How is tolerance avoided when using the nitroglycerin patch?

Abstract

During the last century, nitroglycerin has been the most commonly used antiischemic and antianginal agent. Unfortunately, after continuous application, its therapeutic efficacy rapidly vanishes. Neurohormonal activation of vasoconstrictor signals and intravascular volume expansion constitute early counter-regulatory responses (pseudotolerance), whereas long-term treatment induces intrinsic vascular changes, eg, a loss of nitrovasodilator-responsiveness (vascular tolerance). This is caused by increased vascular superoxide production and a supersensitivity to vasoconstrictors secondary to a tonic activation of protein kinase C. NADPH oxidase(s) and uncoupled endothelial nitric oxide synthase have been proposed as superoxide sources. Superoxide and vascular NO rapidly form peroxynitrite, which aggravates tolerance by promoting NO synthase uncoupling and inhibition of soluble guanylyl cyclase and prostacyclin synthase. This oxidative stress concept may explain why radical scavengers and substances, which reduce oxidative stress indirectly, are able to relieve tolerance and endothelial dysfunction. Recent work has defined a new tolerance mechanism, ie, an inhibition of mitochondrial aldehyde dehydrogenase, the enzyme that accomplishes bioactivation of nitroglycerin, and has identified mitochondria as an additional source of reactive oxygen species. Nitroglycerin-induced reactive oxygen species inhibit the bioactivation of nitroglycerin by thiol oxidation of aldehyde dehydrogenase. Both mechanisms, increased oxidative stress and impaired bioactivation of nitroglycerin, can be joined to provide a new concept for nitroglycerin tolerance and cross-tolerance. The consequences of these processes for the nitroglycerin downstream targets soluble guanylyl cyclase, cGMP-dependent protein kinase, cGMP-degrading phosphodiesterases, and toxic side effects contributing to endothelial dysfunction, such as inhibition of prostacyclin synthase, are discussed in this review.

Nitroglycerin (GTN) has been one of the most widely used antiischemic drugs for more than a century. Given acutely, organic nitrates are excellent agents for the treatment of stable-effort angina, unstable angina, in patients with acute myocardial infarction and in patients with chronic congestive heart failure. The chronic efficacy of nitrates, however, is blunted because of the development of nitrate tolerance.1 The problem of tolerance has been raised since the first clinical reports of nitrate therapy for hypertension in Bright’s disease. In 1888, Stewart reported a case of GTN tolerance in a man who required 20 grains of pure GTN to achieve the same hypotensive effect as induced by the initial dose of 1/100 grain. He later stressed that this was a common problem in clinical practice.2 The mechanisms underlying this phenomenon are still poorly defined. Nevertheless, recent data indicate that GTN-induced reactive oxygen species (ROS) formation may inhibit the GTN-metabolizing enzyme.3,4 This combination of adverse effects may explain the phenomenon of the clinically relevant tolerance and cross-tolerance to endothelium-dependent and -independent nitrovasodilators.

Mechanisms of GTN Activation and Action

How Does GTN Induce Vasodilation?

There is a major consent that the principle mechanism of GTN-induced smooth muscle relaxation is the activation of the intracellular NO receptor enzyme, soluble guanylyl cyclase (sGC), subsequent elevation of the cyclic GMP (cGMP) levels, and activation of cGMP-dependent protein kinases, and/or cyclic nucleotide-gated ion channels.5 Thus, GTN and other organic nitrates are believed to use the same signaling mechanism as NO generated by NO synthases. This hypothesis is supported by the finding that specific inhibitors of NO-sensitive sGC, like ODQ and NS 2028, decrease GTN-induced vasodilation.6,7 Furthermore, NO-sensitizing agents such as YC-1 increase the vasodilator potency of GTN dramatically8 and so do inhibitors of cGMP-degrading phosphodiesterases.9 However, the precise mechanism by which GTN activates vascular sGC is still controversially discussed.10,11 Soluble GC is a heterodimeric hemoprotein consisting of α and β subunits.12 The enzyme is strongly activated by binding of NO to the ferrous heme-iron, half maximally at ≈1 nmol/L steady-state concentration of NO.13 GTN requires intracellular (endothelial and/or smooth muscle cells) bioconversion to elicit cGMP formation and vasodilation, and, until recently, NO was regarded as the bioactive metabolite.14 Following the discovery of endothelium-derived NO,15 the idea of NO being the active principle of GTN was very attractive and led to the speculation that GTN may replace a compromised endothelial NO production, such as in patients with coronary heart disease.16 Several studies supported the GTN/NO hypothesis by demonstrating the formation of NO in cells and tissues exposed to GTN, either in vitro or in vivo.16–18 However, because in all of these studies, GTN was applied in concentrations far exceeding the therapeutic range,19 it remained unresolved whether GTN in therapeutically effective low concentrations increases vascular NO levels.

No NO From GTN?

We recently addressed this issue by NO spin trapping and electron paramagnetic resonance (EPR)-detection using colloid iron-diethyldithiocarbamate, Fe(DETC)2.20 Cyclic GMP formation was indirectly assessed by analyzing the phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). VASP is a prominent cGMP-dependent protein kinase (cGK) substrate and a reliable biochemical marker of cGK-I activity.21,22 In endothelium-intact vessels, we found that GTN exhibited a striking dissociation (3 log units of concentration difference) between its vascular activity (increase in P-VASP and vasorelaxation, observed with nanomolar concentrations) and its NO-donor properties (increase in NOFe(DETC)2 formation, observed at micromolar concentrations).23 This finding was in contrast to the coincidence of NO formation and vascular activity seen with isosorbide dinitrate (ISDN) and calcium ionophore (A23187) in the same vessel type and caused doubt on the GTN/NO hypothesis. In support of this finding, the vasodilator activity of GTN was much less susceptible to inhibition by the NO scavenger carboxy-PTIO than that of established NO donors.24 These results challenge the widely accepted GTN/NO hypothesis and suggest that at therapeutically effective (nanomolar) concentrations, GTN activates the vascular sGC/cGK-I pathway independently of a biotransformation to NO. Interestingly, however, in the presence of the endothelium GTN applied in micromolar concentrations23 exhibits lower vasodilator potency, yet higher NO formation than in endothelium-denuded vessels. This finding indicates that metabolism of higher concentrations of GTN to NO, occurs preferentially in endothelial cells.16 Our findings raise the question how GTN can activate sGC and elicit vasorelaxation independent of EPR-detectable NO formation. An explanation may be that the bioactive GTN metabolite influences the activity of sGC indirectly via modulation of its intracellular localization25 and/or its interaction with sGC-binding proteins.26 It is also possible that an sGC-activating factor chemically closely related to NO but not detectable by spin trapping is formed from GTN. This issue is still unresolved.

GTN Downstream Signaling

Following activation of sGC the resulting increase in cGMP triggers further signaling events by activation of protein kinases, cation channels, and cyclic nucleotide-degrading phosphodiesterases. These shape the biological response, ie, inhibition of smooth muscle contraction, of proliferation etc. For details, see page 1 of the online data supplement available at http://circres.ahajournals.org.

GTN Biotransformation

GTN is metabolized by different pathways, which are linked either to its activation or inactivation. Metabolites generated by the inactivating routes are thought to include inorganic nitrite and nitrate, and glycerol-1,3-dinitrate (1,3-GDN). This metabolism is accomplished by glutathione reductase (GR) and glutathione-S-transferase (GST).27 Metabolites generated by bioactivation routes include NO, S-nitrosothiols, inorganic nitrite and glycerol-1,2-dinitrate (1,2-GDN).28 The bioactivation pathway appears to differ between low (therapeutic) and high (pharmacological) concentrations of GTN. As described above, the pathway realized at antiischemic and vasodilating concentrations of GTN (nanomolar range) does not generate measurable amounts of NO.23 Other characteristic features of the so-called high-potency pathway (Figure 1) not shared by the low-potency pathway29 include a susceptibility to inhibition by pertussis toxin30 and by superoxide-generating unspecific inhibitors of sGC such as LY83583 and methylene blue.31 The high-potency pathway is also prone to a rapid GTN-induced tachyphylaxis.29

Figure 1. Pathways of organic nitrate bioactivation in vascular cells. The high-potency nitrates (GTN, PETN, PETriN) are bioactivated by mitochondrial ALDH-2 when used in low doses, generating clinically relevant concentrations (<1 μmol/L). The reductase activity converts the organic nitrates to nitrite and the denitrated metabolite (1,2-glyceryl dinitrate, PETriN, or its dinitrate PEDN). Nitrite requires further bioactivation by either reduction by the respiratory chain (cytochrome oxidase [CytOx]) or by acidic disproportionation in the intermembrane space (H+), finally yielding NO or a related species (NOx), which activate sGC and trigger cGMP signaling via cGK-I. The low-potency nitrates (ISDN, ISMN, GDN, PEDN, and their respective mononitrates glyceryl mononitrate [GMN] and PEMN) are bioactivated by P450 enzyme(s) in the endoplasmic reticulum (smooth ER) directly yielding NO. The latter mechanism also accounts for the high potency nitrates used at high doses.

Low-Potency Pathway for Bioactivation of GTN

The low-potency pathway leads to formation of measurable amounts of NO in vascular tissues in vivo32 and in vitro23 (Figure 1). Therefore, NO is a vasoactive principle of higher concentrations of GTN. Cytochrome P450s (CYPs) are favorable candidates for GTN-derived NO formation in vascular tissues exposed to high concentrations of GTN (Figure 1). For further details, please refer to the online data supplement.

High-Potency Pathway for Bioactivation of GTN

Chen et al suggested that the mitochondrial isoform of aldehyde dehydrogenase (ALDH-2) is responsible for bioactivation of GTN applied in low concentrations (Figure 1),4 because inhibitors of this enzyme in vitro and in vivo and depletion of mitochondria reduced the bioactivity of GTN. ALDH-2 forms inorganic nitrite form GTN, but the bioactive intermediate (NOx), which activates sGC has not yet been identified. For further information, see the online data supplement.

Differences Between Organic Nitrates

Structurally different organic nitrates exhibit different therapeutic profiles with regard to unwanted reactions and the benefit provided to the patients in specific disease states. This is not only because of different pharmacokinetics, pharmacodynamics, dosing regimen, and routes of application, but also because of different bioactivation mechanisms. We recently assessed whether other organic nitrates besides GTN are also bioactivated by ALDH-2 in rat aorta.33 The highly potent organic nitrate esters such as GTN, pentaerythrityl tetranitrate (PETN), and pentaerythrityl trinitrate (PETriN) were bioactivated by ALDH-2, whereas less potent nitrates such as ISDN, isosorbide-5-mononitrate (ISMN), pentaerythrityl dinitrate (PEDN), and pentaerythrityl mononitrate (PEMN) were not. The vasodilator potency of the organic nitrates closely correlated to their susceptibility to inhibition of vasodilator responses, cGMP formation and cGK activity (P-VASP formation) by benomyl, their potency to inhibit ALDH-2-dehydrogenase activity in mitochondria from rat heart, and their potency to increase mitochondrial superoxide formation in vitro.33 The group of the highly potent nitrates could be further differentiated by their capability to block ALDH-2-esterase activity. This activity was not affected by ISDN, ISMN, PETN, or its metabolites PETriN, PEDN, PEMN, whereas it was inhibited by benomyl, GTN (500 μmol/L) applied in vitro, peroxynitrite (500 μmol/L), hydrogen peroxide (10 mmol/L), and in aorta from in vivo GTN-tolerant rats. These findings show that GTN specifically inhibits the ALDH-2 esterase activity, whereas PETN and PETriN do not. This difference suggests that the ALDH-2 esterase activity is required for bioactivation of the highly potent organic nitrates and might explain why GTN elicits nitrate tolerance with much higher potency than PETN or the other nitrates.

The concept that different pathways account for bioactivation of highly potent nitrates at low and high concentrations (see under sections Low-Potency Pathway for Bioactivation of GTN and High-Potency Pathway for Bioactivation of GTN; Figure 1) also extends to the organic nitrates with low vasodilator potency, such as ISDN and ISMN. It appears that the di- and mononitrates are not bioactivated by ALDH-233 but by other routes that are effective only with high concentrations of organic nitrates. A favorable candidate for this low-potency biotransformation is cytochrome P450 (Figure 1), which has been shown to accomplish NO formation from ISDN in the liver34 and in human coronary arteries.35

Also with respect to induction of nitrate headache, organic nitrates show remarkable differences. GTN most frequently induces headache, followed by ISDN, whereas ISMN and PETN clearly induce less headache.36,37 These differences may be related to vessel-type selectivity and pharmacokinetics.

Nitrate Tolerance

Nitrate tolerance is a complex phenomenon, which involves neurohormonal counter-regulation, collectively classified as pseudotolerance,23 as well as intrinsic vascular processes, defined as vascular tolerance. GTN-induced desensitization of vasodilator responses to NO donors and endothelium-derived NO is termed cross-tolerance. A typical phenomenon associated with vascular tolerance is the worsening of anginal symptoms compared with pretreatment state after cessation of nitrate therapy, the so-called withdrawal or rebound effect. All of these states have to be discerned from an acute loss of GTN efficacy at intermediate to high concentrations in in vitro experiments, the so-called tachyphylaxis.

Vascular and Systemic Features of Nitrate Tolerance

Role for Neurohormonal Counter Regulation in Nitrate Tolerance?

GTN therapy is associated with activation of neurohormonal vasoconstrictor forces. This has been demonstrated for intravenous GTN therapy and with GTN patches in patients with coronary artery disease,38,39 heart failure,40 and control subjects.41 The GTN dose used ranged between 0.3 μg/kg per minute in controls and patients with coronary artery disease and 5 to 7 μg/kg per minute in patients with heart failure. GTN-induced drops in blood pressure cause a baroreflex stimulation leading to a variety of neurohormonal adjustments. These include increases in catecholamine levels and release rates,41,42 increases in plasma vasopressin, plasma renin activity (reflecting increased circulating angiotensin II), and aldosterone levels39,41 (Figure 2). These changes are not GTN specific and have also been observed during therapy with other vasodilators. The degree of neurohormonal stimulation depends on the dose of GTN.

Figure 2. Molecular mechanisms of nitrate tolerance. Within 1 day of continuous low-dose GTN therapy, neurohormonal counter-regulation consisting of increased catecholamine and vasopressin plasma levels, increased intravascular volume, and activation of the renin-angiotensin-aldosterone-system (RAAS) reduces therapeutic efficacy (pseudotolerance). After 3 days, endothelial and smooth muscle dysfunction develops (vascular tolerance and cross-tolerance) by different mechanisms: (1) Increased endothelial and smooth muscle superoxide formation from NADPH oxidase activation by PKC and from the mitochondria; (2) Direct inhibition of NOS activation by PKC; (3) Uncoupling of endothelial NOS caused by limited BH4 availability caused by peroxynitrite (ONOO−)-induced oxidation of BH4 and reduced expression of GTP-cyclohydrolase (GTP-CH); (4) Vasoconstrictor supersensitivity caused by increased smooth muscle PKC activity; (5) Impaired bioactivation of GTN caused by inhibition of ALDH-2; (6) Inhibition of smooth muscle sGC by superoxide and peroxynitrite; (7) Increased inactivation of cGMP by PDE; (8) Inhibition of prostacyclin synthase (PGI-S) by peroxynitrite, leading to reduced PGI2 formation. For sake of clarity, tolerance-induced radical generation in endothelial mitochondria was omitted from the scheme.

GTN therapy is also associated with a marked increase in intravascular volume, which may attenuate the preload effect of GTN. A decrease in hematocrit during long-term GTN treatment39,43 very likely reflects intravascular volume expansion secondary to a transvascular shift of fluid caused by an alteration in Starling forces and/or aldosterone-mediated salt and water retention.39,41 All of these neurohormonal counter-regulatory mechanisms are regarded as pseudotolerance.

Enhanced Sensitivity to Vasoconstriction Contributes to Nitrate Tolerance

A further mechanism contributing to vascular tolerance is the increased sensitivity to receptor-dependent vasoconstrictors (Figure 2). This has been shown in rabbits treated with GTN for a 3-day period in a clinically relevant concentration of 1.5 μg/kg per minute44 and in rats chronically infused with GTN. Reports from patients with coronary artery disease also indicate that this observation may have clinical significance. Heitzer et al observed that reductions in forearm blood flow in response to intraarterial (brachial artery) angiotensin II and phenylephrine were markedly enhanced in patients pretreated with GTN for a 48-hour period (0.5 μg/kg per minute).45 These hypercontractile responses could be blocked by concomitant treatment with the angiotensin-converting enzyme inhibitor captopril, suggesting that an activated renin angiotensin system is responsible, in part, for this phenomenon. Therefore, an increase in sensitivity to vasoconstrictors represents a major mechanism responsible for the attenuation of the vasodilator effects of GTN.45

In nitrate tolerant rabbits, we could normalize vasoconstrictor responses in vitro by inhibitors of protein kinase C (PKC),44 an important enzyme for maintenance of agonist-induced smooth muscle contraction. Furthermore, treatment of rats with PKC inhibitors prevented, in parallel, nitrate tolerance and the sensitization to vasoconstrictors.46 Interestingly, increased sensitivity was not demonstrated for a classical activator of PKC, such as endothelin-1 (ET-1). We speculated that locally produced ET-1 already occupied the receptors. Indeed, vessels from GTN-treated rabbits, in contrast to controls, exhibited strong ET-1 and big-ET-1 immunostaining within the media.44 Thus, increases in local ET-1 may either downregulate or occupy ET-1 receptors, making them unavailable for activation by exogenous ET-1.44 In support of these findings, oxidative stress markedly stimulated the expression of ET-1 in cultured endothelial and smooth muscle cells.47,48 Because in the setting of tolerance, increased ROS production is observed in all layers of the vascular wall,49 it is very likely that the increase in ET-1 expression in tolerant tissue is elicited by oxidative stress.

GTN Treatment Induces Endothelial Dysfunction: The Phenomenon of Cross-Tolerance

A phenomenon related to nitrate tolerance is cross-tolerance to the vasodilator action of other organic nitrates, NO donors, and endothelium-derived NO. This has been observed most commonly when GTN was administered chronically in experimental animal models50–52 and not when nitrate tolerance was produced in vitro by short-term exposure (1 hour) of isolated vascular segments to high concentrations of GTN (0.55 mmol/L).53 Endothelial dysfunction (ED) can be observed in humans during prolonged GTN therapy. In large coronary arteries, Caramori et al found that continuous treatment (5 days) with GTN patches leads to enhanced acetylcholine (ACh)-induced paradoxical constriction, instead of endothelium-dependent vasodilation, which was taken as a surrogate parameter for ED.54 By using strain gauge plethysmography Gori and coworkers showed that chronic (6-day) GTN treatment (0.6 mg/h, GTN patches) resulted in a marked reduction of ACh-infusion-induced increases in forearm blood flow of healthy volunteers.55 Likewise, the vasoconstriction elicited in control subjects by l-NMMA (NOS inhibitor) infusion, which unmasks a tonic reduction in vascular tone by basal NOSIII-derived NO, was significantly blunted in volunteers treated with GTN. In the lowest concentration, l-NMMA even caused a paradoxical dilation. The authors concluded that GTN treatment reduces basal as well as agonist-stimulated vascular NO bioavailability and that this may, at least in part, be attributable to abnormalities in NOS function, eg, that NOS generates a vasoconstrictor agent.55 Taken together, we believe that chronic GTN treatment causes ED (Figure 2), which may have important clinical implications, because ED has been shown to be a predictor on adverse long-term outcome in patients with coronary artery disease.56

Nitrate Tolerance: Molecular Mechanisms

Does Oxidative Stress Account for Nitrate Tolerance and Cross-Tolerance?

In 1995, we defined a new molecular mechanism accounting for GTN tolerance and cross-tolerance.52 We then found that aortic segments from 3-day GTN-exposed rabbits were tolerant to the vasodilator action of GTN in vitro and exhibited cross-tolerance to ACh and the sydnonimine SIN-1, confirming previous reports by Murad and coworkers.51 The removal of the endothelium, however, markedly attenuated tolerance to GTN and cross-tolerance to SIN-1, suggesting a substantial role of the endothelium in mediating tolerance, as recently confirmed by de la Lande et al.57 We hypothesized that the endothelium is either chronically releasing a vasoconstrictor and/or that NO (or the NO-like bioactive principle of GTN) becomes chemically inactivated before it can stimulate the sGC in vascular smooth muscle. In support of this hypothesis, we found that the superoxide levels in tolerant vessels amounted to approximately twice that in the controls and were normalized by removal of the endothelium.52 Because diphenylene iodonium acutely inhibited superoxide formation, we suggested a flavin-containing oxidase as a likely superoxide source52 and detected an increased activity of membrane-bound NADH/NADPH oxidase in tolerant vascular tissue.58 Similarly, in nitrate tolerant human volunteers and chronically GTN-infused dogs, an increase in platelet NAD(P)H oxidase activity and superoxide formation has been detected.59,60 So far, it is not known whether nitrate tolerance increases the expression of subunits critical for NADPH oxidase activity or whether it stimulates an association of cytosolic subunits with the membrane-bound cytochrome b5/p22phox oxidase components.

Subsequently, we demonstrated that GTN treatment stimulates the vascular production of peroxynitrite,61 an ROS generated from a rapid reaction of NO with superoxide62 (Figure 2). A stable metabolite of peroxynitrite, nitrotyrosine, is formed by nitration of tyrosine, either free or protein bound.63 In vitro and in vivo data indicated that GTN treatment increased vascular49,64 and urinary nitrotyrosine levels,61 which can be taken as a semiquantitative indicator of increased peroxynitrite formation. Increased vascular peroxynitrite formation may affect the proper function of NOSIII and thus induce ED by different mechanisms.

Peroxynitrite can oxidize the NOSIII cofactor tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2)65 via intermediate formation of trihydrobiopterin (BH3) radicals66 (Figure 2). Provided that dihydrobiopterin reductase activity is not sufficient, the resulting intracellular BH4 deficiency may lead to dysfunctional NOSIII.65,67 Dysfunctional NOSIII can bind and transfer electrons to molecular oxygen, but further reaction with l-arginine is not possible. Consequently, NOSIII becomes uncoupled and releases superoxide (figure 2). Thus, GTN therapy may switch NOSIII from an NO to a superoxide-producing enzyme, which may further increase oxidative stress in vascular tissue in a positive feedback fashion. Indeed, we recently demonstrated increased expression of an uncoupled NOS in an animal model of nitrate tolerance, by showing that an inhibitor of NOS, l-NNA, significantly reduced vascular superoxide production in tolerant vessels.68 In addition, supplementation of GTN-treated rats with BH4 reversed GTN-induced ED,69 further indicating that ED induced by chronic GTN treatment is, at least in part, secondary to intracellular depletion of BH4. The clinical relevance of this experimental finding has been recently highlighted.70 In these studies, the authors could not only demonstrate that GTN-induced ED responded well to treatment with folic acid, a substrate for BH4 synthesis, they also found a large improvement of nitrate tolerance in forearm vessels of healthy volunteers. In support of theses findings, recent in vitro studies indicate that folic acid restores NOSIII function by increasing depleted intracellular BH4 levels.71 Furthermore, the laboratory of Fung addressed the effect of nitrate tolerance in rats (10 μg/min intravenously for 8 hours) on vascular gene expression and observed a marked 53% decrease in GTP-cyclohydrolase I feedback regulatory protein mRNA.72 This protein controls the rate of BH4 synthesis by GTP cyclohydrolase (Figure 2). Provided that mRNA and protein expression are directly correlated (which we do not know), the rate of BH4 synthesis will be reduced by half in nitrate-tolerant tissue.

Interestingly, exogenously applied l-arginine improved tolerance in rat aorta73 and humans.74 An explanation for this unexpected finding may be provided by the observation that l-arginine addition can prevent immediate GTN-induced superoxide formation in cultured bovine aortic endothelial cells.75 However, the significance of this finding for in vivo tolerance is unclear.

GTN (1 hour, 10 μmol/L) has been shown to activate PKC in cultured endothelial cells, as indicated by a transient membrane translocation of PKCα and PKCε. This response was associated with increased tyrosine nitration,76 which could be blocked by peroxynitrite scavengers (uric acid), superoxide dismutase, NG-nitro-l-arginine methyl ester, and the PKC inhibitor chelerythrine. The authors concluded that GTN-induced activation of specific PKC isoforms triggers intracellular events leading to NOS uncoupling. Activation of endothelial PKC induces phosphorylation of NOSIII, leading to an inhibition of NO production by the enzyme,77,78 all of which may also contribute to GTN-induced ED. Because activation of PKC is induced by superoxide79 and peroxynitrite,80 a vicious cycle is set up by GTN, involving mutual activation of PKC, increased ROS production, depletion of intracellular BH4, and uncoupling of NOSIII (Figure 2). The GTN-induced increase in oxidative stress stimulates production of endothelin-1 within endothelial and smooth muscle cells,47,48 leading to further PKC activation, which, in turn, may trigger enhanced constrictor responses to almost every receptor-dependent agonist44 (Figure 2). PKC may also activate NADPH oxidases in the vasculature, contributing to increased vascular superoxide formation.

The finding that the degree of GTN tolerance was similar in NOSIII knock-out and wild-type mice72 does not disqualify uncoupled NOSIII from the mechanism of GTN tolerance, because neuronal-type NOSI can functionally substitute for NOSIII in NOSIII knock-out mice.81 It is conceivable that in these mice, vascular NOSI will be uncoupled in the nitrate-tolerant state.

Sage et al82 showed that nitrate tolerance in patients is causally related to increased superoxide formation and reduced GTN biotransformation in human blood vessels. Rings prepared from the internal mammary artery and saphenous vein of patients treated for 24 hours with GTN (10 μg/min intravenously) before elective bypass surgery were tolerant to GTN, exhibited increased superoxide formation as detected by lucigenin (10 μmol/L) chemiluminescence, and generated 40% less 1,2-dinitroglycerin, the metabolite derived from bioactivation of GTN82 (Figure 1). They failed, however, to demonstrate cross-tolerance to endothelium-dependent (A23187) and -independent (sodium nitroprusside) vasodilators. Also, an acute 3-fold increase in vascular superoxide production by exposure to the superoxide dismutase (SOD) inhibitor diethyldithiocarbamate did not modify the GTN dose-response relationship. Therefore, the authors concluded that impaired GTN biotransformation more likely accounts for tolerance than vascular superoxide formation and that the endothelial function is preserved in the tolerant state.82

In contrast, using higher GTN doses (35 μg/min intravenously) and longer treatment periods (48 hours), we could demonstrate endothelial dysfunction in the arteria mammaria and arteria radialis in patients undergoing coronary bypass surgery and markedly increased superoxide production in these vessels,22 confirming our previous findings in patients with stable coronary artery disease.39 The failure of ED development in the study by Sage et al82 also contrasts to the results from Gori et al55 and Caramori et al.54 The discrepant findings are very likely to be explained by the different dose and duration of GTN application. After 1 day of GTN exposure, pseudotolerance may still prevail over vascular tolerance.

Further support for the oxidative stress concept was provided by the demonstration that nitrate tolerance achieved by 7-day GTN treatment (0.6 mg/h) of healthy volunteers increased the plasma levels of cytotoxic aldehydes and isoprostanes, which are considered as sensitive markers for free radical-induced lipid peroxidation.83 Similarly, in isolated platelets of healthy volunteers exposed to 0.4 mg/h GTN for 3 days, McGrath et al detected increased esterified 8-epi-PGF2α, a marker of oxidative stress and COX activation.84 To address the role of mitochondrial oxidative stress in nitrate tolerance, we recently assessed the effect of GTN on wild-type versus heterozygous Mn-SOD deficient (Mn-SOD+/−) mice. Mn-SOD is the mitochondrial SOD isoform. We detected increased ROS formation and decreased ALDH-2 activity in isolated mitochondria from MnSOD-deficient mice. The aorta of these animals exhibited an increased sensitivity for development of nitrate tolerance on acute challenges of isolated vessels with GTN and on chronic GTN infusion of these mice.85 These findings suggest that increased mitochondrial oxidative stress could be a decisive component in nitrate tolerance development.

Effect of GTN-Induced Superoxide/Peroxynitrite Production on Prostacyclin Synthase

Recent findings show that prostacyclin synthase is a highly vulnerable target of peroxynitrite and that chronic GTN application in vivo inhibits this enzyme via peroxynitrite-induced tyrosine nitration.22,49,86,87 Consequently, prostacyclin (PGI2) formation is decreased and prostaglandin H2 (PGH2) formation increased in nitrate tolerance, leading to increased vascular tone. For details, see page 2 of the online data supplement.

GTN Signaling Targets Hit by Superoxide and Peroxynitrite

Effects of GTN Tolerance on sGC Activity and Expression

In nitrate tolerance, not only reduced bioactivation of GTN will decrease stimulation of vascular sGC, but GTN tolerance-induced superoxide and peroxynitrite may also directly interfere with nitrovasodilator action at the level of sGC (Figure 2). Both superoxide and peroxynitrite are potent direct inhibitors of NO-sensitive sGC,8,88,89 and reduced basal and NO-stimulated sGC activities were detected in peroxynitrite exposed cells and vascular tissues.89 We also found that in the absence of glutathione very low concentrations (<1 μmol/L) of peroxynitrite nearly abolished NO-dependent, as well as NO-independent (YC-1), activation of the purified enzyme (A.M., unpublished results, 2000). Interestingly, exposure of the purified enzyme to a fully inhibitory concentration of peroxynitrite (1 μmol/L) did not induce formation of immunodetectable nitrotyrosine on sGC subunits, suggesting that this inhibition is not accomplished by nitration of tyrosine residues. Similarly, in homogenates from GTN-tolerant rabbits, sGC was not detected by a 3-nitrotyrosine antibody (A.M., unpublished results, 2000), indicating that sGC is not tyrosine nitrated in nitrate tolerance. Provided that peroxynitrite and superoxide formation is sufficiently high in nitrate tolerant tissues, tonic inhibition of sGC may contribute to cross-tolerance to other nitrovasodilators, NO-donors and endothelium-dependent agonists, as observed previously51 (Figure 2). Our (unexpected) recent observation that expression of sGC subunits α1 and β1 was increased in nitrate-tolerant vascular tissue86 may be interpreted as a biological counter-regulatory mechanism compensating partially for lower sGC activity and cGMP formation.

Effect of GTN-Induced Superoxide/Peroxynitrite Production on the Activity and Expression of the cGMP-Dependent Protein Kinase

How does GTN-induced stimulation of superoxide/peroxynitrite production influence intracellular cGMP downstream signaling? Previous studies with mice deficient in cGMP-dependent protein kinase (cGK-I) highlighted the crucial role of this enzyme in mediating cGMP-stimulated vasodilation.90 The phosphorylation of the vasodilator stimulated phosphoprotein (VASP) at Ser239 (P-VASP) has been shown to be useful for monitoring cGK-I activity in vascular tissue. Changes in vascular P-VASP levels are closely related to endothelial function and oxidative stress,21 suggesting that P-VASP can be used as a novel, biochemical surrogate parameter for vascular NO-bioavailability and/or efficiency of cGMP downstream signaling.22,49,86,87 We could not detect any changes in cGK-I expression in aortas from GTN-tolerant rats and rabbits compared with controls, but a striking reduction of phosphorylated VASP when compared with untreated controls was detected.

To address specifically the role of oxidative stress in inhibiting the NO-signaling, the phosphorylation level of VASP in GTN tolerant vascular tissue was quantified in response to in vitro and in vivo treatment with the superoxide/peroxynitrite scavenger vitamin C, ebselen, and uric acid. We could demonstrate that all of these antioxidants markedly restored P-VASP, decreased superoxide levels and nitrotyrosine formation to control, and, accordingly, restored GTN sensitivity in vessels from GTN-tolerant animals.86,87 These observations were recently confirmed with studies testing the effects of long-term GTN infusion on cGMP/cGK-I signaling in patients with coronary artery disease.22 For details on beneficial effects of antioxidants on the development of nitrate tolerance, see pages 15 to 18 in the online data supplement.

Effect of GTN Therapy on the Activity and Expression of Cyclic Nucleotide-Metabolizing Phosphodiesterases

Because vasorelaxation to GTN is mediated by increased cGMP formation, this response is sensitive to regulation by cGMP-metabolizing phosphodiesterases (PDE) (Figure 2). It is, therefore, not surprising that several previous studies showed a relief from nitrate tolerance in vitro and in vivo by application of PDE inhibitors such as the PDE5A1-specific compound zaprinast.9,91 These findings fostered the speculation that nitrate tolerance might be based on an increase in cGMP-degrading PDE activity and/or expression in the vascular smooth muscle. Indeed, in chronically GTN-infused rats, we detected a marked increase in the activity and expression of the Ca2+/calmodulin-dependent PDE1A1 isoform, whereas expression of Ca2+-independent PDE5A1 was not appreciably altered.92 Accordingly, the PDE1-specific inhibitor vinpocetine partially restored nitrate sensitivity of the tolerant vasculature. The increase in the expression of this enzyme would, at least in part, explain the decreased responsiveness of the tolerant vasculature to GTN and to endothelium-dependent vasodilators (cross-tolerance), the decreased activity of the cGMP-dependent kinase/NO signaling, as assessed by P-VASP, and the increase in sensitivity to intracellular Ca2+-eliciting vasoconstrictors observed in animal and in human studies. Interestingly, angiotensin II infusion had a similar effect on PDE1A1 activity and expression as GTN infusion,92 which would explain why angiotensin-converting enzyme inhibitors positively influence GTN-induced hypersensitivity to vasoconstrictors and GTN-induced tolerance and cross-tolerance. A recent study addressing alterations in vascular gene expression in nitrate tolerant state (8 hours of GTN infusion in rats) detected a 1.8-fold increased expression of cGMP-stimulated cAMP-metabolizing PDE2A2 mRNA in tolerant rat aorta, whereas an increase in PDE1A1 mRNA was not reported.93 This may provide a hint that cross-talk between cAMP and cGMP metabolism might also be affected in nitrate tolerance. On the other hand, the gene expression data of the study by Wang and Kim were collected at different stages of nitrate tolerance (8 hours versus 3 days), and tolerance was induced by different GTN doses (10 μg/min intravenously versus 2.5 to 3 μg/min subcutaneously), which may well explain the different findings.

Mechanisms Underlying Tolerance: Impaired Biotransformation Versus Oxidative Stress Concept

Within the last decades, several concepts concerning the mechanisms underlying nitrate tolerance have been intensively discussed. One favorite hypothesis originating from the earlier work by Needleman et al94 and later modified by others was that impaired GTN bioactivation leads to decreased GTN sensitivity in the tolerant vasculature. The other concept, discovered by us,52 claimed increased oxidative stress and reduced NO bioavailability as the mechanism underlying nitrate tolerance. Pros and cons have frequently been raised over the years, without conclusion. For instance, impaired bioactivation of GTN may not explain associated phenomena such as endothelial dysfunction, increased sensitivity to vasoconstrictors, and/or increased vascular superoxide production.

A clue to these apparently contradicting concepts appeared recently by the identification of ALDH-2 as the enzyme responsible for GTN bioactivation4,3,33,95–97 (see preceding paragraph). Chen et al showed in in vitro studies that tolerance-inducing high concentrations of GTN (0.3 mmol/L, 30 minutes) inhibited ALDH-2 dehydrogenase activity and GTN biotransformation to 1,2-glyceryl dinitrate (GDN) (GTN reductase activity), frequently taken as a monitor for bioactivation of GTN. Similarly, in vitro high-dose GTN exposure attenuated cGMP increases in response to acute GTN challenge.4 We assessed whether inhibition of ALDH-2 also accounts for nitrate tolerance in vivo. We found that the aortas from tolerant (3-day GTN-infused) rats exhibited reduced GTN vasodilator responses, but in contrast to nontolerant controls, in vitro treatment of in vivo tolerant aortic rings with ALDH-2 inhibitors did not affect the GTN concentration-response curve.3 Total ALDH activity in vascular homogenates and in mitochondria isolated from tolerant rat aorta and heart was reduced by more than 50% compared with controls. Daidzin, a more specific ALDH-2 inhibitor, caused a similar decrease in isolated control tissues and mitochondria.3 Furthermore, acute exposure of isolated mitochondria to a higher concentration of GTN (5 to 500 μmol/L) decreased ALDH activity to a similar extent, and, in addition, stimulated mitochondrial superoxide formation. Inhibition of complex III by antimycin A similarly elicited superoxide formation and inhibition of ALDH activity. In addition, mitochondria isolated from tolerant animals generated ROS at ≈50% higher rate than control mitochondria, and this was entirely blocked by acute addition of DTT, uric acid, or ebselen. The effects of nitrate tolerance on classical ALDH activity were mirrored by a similar reduction of GTN reductase activity, as detected by formation of 1,2-GDN.3 In conclusion, these findings show that in vivo nitrate tolerance is caused by inhibition of ALDH-2 and suggest that GTN metabolism triggers superoxide production within mitochondria (Figure 2).

These data actually confirm previous observations published by Needleman and Hunter showing that incubation of isolated heart mitochondria with high concentrations of nitrates induced swelling of mitochondria, stimulated oxygen consumption, and uncoupled oxidative phosphorylation,98,99 consistent with a mitochondrial source of nitrate-elicited ROS. Regardless of the exact mechanism by which GTN stimulates ROS production (eg, premature release of partially reduced oxygen from complex IV, accumulation of toxic aldehydes, initiation of lipid peroxidation, depolarization of mitochondrial membrane potential, mitochondrial swelling, etc), loss of ALDH-2 activity should cause GTN to accumulate in mitochondria and, thus, amplify the effect (Figure 2). Chen et al proposed that oxidation of essential thiol groups in the active site of the enzyme underlies the molecular mechanism of tolerance, as supported by our study.3,33 However, it is not known whether the artificial substrate GTN and/or ROS cause inhibition of ALDH-2. In studies with purified yeast ALDH, we found that GTN, superoxide, and peroxynitrite were all capable of directly inhibiting the enzyme (A.D., unpublished observations, 2004). These findings support the idea that oxidative stress may contribute directly to mechanism-based tolerance, either by oxidative inhibition of ALDH-2 and/or perhaps by depleting essential cofactors required for reactivation of oxidized ALDH-2, such as lipoic acid. Irrespective of the exact sequence of events, incubation of tolerant tissue with various disulfide-reducing agents (dithiothreitol) and antioxidants (vitamin C) completely restored vascular ALDH activity and simultaneously normalized mitochondrial ROS production. Inasmuch as nitrate tolerance may underlie the increases in cardiac morbidity seen with chronic nitrate use (based on metaanalysis),100 these observations may have therapeutic implications. Earlier observations that ALDH dehydrogenase activity is reduced in nitrate tolerant patients101 provide support for the clinical relevance of this mechanism.

Do All Nitrates Induce Tolerance?

Although tolerance development occurs with all nitrates, depending on dose and duration of treatment, PETN is a remarkable exception. This organic nitrate exhibits considerably less tolerance-inducing activity than all of the others. This property could be related to the unique antioxidative defense protein-inducing properties of PETN and its metabolites. For details, see page 3 of the online data supplement.

Old and New Strategies to Prevent the Development of Tolerance and Cross-Tolerance

Several clinical approaches to prevent nitrate tolerance were tested in the past; however, none of them has yet been accepted as a gold standard. For details, see page 4 of online data supplement.

Summary and Clinical Implications

In summary, continuous systemic therapy with organic nitrates induces tolerance and endothelial dysfunction in patients with coronary artery disease54,102 and even in healthy controls.55 Mechanisms contributing to this phenomenon may be a nitrate-induced stimulation of vascular (mitochondrial) superoxide and/or peroxynitrite production and the ensuing inhibition of ALDH-2, leading to impaired biotransformation of GTN. Several studies indicate that chronic GTN treatment worsens endothelial function and a recent (ex-post) metaanalysis indicates that nitrates may worsen prognosis in patients with ischemic heart disease.100 Further studies are required, however, to understand the precise nature of mechanisms underlying GTN-induced endothelial dysfunction to develop strategies to prevent these GTN-induced side effects (see online data supplement, pages 8 to 18), although there are probably significant differences between organic nitrates with respect to bioactivation pathways, induction of ROS formation and tolerance development. More generally, mitochondrial injury and the ensuing oxidative stress unify concepts of tolerance and cross-tolerance and provide a molecular rationale for the range of agents that seemingly prevent the development of nitrate tolerance. It is likely that agents ameliorating oxidative stress (eg, angiotensin converting enzyme inhibitors, angiotensin II type 1 receptor blockers, statins, l-arginine, BH4, ascorbate) will restore sensitivity to GTN, but it remains to be seen whether they are able to prevent inhibition of ALDH-2 during prolonged GTN treatment. Likewise, it will be of interest to see whether ebselen and uric acid, which lessen mitochondrial ROS production and preserve ALDH-2 activity in vitro, can also do so in vivo and whether this confers protection from tolerance.

Original received April 26, 2005; resubmission received July 1, 2005; revised resubmission received August 8, 2005; accepted August 19, 2005.

We thank the German Research Foundation for continuous financial support for our nitrate tolerance studies (Mu 1079/6-1 and SFB533-C17 to T.M.).

Footnotes

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