EDITORIAL COMMENT
Amelioration of nitrate tolerance: matching strategies with mechanisms*
John D. Horowitz, MBBS, PhD*,*
* Cardiology Unit, North Western Adelaide Health Service, University of Adelaide, Adelaide, Australia
* Reprint requests and correspondence: Prof. John D. Horowitz, Cardiology Unit, Queen Elizabeth Hospital, NWAHS, Woodville Road, Woodville SA 5011, Australia.
john.horowitz{at}adelaide.edu.au
The therapeutic promise of the organic nitrates remains intriguingly unfulfilled after a century of clinical experience. Two recent studies provide a glimpse of potential efficacy, both in the management of congestive heart failure and of evolving myocardial infarction (1,2). For example, Cotter et al. (1) demonstrated that intravenously infused isosorbide dinitrate (ISDN) improves symptomatic status in patients with acute pulmonary edema, possibly via reduced risk of development of acute myocardial infarction. Furthermore, in the ISIS-4 trial (Fourth International Study of Infarct Survival) (2), oral isosorbide mononitrate (ISMN) therapy was associated with approximately a 20% reduction in mortality during the first 24 h after acute myocardial infarction.
On the other hand, numerous studies attest to the observation that the various actions of organic nitrates, whether vasodilator or anti-aggregatory, tend to diminish during long-term nitrate therapy, and this phenomenon, loosely termed nitrate tolerance, is associated with progressive attenuation of the therapeutic effects of nitrates, irrespective of whether the end point measured is hemodynamic (in congestive heart failure), anti-ischemic (in angina pectoris) or associated with post-infarct outcome.
Considerable progress has been made on the basis of recent studies delineating the major factors restricting efficacy of organic nitrate therapy. These may be categorized as: 1) nitrate resistance: de novo impairment of tissue responsiveness to organic nitrates and to nitric oxide, 2) "true" nitrate tolerance: progressive desensitization of blood vessels and/or platelets to biological effects of organic nitrates, and 3) "pseudo-tolerance": algebraic attenuation of the effects of organic nitrates in vitro through increased secretion of substances exerting biologically opposing effects.
There is general agreement that "nitrate resistance" shares pathogenetic mechanisms with endothelial dysfunction, and is manifest both in vasculature (3) and in platelets (4). Increased secretion of catecholamines, angiotensin II and endothelin have been demonstrated in various models (5,6): irrespective of mechanism(s), it is generally agreed that pseudo-tolerance underlies the phenomenon of "rebound" ischemia after sudden cessation of nitrate therapy (7). On the other hand, the mechanism(s) underlying the development of "true" nitrate tolerance remain controversial, with no-clear cut strategies available for circumvention of this problem.
As recently summarized in a number of reviews (8–10), two major categories of mechanism have been proposed for the development of "true" nitrate tolerance: impaired enzymatic release of nitric oxide (NO) from organic nitrates ("impaired bioconversion") and increased endothelial generation of superoxide, impairing both responses to organic nitrates and to agents stimulating release of NO from vascular endothelium. While it has recently been proposed that these two categories may be linked (10), little evidence of a single over-arching mechanism is currently available.
Part of the problem associated with evaluation of mechanism of nitrate tolerance lies in the choice of available methodology. While clinical studies (e.g., measuring exercise performance) may document attenuation of therapeutic effect, it is difficult to delineate the relative role(s) of "true" versus pseudo-tolerance in this attenuation. Furthermore, the choice of subjects may be critical: e.g., studies in normal subjects may be used to demonstrate progressive impairment of endothelial function during chronic nitrate therapy, but the relevance of this change is probably exaggerated relative to that potentially occurring patients in whom endothelial dysfunction is likely to be present at the time of initiation of nitrate therapy.
Major evidence in favor of the "impaired bioconversion" hypothesis includes the fact that nitrate tolerance can be induced in vitro, under which circumstances it is largely, although not entirely, specific for organic nitrates, with minimal cross-tolerance to direct NO donors or authentic NO (11,12). Furthermore, under a number of circumstances in both human and animal models, induction of nitrate tolerance is associated with little if any change in measures of oxidative stress and/or endothelial function (13–16). Furthermore, induction of tolerance by glyceryl trinitrate (GTN) in patients with stable angina pectoris was associated with impairment of biotransformation of GTN to glyceryl 1,2-dinitrate (1,2-GDN) (15). Finally, Chen et al. (17) have recently identified aldehyde dehydrogenase as the major enzyme system catalysing nitrate bioconversion to 1,2-GDN in rabbit aortae and mouse macrophage cells. Purified aldehyde dehydrogenase, which has an active site thiol group, catalyzes 1,2-GDN formation only in the presence of additional thiols, consistent with previous findings that the effects of GTN in stimulating activation of soluble guanylate cyclase were sulfhydryl-dependent in vitro (18), and that certain sulfhydryl agents, such as N-acetylcysteine (NAC) potentiate the hemodynamic and anti-aggregatory effects of organic nitrates (19,20). It was therefore postulated by Chen et al. (17) that nitrate tolerance reflected progressive inhibition of aldehyde dehydrogenase activity. Indeed, GTN and isosorbide dinitrate have been reported to inhibit aldehyde dehydrogenase both in vitro and in vivo (21).
There is also considerable evidence that nitrate tolerance is sometimes associated with incremental impairment of endothelial function, largely due to generation of superoxide (O2–) free radical. While it is true that some NO donors, such as GTN, generate O2– during enzymatic bioconversion (22), the majority of studies suggest that chronic continuous in vivo exposure to high doses of GTN both in animal models and in humans, sometimes induces incremental O2– generation and resultant evidence of redox stress (23–25). While the endothelium represents the principal source of O2–, both the NAD(P)H oxidase system (23) and dysfunctional endothelial nitric oxide synthase (eNOS)(24) appear to contribute to O2– generation. Both of these findings are of potential therapeutic importance as NAD(P)H oxidase expression is stimulated by angiotensin II while "uncoupling" of eNOS (to generate O2– rather than NO) may occur in the absence of its cofactor tetrahydrobiopterin (BH4) (26) or with depletion of arginine levels. On the other hand, eNOS knockout mice do not display altered susceptibility to induction of GTN tolerance (27). Irrespective of precise mechanism, the implication of a central role of O2– generation in nitrate tolerance would be attenuation of endogenous endothelium-dependent NO physiological effects, whether vasodilator or anti-aggregatory. This cross-tolerance to endogenous NO, which has been documented in many animal models of GTN-induced tolerance and in some human studies, would imply that nitrate therapy might be potentially harmful to long-term clinical outcomes.
In the this issue of the Journal, Müller et al. (28) reported the characteristics of nitrate tolerance induced in rabbits via 4 months eccentric dosing with high doses (200 mg/kg/day) of ISMN. Tolerance was assessed by examination of reactivity of aortic rings in comparison with those of control animals. The main findings were that in the presence of a moderate level of tolerance to ISMN (approximately a five-fold increase in ISMN dose to produce 50% relaxation responses) there was neither cross-tolerance to the direct NO donor S-nitroso-N-acetyl penicillamine, nor any diminution of relaxations to the endothelial-dependent vasodilator acetylcholine. Furthermore, superoxide production was unchanged in vessels from tolerant animals. Hence it can be concluded that in this model of nitrate tolerance induced by long term ISMN administration there is neither induction of endothelial dysfunction nor cross-tolerance to NO. Although not specifically assessed, the findings are consistent with impairment of nitrate bioconversion as a mechanism for tolerance induction.
What are the implications of these findings? First, this provides incremental evidence that tolerance induction in vivo may be relatively nitrate-specific, that is, similar to in vitro tolerance, under some circumstances; this finding is consistent with some, but not all, previous findings in man. It also follows that the induction or aggravation of endothelial dysfunction is a variable, rather than constant, component of nitrate-tolerant states. Nevertheless, some important issues remain unanswered. For example, aortic smooth muscle may not be fully representative of regional vasomotor sites for organic nitrates: in a recent human study, one to two days of continuous high-dose intravenous GTN infusion induced nitrate-specific tolerance in saphenous veins, but there was impairment of endothelial function in internal mammary and radial arteries (29). Second, it is not clear to what extent the findings depend on the nitrate inducing tolerance and/or the nitrate examined in the tolerant setting. Based upon previous in vitro results (11,12), it is possible that the small shift in the ISDN concentration-response curve in the study by Müller et al. (28) may have corresponded with far greater inhibition of GTN responses; however this was not specifically examined. Furthermore, the majority of in vivo studies in which tolerance induction was associated with increased oxidative stress have utilized GTN as a tolerance-inducing agent, and it certainly remains possible that continuous high-dose GTN therapy induces a different form of tolerance, as well as a greater degree of tolerance, compared to some other nitrates.
An understanding of the mechanism(s) underlying nitrate tolerance induction must be a precursor to clinical studies evaluating possible means of circumvention of tolerance; these studies should also extend to evaluating the efficacy of tolerance-modifying regimes in the clinical setting. To date, a number of agents have been evaluated clinically as regards interactions with nitrate tolerance induction. Data are limited and inconsistent (Table 1). In particular, utilization of angiotensin-converting enzyme (ACE) inhibitors (and ATI antagonists) has not been associated with marked amelioration of nitrate efficacy, despite the known effects of ACE inhibitors in reversing endothelial dysfunction. Folic acid, which clearly improves endothelial dysfunction in many settings (30), also potentiates hemodynamic responses to GTN (31). However, it is not clear that this potentiation involves a specific interaction with the process of tolerance induction, and the therapeutic implications remain untested. N-acetylcysteine potentiates the vasodilator and anti-aggregatory effects of GTN in many settings, and appears useful in combination with GTN in unstable angina (32) as well as in acute pulmonary edema and possibly acute myocardial infarction. However, NAC both potentiates GTN-induced activation of soluble guanylate cyclase (33) (suggesting an effect on bioconversion) and also exerts well-documented anti-oxidant effects. It remains controversial to what extent the clinical efficacy of NAC reflects a specific interaction with nitrate tolerance.
In summary, we can conclude that nitrate tolerance induction involves a nitrate-specific (bioconversion?) component in most cases, with a variable component of increased oxidative stress and incremental endothelial dysfunction. It can be anticipated that the biological role of aldehyde dehydrogenase in catalyzing NO release from organic nitrates in humans will soon be more completely understood. With this will emerge a biochemical means for "on-line" evaluation of the efficacy of nitrates during chronic therapy, which in turn should facilitate clinical efficacy studies. This progress has been a long time coming.
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Footnotes
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* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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