998, about 130 hypotheses was activation of cytosolic important exception that nitrite (NO2 ) Ϫ years after Alfred Nobel’s invention of guanylate cyclase in enzyme reaction mix- rather than nitrate (NO3 ) was a product dynamite and the first observed clinical tures and not vascular smooth muscle re- of the enzymatic reaction. Thus, these benefit of nitroglycerin, the Nobel Prize in laxation (3). Isolated enzyme reaction observations were in agreement with the Physiology or Medicine was awarded for mixtures or broken cell preparations are earliest biological findings that nitroglyc- ‘‘Nitric Oxide as a Signaling Molecule in very different from intact cells or tissues. erin is metabolized by tissues to inorganic Ϫ the Cardiovascular System’’. Despite these The early work with cellular extracts did nitrite or NO2 (3–5, 7–9). The classical achievements, the precise molecular not address the likely possibility that the sulfhydryl requirement for vascular mechanism by which NO is generated reaction between nitroglycerin and thiol smooth muscle relaxation by nitroglycerin from nitroglycerin remained elusive until might be enzymatic in nature. In fact, the (7) was explained as a chemical reaction the work of Chen et al. (6), reported in this evidence was in favor of a nonenzymatic between nitroglycerin and thiol sulfhydryl issue of PNAS. chemical reaction (3). Subsequent studies group to generate an intermediate S- Previous studies showed that the bioac- suggested that one or more enzymatic nitrosothiol species, which then decom- tivation of nitroglycerin somehow in- mechanisms might be responsible for the posed with the liberation of NO (3). Other volved thiols or sulfhydryl-containing bioactivation of nitroglycerin (10–16). explanations and hypotheses were of- compounds, and that NO or NO-contain- However, none of these enzyme systems fered, but none of them could be repli- ing compounds constituted the biologi- could catalyze the selective formation of cated or confirmed across different tissues cally active species (1–5, 7). The earliest 1,2-glyceryl dinitrate from nitroglycerin (18–19). Therefore, the selective conver- studies suggested that an interaction be- and no correlation could be found be- tween nitroglycerin and sulfhydryl (-SH)- tween tolerance to nitroglycerin action containing cellular receptors was neces- and tolerance to enzyme activities. The See companion article on page 8306. sary for vascular smooth muscle relaxation article by Chen et al. (6) uncovers the role *E-mail: [email protected].

7816–7817 ͉ PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.132271799 Downloaded by guest on September 27, 2021 sion of 1,2,3-glyceryl trinitrate (nitroglyc- from the thionitrite intermediate and is effects of NADϩ in catalyzing the appar- erin) to 1,2-glyceryl dinitrate plus nitrite, reduced to NO. These observations are ent organic nitrate ester reductase activity together with the dependence on a reduc- analogous to the earlier views of a ‘‘thiol of mitochondrial aldehyde dehydroge- ing thiol cofactor, made mitochondrial receptor model,’’ where nitroglycerin bio- nase? Second, what is the influence of aldehyde dehydrogenase a compelling activation required the presence of thiols, NADϩ on the kinetic parameters associ- Ϫ choice for the elusive enzyme pathway NO2 was generated as an intermediate ated with the organic nitrate reductase responsible for nitroglycerin bioactivation (in the production of NO), and tolerance activity? Third, because glutathione does in vascular smooth muscle. was explained as thiol depletion (3–5, not ‘‘reactivate’’ the enzyme in vitro, what Repeated and prolonged administra- 7–9). Since nitroglycerin was discovered to thiol is responsible for reactivation in tion of nitroglycerin and other organic elicit vascular smooth muscle relaxation vivo? Fourth, because nitroglycerin is well nitrate esters causes the development of via mechanisms involving conversion to known to be a more potent and effective tolerance or desensitization of vascular NO and stimulation of cGMP production, venodilator than arteriodilator, what are smooth muscle to further vasorelaxation experiments were conducted to ascertain the differences in distribution and activi- by nitroglycerin. This phenomenon has exactly how nitroglycerin and NO activate ties of mitochondrial aldehyde dehydro- become a serious limitation to the chronic cytosolic guanylate cyclase (22). Using genase in venous vs. arterial smooth mus- use of organic nitrate esters to treat an- unpurified sources of enzyme, NO acti- cle? Answers to these important questions gina pectoris. Understanding the molecu- vated guanylate cyclase in the absence of will enable an even deeper understand- lar mechanisms associated with the devel- further additions, whereas nitroglycerin ing of the mechanism of nitroglycerin opment of ‘‘nitroglycerin tolerance’’ required the addition of cysteine to cause bioactivation. would undoubtedly lead to the discovery the enzyme activation. Additional exper- The study of Chen et al. (6) teaches us either of ways to avoid tolerance or of new iments revealed that nitroglycerin can un- that mitochondrial aldehyde dehydroge- NO-generating drugs that do not cause dergo chemical reactions with cysteine to Ϫ nase is at least partially responsible for the tolerance. The studies of Chen et al. (6) form NO and S-nitrosocysteine (3). 2 bioactivation of nitroglycerin and is likely demonstrate that in vascular tissue made Several S-nitrosothiols, including S- to be the target of nitroglycerin tolerance. tolerant to the vasorelaxant effect of ni- nitrosocysteine, were synthesized and Moreover, by understanding the molecu- troglycerin, a comparable decrease occurs found to activate guanylate cyclase in the lar mechanism of nitroglycerin bioactiva- in both mitochondrial dehydrogenase ac- absence of further additions. No other tion and tolerance, it may now be possible tivity and tissue cGMP accumulation. thiol (including DTT or glutathione) or Consistent with this observation is the reducing agent could substitute for cys- to design and develop novel nitrovasodi- report that aldehyde dehydrogenase activ- teine in enabling nitroglycerin to activate lator drugs that do not cause tolerance. ity is markedly inhibited in patients un- guanylate cyclase. Interestingly, the reac- One approach might be to develop drugs dergoing chronic administration of nitro- tion between nitroglycerin and cysteine to that do not engage mitochondrial alde- Ϫ hyde dehydrogenase for the generation of glycerin and other organic nitrate esters form NO2 occurred best at pH 9.6, which (20). These findings also are consistent is near the pH optimum of mitochondrial NO. Ideally, the most appropriate kind of with previous reports that nitroglycerin aldehyde dehydrogenase. However, the NO-donor drug might be one that is tar- tolerance in patients can sometimes be pH-dependent chemical reaction between geted to an enzyme that is selectively COMMENTARY overcome by administration of N-acetyl- nitroglycerin and cysteine occurred in the distributed to the vascular smooth muscle cysteine (5, 21). absence of any aldehyde dehydrogenase or and acts as a substrate with only limited The authors reveal that mitochondrial in the absence of any tissue extract, for capacity to inhibit catalytic activity. Such aldehyde dehydrogenase functions also as that matter. Therefore, this reaction was a a drug would be an effective vasodilator a nitroglycerin reductase, where nitroglyc- nonenzymatic reaction that was responsi- that could be used in combination with erin acts as a substrate for the enzyme’s ble for the activation of guanylate cyclase other drugs for the symptomatic treat- reductase activity. As illustrated in by nitroglycerin. These earlier observa- ment of hypertension. To be useful for the Scheme 1 of the article (6), the authors tions taken together with the recent find- symptomatic treatment of angina pectoris, suggest that nitroglycerin binds to one of ings of Chen et al. (6) indicate that both however, the drug would need to be tar- the two cysteine sulfhydryl groups adja- enzymatic and nonenzymatic mechanisms geted more to venous than arterial smooth cent to the active-site thiol to form a may play roles in the bioactivation of muscle. Despite the desire to avoid toler- thionitrite-enzyme complex intermediate nitroglycerin. ance, it may be a difficult task, indeed, to plus the product 1,2-glyceryl dinitrate. Several key questions arise from these come up with an overall better antianginal Ϫ Then, presumably, the NO2 is released findings. First, what is the role and precise drug than the 130-year-old nitroglycerin.

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