Neurohormonal Activation and Nitrate Tolerance: Implications for Concomitant Therapy With Angiotensin-Converting Enzyme Inhibitors or Angiotensin Receptor Blockers

Neurohormonal Activation and Nitrate Tolerance: Implications for Concomitant Therapy With Angiotensin-Converting Enzyme Inhibitors or Angiotensin Receptor Blockers Thomas Mu¨nzel,

MD,

Thomas Heitzer, and Carsten Brockhoff

The hemodynamic and anti-ischemic effects of nitroglycerin are rapidly blunted due to the development of nitrate tolerance. With initiation of nitroglycerin therapy, one can detect an increase in plasma renin activity (reflecting increased circulating angiotensin II levels), increases in circulating vasopressin, catecholamine, and aldosterone levels, and signs of intravascular volume expansion. These so-called pseudotolerance mechanisms may compromise nitroglycerin’s vasodilating effects. Long-term treatment with nitroglycerin is also associated with a decreased responsiveness of the vasculature to nitroglycerin’s vasorelaxant potency suggesting changes in the intrinsic mechanisms of the tolerant vasculature itself. The issue of tolerance is even more complicated due to the differences in the susceptibility of arterial resistance versus conductance vessels and veins to develop tolerance. More recent experimental work defined new tolerance mechanisms such as increased

vascular superoxide production and increased sensitivity to vasoconstrictors secondary to an activation of protein kinase C. Both phenomena are prevented by concomitant treatment with angiotensin-1 (AT1)-receptor blockers or angiotensin-converting enzyme (ACE) inhibitors suggesting a causal involvement of the renin–angiotensin system in mediating these phenomena. Despite these encouraging results in animals studies, the clinical reports concerning concomitant treatment of nitrates with ACE inhibitors are quite conflicting. With the present review, we want to summarize new aspects concerning the vasodilator mechanism of nitroglycerin, the role of circulating vasoconstrictor forces in mediating tolerance, and in particular we want to give a brief review about positive and negative results concerning the efficacy of ACE inhibitors in preventing nitrate tolerance. Q1998 by Excerpta Medica, Inc. Am J Cardiol 1998;81(1A):30A– 40A

vivo, nitroglycerin releases nitric oxide via both enzymatic and cellular-indepenIdentncellular-dependent nonenzymatic processes in endothelial and

laxation was markedly inhibited by iberiotoxin (a specific inhibitor of Ca21-dependent potassium channels), while in endothelium-denuded vessels, iberiotoxin was not effective at all. These observations indicate that in the presence of the endothelium, nitroglycerin relaxes the vasculature mainly by activating endothelial KCa21 channels7 while in the absence of the endothelium the mechanisms of nitroglycerin’s vasodilation are completely different. Interestingly, more recent work by Feelisch and Kelm8 indicates that endothelial cells as well as smooth muscle cells are capable of metabolizing glyceryl trinitrate (GTN) to glyceryl dinitrate (GDN) to release the vasodilator nitric oxide. Therefore it is tempting to speculate that in the presence of the endothelium, GTN-induced vasodilation depends, at least in part, on the metabolism of GTN to GDN within endothelial cells while in the absence of the endothelium nitroglycerin metabolism within smooth muscle cells represents the decisive nitric oxide source. This would mean that in contrast to other nitrovasodilators such as sodium nitroprusside or the sydnonimine SIN-1, GTN is, in the presence of the endothelium, a nitrovasodilator with a dilator action that is, at least in part, endothelium-dependent. There is little evidence supporting the concept that the vasodilator effects of nitroglycerin are mediated via inducing vascular prostaglandin I2 (PGI2, prostacyclin) synthesis since the

smooth muscle cells. NO activates the cytosolic guanylyl cyclase,1 resulting in an increase of intracellular cyclic guanosine 39,59-monophosphate and subsequently in a decrease of cytoplasmic Ca21 concentration and relaxation of the vascular smooth muscle. The mechanisms whereby cyclic guanosine monophosphate lowers cytoplasmic Ca21 are still not well understood but may involve activation of a sarcoplasmic Ca21-pump adenosine triphosphatase,2 stimulation of Ca21-activated K1 channels in smooth muscle cells,3 inhibition of inositol triphosphate (IP3)-receptor activity,4 or a decreased sensitivity of contractile proteins to the effects of Ca21.5 Recently, Richard Cohen’s group also described cyclic guanosine monophosphate-independent vasodilator actions of nitric oxide such as the stimulation of Ca21-dependent potassium channels within smooth muscle cells.6 Preliminary experiments indicate that the mechanism of nitroglycerin-induced vasodilation differs greatly depending whether the endothelium is present or not.7 In endothelium-intact coronary arteries, nitroglycerin reFrom the Division of Cardiology, Eppendorf University of Hamburg, Hamburg. Address for reprints: Thomas Mu¨nzel, MD, Universitätskrankenhaus Eppendorf, Abteilung fu¨r Kardiologie, Martinistr. 52, 20246 Hamburg, Germany.

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vasodilator action of nitroglycerin is not attenuated after pretreatment with cyclooxygenase inhibitors.9

MECHANISMS UNDERLYING THE HETEROGENEOUS EFFECTS OF NITROGLYCERIN ON THE VASCULAR TREE One of the fascinating aspects of the pharmacology of nitroglycerin is its different effects on veins, large conductance, and the peripheral and coronary microcirculation. In clinically relevant concentrations, nitroglycerin potently dilates large coronary arteries and veins while having only submaximal and transient effects on coronary and peripheral blood flow.10 –12 David Harrison’s group13 has examined the effects of nitroglycerin on different sized coronary microvessels using in vitro techniques. In these studies, coronary microvessels of different sizes were studied using a microvessel imaging apparatus. Interestingly, it was found that vessels with a diameter .200 mm were potently dilated by nitroglycerin, whereas those with a diameter ,100 mm dilated only minimally, even at the highest nitroglycerin concentrations. In striking contrast, however, putative metabolites of nitroglycerin, such as nitric oxide or the nitrosothiol S-nitrosocysteine, potently dilated the microvessels. In addition, endothelium-dependent vasodilation to bradykinin was similar in all size vessels studied.14 Based on these findings, they concluded that these smaller coronary microvessels were incapable of converting nitroglycerin to its vasodilator metabolite, and that this likely determined the relative selectivity of nitroglycerin as a large vessel vasodilator. In view of the suggested critical role of thiols in the biotransformation of nitroglycerin, one explanation for the insensitivity of small arterioles could have been a deficiency of reduced sulfhydryl groups in the vascular smooth muscle of these vessels. Indeed, preincubation of small arterioles with L-cysteine markedly enhanced the response of smaller vessels. This effect was small vessel-specific since the effects on larger arterioles remained unchanged. Subsequently it was demonstrated that this interaction was specific for the L- and not the D-form of cysteine, and that the L-cysteine probably needed to be incorporated into glutathione to have this effect.15,16

ROLE FOR NEUROHORMONAL COUNTERREGULATION IN NITRATE TOLERANCE? GTN therapy has been shown to be associated with activation of neurohormonal vasoconstrictor forces. This has been demonstrated for intravenous GTN therapy and therapy with GTN patches in patients with coronary artery disease,17,18 patients with heart failure,19 and control subjects.20 The GTN dose was between 0.3 mg/kg per min in controls and patients with coronary artery disease, and 5–7 mg/kg per min in patients with heart failure. GTN-induced blood pressure decreases cause baroreflex stimulation leading to a variety of neurohumoral adjustments. These include increases in catecholamine levels and release

rates,20,21 increases in plasma vasopressin,18,20 plasma renin activity,18,20 (reflecting increased circulating angiotensin II levels), and aldosterone levels.18,20 These changes are not nitroglycerin specific and have also been observed during therapy with other vasodilators. The degree of neurohumoral stimulation depends on the chosen nitroglycerin concentration. GTN therapy is also associated with a marked increase in intravascular volume which may attenuate the preload effects of GTN. During continuous nitroglycerin infusion there is a consistent decrease in hematocrit in patients treated with nitroglycerin for 72 hours. A decrease in hematocrit during long-term nitroglycerin has been demonstrated by several groups19,20,22 and very likely reflects intravascular volume expansion secondary to a transvascular shift of fluid due to an alteration in Starling forces and/or a phenomenon related to an aldosterone-mediated salt and water retention.

DISSOCIATION OF TOLERANCE DEVELOPMENT AND NEUROHUMORAL ACTIVATION Some insight into the role of the above mentioned mechanisms (pseudo vs true vascular tolerance) can be gained from an examination of the time– course of the development of tolerance in certain vessel regions and its relation to neurohumoral activation. Further, the time-course of tolerance may not be the same in the systemic and coronary vasculature. For example, as mentioned above, it has been shown that a large portion of the intravascular fluid shift occurs within the first hour of treatment.22 During this period however, the effects of nitroglycerin on the pulmonary capillary wedge pressure in patients with coronary artery disease and heart failure are usually preserved.23 Over a longer period of infusion, 24 – 48 hours, the pulmonary capillary wedge pressure increases to the pretreatment value19,24 with little or no additional volume retention, suggesting that mechanisms independent of volume retention are involved. Therefore, a significant decrease in hematocrit may be used more like a marker for nitroglycerin treatment rather than a marker for tolerance development. In addition, the persistent decrease in hematocrit actually lends support to the conclusion that, for example, tolerance to epicardial artery effects does not follow the same timeline as tolerance in other vascular beds. A recent observation published by John Parker20 demonstrated that therapy with nitroglycerin patches was associated with a transient activation of the renin– angiotensin system, with increases in vasopressin and catecholamine levels and with signs of intravascular volume expansion. By using an intravenous infusion of GTN, we have recently found a similar time– course of activation of these neurohumoral parameters but have also found that tolerance in epicardial coronary arteries does not coincide with neurohormonal activation (Figures 1 and 2).18 In these studies, the increase in these parameters during nitroglycerin therapy was also transient, and not observed after 72 hours of therapy. During the period in which these parameA SYMPOSIUM: NITRATE TOLERANCE

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FIGURE 1. Effects of increasing intravenous and intracoronary nitroglycerin (NTG) on large coronary artery diameter (left anterior descending [LAD] and left circumflex [LCx]) in patients without (group I) and with 24-hour and 72-hour NTG pretreatment (groups II and III, respectively). Under ongoing NTG infusions, patients pretreated with NTG for 24 hours did not respond with a further increase in diameter indicating a maximal dilated coronary artery. In contrast, patients pretreated with NTG for 3 days responded with a diameter increase, which was not statistically different from those of group I, which is strongly suggestive of tolerance development in large epicardial arteries. Increasing concentrations of NTG were given intravenously mg/kg per min for 7 minutes each. B, 0.2 mg NTG bolus intracoronary, data are given as mean 6 SEM. *Significantly different versus baseline values (after Bonferroni correction for the numbers of comparisons [n 5 4]). (Adapted with permission from J Am Coll Cardiol.18)

ters were highest (24 – 48 hours), the epicardial coronary artery response to nitroglycerin was preserved. Long after these neurohumoral parameters had returned to normal, however, the vasodilation of the epicardial coronary arteries to nitroglycerin was virtually lost. These findings indicate that neurohumoral activation is unlikely to be responsible for the loss of effect of nitroglycerin on the epicardial coronary arteries, and strongly suggest changes intrinsic to the vasculature.

VASCULAR HETEROGENEITY OF TOLERANCE DEVELOPMENT Considering the great variability of different vessel regions and the sensitivity to GTN,13 it is not surprising that there are great differences in the susceptibility of veins, arteries, and arterioles to develop nitrate tolerance. Of particular interest is also whether pseudotolerance or true tolerance mechanisms may affect arterioles, veins, or large arterial conductance vessels differently. In a plethysmographic study, Zelis and Mason25 showed the development of preferential tolerance to the venous effects of nitroglycerin in healthy subjects on long-term treatment with high dose isosorbide dinitrate, but observed a maintenance of its effects on resistance vessels in this localized 32A THE AMERICAN JOURNAL OF CARDIOLOGYT

vascular bed. Since then, the relative susceptibility of the arteriolar, arterial, and venous bed has been a matter of dispute. Results in patients with congestive heart failure, in particular, have been interpreted as suggesting arteriolar rather than venous tolerance on sustained nitrate exposure.27 The rapid attenuation of nitrate effects on blood pressure, which has been described in many studies, has strengthened this impression.26,27 Few studies, however, have measured the venodilator effects of nitroglycerin effects directly. Manyari et al28 reported that long-term treatment with nitroglycerin markedly attenuates the nitroglycerin effects on vascular capacitance. More recent investigations from Ghio et al29 seem to confirm this concept. Patients treated with intravenous nitroglycerin developed a rapid tolerance to the venous circulation but remained responsive on the arteriolar side. These clinical observations are confirmed by experimental findings demonstrating preferential venous tolerance in a dog model for nitrate tolerance within 4 days of continuous nitroglycerin treatment.21 Simultaneously, Stewart et al21 found that in an animal model of nitrate tolerance the hypotensive effects of nitroglycerin were preserved even in the presence of venous tolerance. Since the hypotensive effects of nitroglycerin are, in

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FIGURE 2. Effects of 24-hour and 72-hour nitroglycerin (NTG) infusion respectively on plasma renin activity (PRA), plasma aldosterone levels (ALDO), plasma vasopressin levels (ADH), and hematocrit (HCT). Long-term NTG infusion caused a transient increase in plasma vasopressin and aldosterone levels and a transient increase in plasma renin activity, but a persistent decrease in hematocrit indicating intravascular volume expansion. Data are presented as mean 6 SEM. (Adapted with permission from J Am Coll Cardiol.18)

general, a combination of veno- and arteriolar dilation, Stewart concluded that in the presence of venous tolerance the systemic arterioles remained nitrate sensitive, pointing to tolerance affecting specifically the venous system. He also demonstrated that in nitroglycerin-treated animals the effects of ganglionic blockade on mean arterial pressure were enhanced as compared with animals without nitroglycerin pretreatment. This observation would suggest that in the presence of venous tolerance neurohormonal counterregulatory mechanisms (pseudotolerance) mainly affect arteriolar dilator effects of nitroglycerin.

WHAT MECHANISMS ARE RESPONSIBLE FOR ATTENUATION OF NITROGLYCERIN ACTION WITHIN THE 24 HOUR AND 72 HOUR TREATMENT PERIOD? Numerous studies have documented an early loss (within 24 hours) of the nitroglycerin effects during continuous treatment.24,30 It remains to be determined whether neurohormonal counterregulatory mecha-

nisms or changes intrinsic to the tolerant vasculature itself play a major causal role in this phenomenon. As mentioned above, within 24 hours of continuous nitroglycerin treatment, the vasodilator effects of organic nitrates on large arterial conductance vessels are, in general, preserved even in the presence of neurohormonal activation, while tolerance to nitroglycerin-induced changes in coronary flow (reflecting resistance vessel effects) is usually established.31,32 These observations would seem to indicate that increased levels of vasopressin and angiotensin II encountered with the initiation of nitroglycerin therapy cannot override the vasodilator effects of nitroglycerin on large arterial conductance vessels but may induce vascular constriction at the arteriolar level33,34 in a vessel region, which has been demonstrated to be, per se, nitrate-insensitive.13,14 Therefore, it is tempting to speculate that within 24 hours of continuous nitroglycerin treatment, pseudotolerance in systemic and/or coronary arterioles rather than pseudotolerance in large arteries is responsible for the well-known rapid loss of the nitroglycerin vasodilator effects within this time frame (Figure 3). Within 3 to 4 days of continuous nitroglycerin or isosorbide dinitrate infusion, however, tolerance in large epicardial arteries occurred even in the absence of further neurohormonal activation.18,35 This observation indicates a desensitization of large artery conductance vessels to nitroglycerin, which might be at least in part related to cellular tolerance mechanisms such as an enhanced destruction of nitric oxide36 due to vascular superoxide anion production, or may be secondary to an increase in sensitivity to vasoconstrictors.37

ACE INHIBITORS IN NITRATE TOLERANCE As mentioned above, therapy with nitroglycerin induces neurohormonal activation and tolerance and a nitrate-free interval prevents neurohormonal activation and tolerance. These observations were actually the rationale to test whether concomitant treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II-receptor blockers is able to prevent the development of nitrate tolerance. Given acutely, ACE inhibitors have been demonstrated to augment nitrate actions in patients with stable angina pectoris. Combined therapy with isosorbide dinitrate and captopril (50 –100 mg) resulted in a more expressed antianginal and anti-ischemic effect as compared with patients receiving isosorbide dinitrate alone.38 Similarly, Meredith et al32 showed that acute administration of captopril enhanced nitroglycerin vasodilation of arterioles in patients with stable coronary artery disease. This potentiation of nitroglycerin action was seen in patients with and without nitroglycerin pretreatment suggesting again that pseudotolerance mechanisms such as an activated renin–angiotensin system account, at least in part, for the rapid loss of the nitroglycerin vasodilator effects in arterioles. The clinical data concerning the efficacy of ACE inhibitors in preventing tolerance, however, are quite A SYMPOSIUM: NITRATE TOLERANCE

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FIGURE 3. Proposed time-course of tolerance in different vessels regions in response to chronic nitroglycerin therapy. Within 24 hours of continuous nitroglycerin, treatment tolerance is established in veins and resistance vessels but not in arterial conductance vessels. In this particular time frame, there is also evidence for neurohormonal activation, which is at least in part responsible for tolerance at the resistance vessel level. Within 3 days of continuous nitroglycerin treatment, neurohormonal stimulation is less pronounced and tolerance at the cellular level predominates. Proposed mechanisms may include a protein kinase C (PKC)mediated increase in sensitivity to vasoconstrictors and an increase in vascular superoxide production all of which may attenuate nitroglycerin’s vasodilator effects.

FIGURE 4. Values of mean right atrial pressure as measured with captopril (CAP) alone during day 1 and with captopril and isosorbide dinitrate (ISDN) on day 2. Open symbols indicate p <0.05 versus baseline. Captopril treatment almost completely prevented the development of tolerance. Schedule of drug administration on both days is indicated by arrows. (Adapted from Circulation.40)

contradictory. Uri Elkayam’s group39 recently showed that in patients with congestive heart failure the effects of oral isosorbide dinitrate (40 –120 mg, q6h) were sustained in patients treated concomitantly with captopril (with an average dose of 90 mg/day), whereas patients receiving isosorbide dinitrate alone rapidly developed hemodynamic tolerance (Figure 4). Studying normal subjects and measuring forearm volume, Katz et al40 found that tolerance to the venous forearm circulation was prevented by concomitant administration of captopril or enalapril, suggesting that the sulfhydryl group moiety in ACE inhibitors is not respon34A THE AMERICAN JOURNAL OF CARDIOLOGYT

sible for the positive effects with respect to nitroglycerin tolerance. Similarly, Muiesan et al41 showed that the combination therapy of benazepril with transdermal nitroglycerin induced a significant increase in exercise duration 22 hours postdosing, whereas nitroglycerin given alone was no longer effective at this time point. Pizzulli et al42 showed that ACE inhibition with captopril (50 mg/day) effectively prevented the development of hemodynamic tolerance in response to high-dose nitroglycerin treatment (1.5 mg/kg per min) in patients with coronary artery disease. On the other hand, several groups failed to dem-

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TABLE I Interactions Between Nitrates and ACE Inhibitors in Vivo Reference

Patient Population

Nitrate/Dose

Acute effects of ACE inhibition on nitrate action Meredith CAD NTG (0.175 mg/kg et al32 per min 20 h i.v.) Metelitsa CAD ISDN (50–100 et al38 mg, p.o.)

Chronic effects of ACE inhibition on tolerance Katz Normal nitroglycerin et al40 volunteers (transdermal, 10 mg/day) 3 day treatment Mehra et al39

CHF

ISDN (p.o.), 40–120 mg

Pizzuli et al42

CAD

nitroglycerin (1.5 mg/kg per min)

Muiesan et al41

CAD

nitroglycerin (transdermal, 10 mg/day)

ACE Inhibition Captopril Captopril

Captopril (75 mg/day) Enalapril (10 mg/day) Captopril (90 mg/day)

Parameters

Effects of ACE Inhibition/Comments

Coronary sinus flow (resistance vessels) Exercise test Onset of AP, STsegment depression

Potentiation of nitroglycerin effects on coronary sinus blood flow Enhancement of anti-ischemic effects most pronounced in patients with an initially poor response to ISDN

Venodilation, forearm plethysmography

Patients pretreated with ACE inhibitor

Hemodynamic effects RAP, PCP and PAM

Prevention of tolerance to venodilator effects with captopril and enalapril Patients already pretreated with ACE inhibitor Almost complete prevention of tolerance

Captopril (50 mg/day)

Hemodynamic effects RAP, PCP and MAP

Patients already pretreated with ACE inhibitor Complete prevention of tolerance

Benazepril (20 mg/day)

Exercise test

Combination therapy superior to nitroglycerin alone

Exercise duration Negative studies Dakak CHF et al44

Parker et al43

Healthy volunteers

nitroglycerin (6.6 mg/kg per min, 24 h i.v.)

Captopril (50 mg/day)

Hemodynamic effects

No ACE inhibitor pretreatment No prevention of tolerance

nitroglycerin (transdermal, 10 mg/day) 6 days

Benazepril

Blood pressure response

No ACE inhibitor pretreatment

Neurohumoral parameters

No prevention of tolerance

ACE 5 angiotensin-converting enzyme; CAD 5 coronary artery disease; CHF 5 congestive heart failure; NTG 5 nitroglycerin; AP 5 angina pectoris; RAP 5 right atrial pressure; PC 5 pulmonary capillary wedge pressure; PAM 5 mean pulmonal artery pressure; MAP 5 mean arterial pressure.

onstrate beneficial effects of ACE inhibition on tolerance. Parker and Parker43 showed tolerance to the blood pressure-lowering effects with nitroglycerin patches within 6 days of continuous treatment and a consistent decrease in hematocrit suggesting continued volume expansion. Concomitant therapy with benazepril, a nonthiol-containing ACE inhibitor did not modify the hemodynamic responses to nitroglycerin and also could not modify the significant decrease in hematocrit. Dakak et al44 tested the effects of ACE inhibition on tolerance with respect to hemodynamic parameters such as right atrial pressure and pulmonary capillary wedge pressure in patients with congestive heart failure. In this particular study, captopril in an average concentration of 60 mg/kg had no effect at all on tolerance development. Taken together, the results of these clinical studies indicate that in most of these trials ACE inhibitors were able to prevent tolerance development. One possible explanation of these conflicting results may be whether or not patients were pretreated with the ACE inhibitor before starting nitrate therapy (see Table I). Hemodynamic tolerance, tolerance in local vascular beds, and tolerance with respect to ischemic threshold

was prevented when patients were pretreated for at least 1 week before starting nitrate therapy. Another important aspect may be related to the dose of ACE inhibitor employed. Tolerance was prevented with captopril in patients receiving an average of 90 mg/ day39 whereas no prevention was reported in patients receiving an average of 50 mg/day.44 Indeed, recently we demonstrated that high- rather than low-dose ACE inhibition is required to retain nitrate sensitivity during prolonged treatment with organic nitrates. By using a well-characterized animal model of nitrate tolerance, we observed that high doses of enalapril (1 mg/kg) prevented the development of tolerance in large coronary arteries after cessation of long-term therapy with nitroglycerin.45 Low concentrations of enalapril (0.2 mg/kg per day) failed to prevent tolerance but also failed to increase plasma renin activity for a 24 hour period, although this concentration already had a marked effect on angiotensin I pressor responses. This may indicate that a persistent inhibition of angiotensin II formation might be a prerequisite for the observed beneficial effects of high-dose enalapril on tolerance development. Furthermore, the demonstration of positive effects of ACE inhibitors A SYMPOSIUM: NITRATE TOLERANCE

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FIGURE 5. Effects of liposome-encapsulated superoxide dismutase on relaxation to nitroglyerin (NTG) in vessels from shamoperated and angiotensin II-infused rats. NTG vasodilator responses were markedly attenuated in angiotensin II-pretreated animals and completely normalized via pretreatment with a liposomal superoxide dismutase (SOD) preparation. (Adapted with permission from Z Kardiol.52)

even in the absence of a functional sulfhydryl group suggests that, in agreement with previous observations, sulfhydryl group depletion does not represent the decisive mechanism responsible for nitrate tolerance.46,47

BY WHICH MECHANISMS DO ACE INHIBITORS OR AT1RECEPTOR BLOCKERS PREVENT THE DEVELOPMENT OF NITRATE TOLERANCE?

More on superoxide and enhanced vasoconstriction in nitrate tolerance: An interesting new aspect of long-

Recent experimental data indicate that pseudotolerance mechanisms such as increased circulating angiotensin II levels may be, at least, in part responsible for tolerance at the cellular level. In an animal model of nitrate tolerance we found that aortic segments from rabbits demonstrated a great degree of tolerance secondary to an NADH-oxidase mediated increase in endothelial superoxide36,48,49 and a marked increase in sensitivity to vasoconstrictors secondary to an activation of protein kinase C. Preliminary data seem to indicate that losartan, a specific blocker of the angiotensin II receptor subtype AT1, significantly reduced nitroglycerin-induced increases in vascular superoxide production, preserved, at least in part, nitrate sensitivity in nitrate-treated animals and normalized vasoconstrictor responses to phenylephrine, potassium chloride, and serotonin.50 These observations suggest that angiotensin II may indeed represent an important stimulus for increased vascular superoxide production and protein kinase C activation. Further support for a potential role for angiotensin II in nitrate tolerance was provided by studies showing that via angiotensin II infusion one can mimic all vascular changes seen in response to prolonged treatment with organic ni36A THE AMERICAN JOURNAL OF CARDIOLOGYT

trates.51 Angiotensin II increases vascular superoxide production via activation of the membrane associated NADH-oxidase,51 increases sensitivity to vasoconstrictors due to increased autocrine endothelin production within vascular smooth muscle, and simultaneously impairs the vasodilator potency of nitroglycerin51 (Figure 5). Preliminary data indicate that nitroglycerin therapy leads to an increase in the expression of the endothelial subunit of the NADPH oxidase, gp 91phox52 while angiotensin II infusion is associated with increased expression of the p22 phox subunit in smooth muscle cells,53 raising some doubt whether angiotensin II is responsible for increased superoxide production in the setting of nitrate tolerance. One cannot exclude completely, however, that the beneficial effects of ACE inhibition on tolerance are secondary to an additional endothelial-derived nitric oxide-mediated vasodilator stimulus. ACE is identical to kininase II, an enzyme involved in the breakdown of the endothelium-dependent vasodilator bradykinin.54,55 Therefore, chronic ACE inhibition during nitroglycerin infusion may be associated with an accumulation of locally generated kinins, which in turn may cause an activation of endothelium-mediated vasodilator effects. Additional experiments using bradykinin antagonists are required to address to what extent bradykinin may contribute to the beneficial effects of ACE inhibitors in tolerance. The combination of AT1-receptor blockade and nitroglycerin treatment causes large increases in circulating angiotensin II levels. Angiotensin II may, therefore, strongly stimulate the AT2-receptor. Therefore, one cannot exclude that in the presence of AT1-receptor blockade, the positive effects seen with respect to tolerance might be secondary to unopposed AT2-receptor stimulation.

term therapy with organic nitrates was the demonstration of increased sensitivity to vasoconstrictors such as serotonin, phenylephrine, angiotensin II, and thromboxane. This has been shown to occur in animals treated with nitroglycerin for a 3-day period in a clinically relevant concentration of 1.5 mg/kg per min,37 and in rats with long-term nitroglycerin infusion.56 Preliminary data in patients with coronary artery disease also indicate that this observation may have clinical relevance. Heitzer et al57 observed that reductions in forearm blood flow in response to intraarterial (brachial artery) angiotensin II and phenylephrine were markedly enhanced in patients pretreated with nitroglycerin for a 48-hour period (0.5 mg/kg per min). Interestingly, these hypercontractile responses could be blocked by concomitant treatment with the ACE inhibitor captopril, suggesting that the renin– angiotensin system is, at least in part, responsible for this phenomenon. The demonstration of such hypercontractile responses of the forearm microcirculation within 48 hours of continuous nitroglycerin treatment may indicate that increases in sensitivity to constrictors represent a major mechanism responsible for the attenuation of the vasodilator effects of nitroglycer-

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FIGURE 6. Effects of nitroglycerin treatment (3 days, 1 mg/kg for 72 hours) on the relaxation of rat isolated aorta, precontracted with 1027 M phenylephrine, expressed as either absolute tension (a and c) or as a percentage & baseline (b and d). Treatment with nitroglycerin markedly increased potency of phenylephrine (a), which was completely corrected by in vivo treatment with the protein kinase C (PKC) antagonist N-benzoyl-staurosporine (30 mg/ kg, p.o. daily) (c). Similarly, 3 day treatment with nitroglycerin resulted in a rightward shift of the nitroglycerin dose–response, indicating nitrate tolerance (b). Tolerance was prevented by in vivo treatment with the PKC antagonist (d). (Adapted with permission from Circulation.57)

in.57 Using an animal model of nitrate tolerance we could normalize vasoconstrictor responses using in vitro inhibitors of protein kinase C, an important second messenger for smooth muscle contraction.37 This concept is supported by more recent data demonstrating that in vivo treatment with protein kinase C inhibitors prevented the increase in sensitivity to vasoconstrictors and, in parallel, nitrate tolerance (Figure 6).56 Based on the demonstration of increased superoxide production in tolerance we hypothesized that superoxide may inactivate nitric oxide derived from the organic nitrate to form the highly toxic intermediate peroxynitrite, which is much less powerful in activating the smooth muscle guanylyl cyclase than authentic nitric oxide. Recently, Mu¨lsch et al58 offered an additional mechanism by which superoxide inhibits vasorelaxation induced by organic nitrates. By using purified, heme-containing guanylyl cyclase, they showed that increasing concentrations of O2 markedly inhibited basal soluble guanylyl cyclase activity (Figure 7). In the presence of the nitric oxide-independent

guanylyl cyclase stimulator YC-1, the inhibitory effect of superoxide on soluble guanylyl cyclase activity was markedly attenuated. This observation suggests that guanylyl cyclase may be regulated in vivo by superoxide, and that concomitant administration of a nitric oxide-independent soluble guanylyl cyclase stimulator such as YC-1 may help to prevent the development of nitrate tolerance. The oxidative stress concept of nitrate tolerance is also supported by more recent data from Eberhard Bassenge’s group.59 In a well-characterized model of nitrate tolerance, tolerance development in epicardial arteries and the venous system was completely prevented by concomitant treatment with vitamin C. They also observed an up-regulation of platelet activity during long-term nitroglycerin therapy as demonstrated by enhanced thrombin-stimulated intracellular Ca21 levels and increases in the microviscosity of platelet membranes (indicating enhanced receptor expression) associated with a progressive impairment in basal, unstimulated cyclic guanosine monophosphate A SYMPOSIUM: NITRATE TOLERANCE

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FIGURE 7. Effect of superoxide radicals on basal and YC-1 stimulated soluble guanylyl cyclase activity (sGC). The basal (open columns) and stimulated (hatched columns) were determined in the presence of xanthine (X) and xanthine oxidase (XO). XO significantly inhibited basal sGC activity at all concentrations tested. In the presence of the NO-independent vasodilator YC-1, the inhibitory effect of superoxide on sGC activity was markedly attenuated. (Adapted from Naunyn Schmiedeberg’s Arch Pharmacol.59)

levels. All these changes could be prevented by cotreatment with ascorbate. A further question which was addressed by the same group was, to what extent peroxynitrite, a possible intermediate product of nitric oxide derived from nitroglycerin and superoxide, is formed during long-term nitroglycerin treatment. As a marker, they used peroxynitrite-induced nitrotyrosine formation. They found that transdermal treatment of patients increased urinary nitrotyrosine content in patients with coronary artery disease, a phenomenon that could be suppressed using the antioxidant vitamin C.60 More recently, Gupte et al61 provided a new possible link between enhanced superoxide production and enhanced vasoconstriction in response to nitroglycerin treatment. In their particular animal model they pretreated an isolated heart preparation with superoxide using xanthine/xanthinoxidase. After a superoxide washout period, they infused intracoronary nitroglycerin and observed a paradoxical increase in coronary perfusion pressure. The increase in perfusion pressure was blocked by cyclooxygenase inhibitors and by a thromboxane-receptor antagonist but enhanced by the administration of arachidonic acid. These results indicate that under conditions of increased oxidative stress, nitroglycerin stimulates the release of PGF2a and TXA2, leading to paradoxical vasoconstriction (Figure 8).61 As long-term nitroglycerin therapy has recently been demonstrated to be associated with increased endothelial superoxide production,36 it is tempting to speculate that nitroglycerin-induced release of vasoconstrictive prostaglandins 38A THE AMERICAN JOURNAL OF CARDIOLOGYT

FIGURE 8. Effects of nitroglycerin (NTG) and NGnitro-L-arginine (L-NNA) on prostaglandin (PG) release during control conditions and washout of superoxide. Changes in PG washout by 4mM NTG in zO22 untreated and pretreated hearts are shown. Left: open bar represents NTG induced changes in PG release in zO22 untreated hearts 10 min after NTG treatment. Solid horizontal lines correspond to mean level of each PG released from zO22 untreated hearts. Right: 2 bars indicate changes in PG release (solid bars) induced by NTG or by L-NNA 10 min into zO22 washout period. Dashed horizontal lines correspond to mean level of release of individual PG from zO22 pretreated hearts during washout of zO22 with normal Krebs solution. NTG treatment of zO22 pretreated hearts significantly increased TXA2 and PGF2 release. (Adapted from Ann Intern Med.62)

may contribute, at least in part, to hypercontractile responses of the vasculature in the setting of nitrate tolerance. That oxidative stress within the vasculature dramatically alters nitroglycerin effects was demonstrated more recently in patients with diabetes mellitus.62 In these studies, the investigators found that, compared with controls, patients with diabetes had increased platelet aggregation to adenosine diphosphate, increased blood viscosity, and decreased blood filterability. In controls, nitroglycerin significantly reduced platelet aggregation, blood viscosity, and increased blood filterability. In patients with diabetes, however, nitroglycerin paradoxically increased platelet aggregation, blood viscosity, and decreased blood filterability. These responses were normalized by pretreating patients with vitamin E and glutathione demonstrating that under conditions with pre-existing oxidative stress such as, for example, diabetes and atherosclerosis, some of the beneficial effects of nitroglycerin can be detected only in the presence of antioxidants.62

PERSPECTIVE During the past two decades, a great deal has been learned about both physiologic and pharmacologic

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regulation of vascular tone. This is particularly true for both the endogenous and exogenous nitrovasodilators. It is now understood that exogenous nitrovasodilators must release nitric oxide to produce vasodilation and a substantial amount has been learned about how nitric oxide causes vessels to dilate. Exciting new observations include the demonstration that the vasodilator actions of nitroglycerin are, at least in part, endothelium-dependent, and that in the presence of increased oxidative stress nitroglycerin can actively cause constriction of resistance vessels via release of PGF2a and TXA2. It is apparent that a great deal more remains to be learned about the mechanisms of action of nitroglycerin and adaptations to long-term therapy. Within 24 hours of continuous nitrate therapy, we encounter an activation of neurohormonal vasoconstrictor forces such as the renin–angiotensin system which may compromise the vasodilator effects of nitroglycerin, in particular at the resistance vessel level. During long-term therapy (3–5 days), tolerance in large arteries develops which may be, at least in part, secondary to increased superoxide production and/or increased sensitivity to vasoconstrictors. Much less is known about the time– course of tolerance development in veins and whether pseudotolerance or true vascular tolerance mechanisms predominate. ACE inhibitors beneficially influence tolerance by inhibiting pseudotolerance mechanisms but also by preventing the activation of angiotensin II-sensitive, superoxideproducing enzymes in endothelial and smooth muscle cells, or by inhibiting increased vasoconstrictor responsiveness. Nitrate tolerance continues to be an area of intense investigation, and much more information is required to get insight into the utility of new vasodilator drugs and strategies for maintaining therapeutic effect despite continuous administration.

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