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(Circulation. 1996;94:2241-2247.)
© 1996 American Heart Association, Inc.

Articles

In Vivo Nitrate Tolerance Is Not Associated With Reduced Bioconversion of Nitroglycerin to Nitric Oxide

Jørn Bech Laursen, MD; Alexander Mulsch, PhD; Søren Boesgaard, MD, PhD; Petr Mordvintcev, PhD; Simon Trautner, MS; Nicolai Gruhn, MS; Jens Erik Nielsen-Kudsk, MD; Rudi Busse, MD, PhD; Jan Aldershvile, MD, PhD

Medical Department B, Division of Cardiology, Rigshospitalet, University of Copenhagen, Denmark, and Center of Physiology, Johann Wolfgang Goethe University Clinic, Frankfurt/Main, Germany.

Correspondence to Alexander Mulsch, Zentrum der Physiologie, Klinikum der Universitat Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.

	Abstract

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Background In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats.

Methods and Results Five groups (n=6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)₂ and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P<.05) increased in all tissues in nitrate-tolerant rats in an NTG dose–dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats.

Conclusions The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.

Key Words: arteries • endothelium-derived factors • free radicals • nitroglycerin • veins

	Introduction

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Nitric oxide is an important endogenous regulator of vascular tone. NO formation from NTG correlates with the NTG-mediated vascular responses and accounts for the clinical effects of organic nitrates.^{1 2 3} Continuous administration of organic nitrates (eg, NTG and isosorbide dinitrate), however, leads to tolerance of their hemodynamic and clinical effects.^{4 5 6} The phenomenon of nitrate tolerance is complex, and several mechanisms may be involved. Both nitrate-induced neurohormonal activation counteracting NTG-mediated vasodilation^{7 8 9} and reduced biological activity of NTG-derived NO¹⁰ contribute to its development. In addition, in vitro results suggest that reduced bioconversion of the vasoinactive NTG to vasoactive NO^{2 11 12 13} may be an important mechanism underlying tolerance development. However, the marked differences between previously reported^{10 14 15 16} in vivo and in vitro studies have raised concern about the in vivo relevance of conclusions drawn from in vitro experiments. Thus, although the mechanism is generally accepted, the in vivo validity of a reduced bioconversion of NTG to NO as a cause of nitrate tolerance is currently not clear.

Therefore, the purpose of this in vivo study was to evaluate whether the impaired hemodynamic effect of NTG after development of nitrate tolerance is associated with a reduced vascular NO availability due to a decreased metabolism of NTG to NO. NTG-derived formation of NO was measured by in vivo spin-trapping of NO and subsequent cryogenic ESR spectroscopy in different isolated tissues of nitrate-nontolerant and nitrate-tolerant rats.

	Methods

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Animals
Female Wistar rats (210 to 250 g) were anesthetized with 1% to 3% halothane and N₂O/O₂ (2:1) during a chronic catheterization procedure. One catheter (medical-grade Tygon tubing) was implanted with its tip in the ascending aorta through the left carotid artery, and two catheters were placed in the superior vena cava via the left jugular vein. The catheters were filled with a solution of glucose (500 g/L) and heparin (500 IU/mL) and plugged with a nylon pin. Each catheter was externalized through the neck skin. After catheter implantation, rats were housed individually and exposed to a 12/12-hour light-dark cycle with free access to standard rat chow and tap water. After surgery, rats were allowed to recover until they appeared healthy and had regained their preoperative weights (6 to 8 days).

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

Induction of Nitrate Tolerance
Baseline hemodynamic response to NTG was determined as the blood pressure–lowering effect of intravenous bolus infusions at the end of the recovery period (day 0). Two bolus doses of NTG (0 mg [placebo] and 5 mg NTG) were given (0.4 mL over 60 seconds) separated by a 20-minute interval. An osmotic minipump (Alza Corp) was then placed subcutaneously and connected to one of the two intravenous catheters. NTG (1 or 0.2 mg/h) or placebo (98% ethanol [NTG vehicle]) was delivered from the minipump at a constant rate of 10 mL/h throughout the study period. In this model, which has been described in detail,¹⁷ tolerance to the blood pressure–lowering effect of NTG develops within 24 hours. However, to confirm development of nitrate tolerance, the blood pressure–lowering effect of NTG-placebo bolus was reexamined at day 3 in all treatment groups.

Blood pressure during baseline infusion conditions (before NTG bolus administration) and blood pressure alterations during NTG bolus challenges were recorded continuously by pressure transducers (Baxter Corp) connected to the arterial catheter. Tracings were displayed on a Graphtec linear recorder (Watanabe Instruments Corp).

Measurements of NO
In vivo NO formation in vascular and organ tissues was measured by an FeDETC NO spin-trapping method as previously described.³ DETC (and other dithiocarbamates) are especially suitable to assess NO formation from organic nitrates because they do not catalyze NO release from nitrates (A. Mulsch, unpublished results), in contrast to certain other thiols (L-cysteine, thiosalicylic acid). In vitro studies have shown that this spin-trapping technique attenuates vasodilator responses to NTG by >80%.¹⁸

To optimize the protocol for application of spin traps with regard to maximal NO spin-trapping in preliminary experiments, various doses of FeSO₄ (0, 20, and 40 mg/kg) and protocols for application of DETC (intravenous versus intraperitoneal; bolus versus continuous infusion) were examined. Fe²⁺ ions were applied to enhance formation of the NO spin-trapping agent, ie, the Fe(DETC)₂ complex. The protocol that gave maximal NO spin-trapping [formation of paramagnetic NOFe(DETC)₂] after bolus injection of NTG was chosen for the present experiments. Thirty-five minutes before they were killed, the rats received bolus injections of freshly prepared saline solutions of DETC (600 mg/kg; 0.5 mL IP) and FeSO₄/sodium citrate (40 mg·kg^-1 FeSO₄/200 mg·kg^-1 sodium citrate) (0.2 mL SC). After the rats were killed, the aorta, vena cava, left and right ventricles, and liver were quickly excised and placed on ice. Vessels were quickly rinsed with saline to remove adhering blood. Wet tissues were weighed, pressed in cylindrical Teflon tubes, and frozen in liquid nitrogen as described previously.^{3 18} The specimens were inserted into a liquid nitrogen–cooled fingertip-like Dewar flask, which was placed inside the cavity of the ESR magnet to expose the specimen to radiofrequency irradiation. ESR spectra were recorded at 77 K on a Bruker EPR 300E spectrometer operated at about 9.5 GHz, modulation frequency 100 kHz, modulation amplitude 5 G, and microwave power 20 mW.

In principle, at least two other paramagnetic species besides NOFe(DETC)₂ can appear in DETC- and NO-exposed tissues in the same magnetic field range. In vascular and nonvascular tissues, competition for DETC by endogenous free Cu²⁺ generates the Cu-DETC complex, which does not bind NO. In myocardial and hepatic tissues, competition for NO by high concentrations of endogenous hemoproteins yields the NO-heme complex. However, the three species can readily be distinguished by their characteristic ESR spectra, as shown in Fig 1. The original tracing (tracing A) is a composite of all three paramagnetic species. Distortion of the baseline from 3300 to 3500 G is due to the NO-heme complex and is corrected for by electronic subtraction of an NO-hemoglobin standard signal (tracing B). From the integrated signal of the fitted standard, the concentration of NO-heme in the tissue is derived. The signal of a Cu(DETC)₂ standard (tracing D) is then subtracted from the corrected signal (tracing C) to yield a pure signal of NOFe(DETC)₂ (tracing E). This signal is then integrated, and the concentration of NO trapped is calculated by comparison with an NOFe(DETC)₂ standard. The detection limit of the ESR method was 0.02 nmol NOFe(DETC)₂/g tissue and 0.5 nmol NO-heme/g tissue. Since NO is trapped as a stable nitrosyl complex, the ESR method detects NO accumulating during the period of exposure to the spin-trapping agent. Although the NOFe(DETC)₂ complex accounts for the entire NO formation in the blood vessel wall, it provides only a lower estimate of total NO formation in organ tissues because of competition for NO by myoglobin in the heart and cytochrome P450 in the liver,¹⁹ besides other heme (eg, blood) targets.²⁰ Tissue concentrations of NOFe(DETC)₂ and NO-heme are listed separately.

View larger version (17K): [in this window] [in a new window]   Figure 1. Evaluation of representative ESR recording for NO-heme and NOFe(DETC)2. Cryogenic ESR spectra were recorded on a heart from a low-dose NTG–tolerant rat as described in "Methods." The original spectrum bearing three major paramagnetic species was successively deconvolved as shown. Tracings: A, Original ESR spectrum. B, NO-hemoglobin standard (2.6 µmol/L in heme); characteristic triplet at g is common to pentacoordinate NO-heme proteins.19 20 C, Difference spectrum A-B. D, Cu(DETC)2 standard (15 µmol/L). Typical hyperfine splitting lines labeled a, b, c, d. E, Difference spectrum C-D represents the pure signal of NOFe(DETC)2 complex (0.6 µmol/L). Characteristic g-factor values (g, g||) are indicated. Typical triplet hyperfine splitting line at g.

Measurements of Vascular Relaxation to NTG In Vitro
Vascular ring segments (OD, 1 to 1.5 mm; length, 2.5 mm) were prepared from isolated rat thoracic aorta. In half the vessels, the endothelium was carefully removed. Each arterial segment was mounted in a precision myograph by insertion of two fine stainless steel pins into the vessel lumen. One of the pins was connected to a highly isometric strain-gauge transducer for recording of isometric tension changes. The vessel myograph has been described in detail previously.²¹ Six myographs were used at the same time, allowing six vascular preparations to be studied simultaneously. Transducer signals were amplified and displayed on an eight-channel recorder (Graphtec Corp). The arterial preparations were submerged in 5-mL tissue baths containing Krebs solution (composition in mmol/L: NaCl 118.0, KCl 4.6, CaCl₂ 2.5, MgSO₄ 1.15, NaHCO₃ 24.9, KH₂PO₄ 1.15, and glucose 5.5) at 37°C and aerated with a mixture of 95% O₂/5% CO₂ (pH 7.4).

Experimental Protocols
Nine different groups of conscious, unrestrained, chronically catheterized rats were divided into three different study groups.

Study 1: NO Formation in Normal Rats and Rats Undergoing Continuous Long-Term Infusion of NTG
Three groups of rats were studied. At 0 hours, two groups of rats received either a long-term high-dose infusion of NTG (1 mg/h, n=8, high-dose NTG group) or a low-dose infusion of NTG (0.2 mg/h, n=6) for 75 hours. In addition, a control group (n=8) of chronically catheterized but otherwise nonmedicated rats was included (control).

The hypotensive effect of an NTG bolus dose was determined immediately before the start of the long-term NTG infusion (the two infusion groups) and repeated after 72 hours of infusion (including the control group). Two hours later, during the ongoing NTG infusion in the NTG groups, NO was trapped in vivo by intraperitoneal injection of DETC and subcutaneous injection of Fe²⁺ citrate as described above. The aorta, vena cava, heart, and liver were subsequently removed and analyzed for their NO content.

Study 2: Effect of Additional NTG on NO Formation in Nitrate-Nontolerant and Nitrate-Tolerant Rats
Four groups of rats were investigated. Three of these groups were treated as described in study 1 (control, n=8; low-dose NTG, n=5; high-dose NTG, n=8). In addition, a fourth group of rats received long-term infusion of NTG vehicle (n=7). Baseline blood pressure and blood pressure responses to NTG at 0 hours and at day 3 (72 hours) before the NO spin-trapping procedure were measured as described.

To investigate the amount of NO produced from a specific dose of NTG in nitrate-nontolerant and nitrate-tolerant rats, a bolus infusion of NTG (6.5 mg/kg IV over 20 minutes) was administered during the last 20 minutes of the NO spin-trapping procedure. Apart from this bolus dose of NTG, other experimental conditions were the same as in study 1. Therefore, any differences in the amount of NO trapped between study 1 and study 2 were consequently interpreted as being related to the NTG bolus infusion.

Study 3: Confirmation of Vascular Nitrate Tolerance
To substantiate that NO measured in aortic tissue from the nitrate-tolerant rats actually was derived from nitrate-tolerant aortic vessels, two other groups of rats received either NTG (1 mg/h) (high-dose NTG, n=8) or NTG vehicle (placebo, n=8) for 72 hours. After confirmation of development of in vivo nitrate tolerance in the NTG-treated group (ie, a reduced blood pressure response to an NTG bolus), rats were decapitated, and the thoracic aorta was rapidly excised, placed in cold Krebs solution, and prepared for myographic experiments as described.

At the outset of each experiment, the vessel preparation was stretched to a resting tension of 1 g to optimize the vasoconstrictor response to K⁺ depolarization. Tissues were allowed to equilibrate for 1 hour. The vessels were then preconstricted by K⁺ (iso-osmolar K⁺-rich [30 mmol/L] Krebs solution), and when a stable contraction plateau was reached, NTG (10^-9 to 3x10^-4 mmol/L) or its vehicle was added cumulatively to the tissue baths at time intervals of 3 minutes. At the end of the experiment, the vessels were rechallenged by K⁺ (30 mmol/L) followed by addition of acetylcholine (1 mmol/L) on top of the K⁺ contraction. A vasodilator response was taken as evidence of a functionally intact endothelium, whereas a missing response was considered the result of successful removal of endothelium.

Drugs
NTG solutions were prepared from a stock solution (100 mg NTG in 1 mL of 98% ethanol). For prolonged infusion, NTG was further diluted with 96% ethanol (20 mg/mL) when necessary. All other solutions were prepared in 0.9% saline and adjusted to pH 7.4. Acetylcholine, L-NAME, DETC, FeSO₄, and sodium citrate were purchased from Sigma Chemical Co.

Calculations and Statistics
MAP was estimated, in mm Hg, as diastolic pressure plus (systolic minus diastolic pressure) divided by 3. The reported changes in MAP to NTG bolus infusions represent the difference between the baseline MAP (immediately before NTG) and the nadir on the blood-pressure curve after NTG. All data are presented as mean±SEM. Within 10 minutes, blood pressure in all rats recovered to the pre–NTG-bolus level. Differences between pretreatment and posttreatment means were determined by Student's paired t test.

In the myograph experiments, NTG responses are expressed as percent relaxation of the contraction induced by K⁺. Reversal of tone back to baseline value was taken as 100% relaxation. Values of EC₅₀ (the concentration that produces half-maximal effect) expressed as pD₂ (log EC₅₀ values) were estimated by fitting the concentration-relaxation curves for NTG to the three-parameter logistic equation (Hill equation) by nonlinear regression analysis. The concentration-relaxation curves for NTG are presented in the concentration range 10^-9 to 10^-5 mmol/L. At higher concentrations (3x10^-5, 10^-4, and 3x10^-4 mmol/L), NTG vehicle itself caused vasorelaxation. Subtraction of the NTG-vehicle effect allowed an estimation of the maximal effect of NTG. In calculation of pD₂ values, maximal effect for NTG was set equal to 80% in endothelium-intact vessels and to 100% in endothelium-denuded preparations. The significance between pD₂ values was assessed by a two-way unpaired Student's t test.

In all experiments, comparisons between more than two treatment groups were performed by ANOVA. Statistical significance was assumed when P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Study 1: NO Formation in Normal Rats and Rats Undergoing Continuous Long-term Infusion of NTG
Infusion of NTG for 72 hours significantly reduced the hypotensive effect of a bolus injection of NTG, suggesting the development of NTG tolerance (Table 1). Furthermore, the response to NTG was significantly (P<.05) more attenuated in the high-dose NTG group than in the low-dose NTG group (Table 1, Fig 2). The hypotensive effect of NTG in the nonmedicated control group was similar to the pre–NTG-infusion responses in the two NTG infusion groups (P>.05). Baseline MAP values (before NTG bolus challenges) were similar in all treatment groups and did not differ between day 0 and day 3 (Table 1).

View this table: [in this window] [in a new window]   Table 1. Baseline MAP and MAP to a Bolus Injection of 5 mg NTG Before Start of Experiments (Day 0) and After 72 Hours of NTG/Placebo Infusions (day 3) in the Different Treatment Groups

View larger version (20K): [in this window] [in a new window]   Figure 2. Top, NO production measured as NOFe(DETC)2 (nmol·g tissue-1·35 min-1, mean±SEM) in aorta and vena cava from a nonmedicated control group (control, n=8), a group infused with 0.2 mg NTG/h for 74 hours (low-dose NTG, n=6), and a group infused with a five times higher dose of NTG (high-dose NTG, n=8) for the same period of time. Bottom, Fall in MAP (mm Hg) to a bolus dose of NTG in the same groups of animals. 1P<.05 compared with control; 2P<.05 compared with the low-dose NTG group.

In the control group not exposed to prolonged NTG infusion, significant amounts of nitrosyl-iron complexes were detected only in the myocardium and liver, whereas the endogenous NO formation in aorta and vena cava was below the detection limit of the ESR technique (Table 2). In contrast, a significant and NTG dose–dependent (high- versus low-dose groups) increase in NO formation was clearly detected in the vasculature [NOFe(DETC)₂] and organs [NOFe(DETC)₂ and NO-heme] of nitrate-tolerant rats (Table 2, Fig 2). The concentration of NO-heme was much higher than that of NOFe(DETC)₂ in the heart but lower in the liver. Total nitrosyl-iron complex formation was about 15-fold higher in the liver and about 30 times higher in the heart than vascular levels. This relation appeared to be independent of the dose of NTG applied.

View this table: [in this window] [in a new window]   Table 2. NO Production in Aorta, Vena Cava, Left Ventricle (Heart), and Liver From Study 1

In summary, even after prolonged (74 hours) continuous infusion of NTG, NO generation from NTG is increased dose dependently (and occurs at a very high rate compared with endogenous NO formation), although the hemodynamic responses to NTG are significantly attenuated.

Study 2: Effect of Additional NTG on NO Formation in Nitrate-Nontolerant and Nitrate-Tolerant Rats
The hypotensive effect of NTG was significantly attenuated after prolonged NTG infusion, substantiating the development of tolerance (Table 1).

Infusion of additional NTG (6.5 mg/kg) during the last 20 minutes of the NO spin-trapping period significantly increased NO formation in all tissues of the control group (P<.05) compared with the control in study 1 (without NTG) (Tables 2 and 3). As in study 1, formation of NO-heme in organs increased in parallel to NOFe(DETC)₂ and exceeded the latter nitrosyl complex in the heart, whereas NOFe(DETC)₂ was the major paramagnetic species in the liver. Inhibition of endogenous NO synthase and NO production by L-NAME (1.5 mg/kg for 60 seconds, n=2) 5 minutes before the NO spin-trapping procedure did not affect the amount of NO produced from NTG in control rats (data not shown). Similarly, infusion of NTG vehicle for 3 days did not affect the formation of NO from NTG (P>.05) (Table 3).

View this table: [in this window] [in a new window]   Table 3. NO Production in Aorta, Vena Cava, Left Ventricle (Heart), and Liver From Study 2

In the two groups receiving both long-term NTG infusion and additional NTG (low- and high-dose NTG groups, study 2), NO formation was significantly increased compared with the corresponding two groups receiving only long-term NTG infusion (study 1) (Tables 2 and 3). Thus, NO formation is significantly increased after NTG bolus supplementation to nitrate-tolerant rats.

NO measured in the nitrate-tolerant groups of study 2 are derived partly from the continuous NTG infusion, partly from the additional NTG administered during the DETC exposure. However, because the amount of NO generated from the continuous infusion of NTG was measured in study 1, a reliable estimate of the amount of NO produced from the additional NTG bolus dose can be obtained (by subtracting the corresponding values in study 1 from study 2; Fig 3). If this is done, the results suggest that the formation of NTG-derived NO after development of tolerance is similar to the amounts of NTG-derived NO produced in the nontolerant control/placebo group (Fig 3). It therefore seems unlikely that nitrate tolerance is caused by a reduced metabolic conversion of NTG to NO.

View larger version (57K): [in this window] [in a new window]   Figure 3. Bar graphs showing total NO [NOFe(DETC)2 and NO-heme] measured in aorta, vena cava, heart, and liver in two NTG-nontolerant [control (n=7), placebo (n=6)] and two NTG-tolerant [low-dose (n=5) and high-dose (n=7) NTG] groups of rats after addition of NTG during the NO spin-trapping procedure. Total height of bars represents amount of NO measured in study 2. This can be divided into two components. Dotted area below horizontal line represents NO contribution from underlying baseline NTG infusion as measured in study 1. Thus, the amount of NO derived solely from the NTG bolus (above the horizontal line) is similar in all treatment groups, suggesting that the attenuated hemodynamic effect of NTG in the tolerant groups is not associated with reduced conversion of NTG to NO.

Study 3: Confirmation of Vascular Nitrate Tolerance
In aortic segments excised from in vivo nitrate-tolerant rats (Table 1), vasorelaxation to NTG was significantly attenuated compared with that observed in vessels isolated from nontolerant animals (NTG vehicle) (Fig 4). pD₂ for NTG was -5.97±0.08 (EC₅₀=1.1 µmol/L) in vessels from NTG-tolerant rats and 6.87±0.08 (EC₅₀=0.14 µmol/L) in arteries from nontolerant rats (P<.05). These values indicate an eightfold rightward shift in the concentration-relaxation curve for NTG in the tolerant compared with the nontolerant state. The difference in responsiveness to NTG in the two groups was not caused by differences in precontraction levels induced by K⁺, because the degree of precontraction was similar in the two groups (1.16±0.21 versus 1.02±0.16 g, respectively). The findings therefore emphasize that measurements of NO in aorta were actually performed in a nitrate-tolerant artery.

View larger version (15K): [in this window] [in a new window]   Figure 4. Concentration-response curves (mean±SEM) showing the relaxing effect of NTG in isolated rings of rat thoracic aorta excised either from animals made tolerant to NTG in vivo or from rats treated by NTG vehicle in vivo. Left, the endothelium of the isolated arteries was intact; right, the endothelium had been mechanically removed. Concentration-response relationships of tolerant and nontolerant endothelium-intact vessels were significantly different (see text). Data from 32 ring experiments in 16 rats.

Intriguingly, removal of the vascular endothelium from arteries excised from nitrate-tolerant animals completely restored the vasodilator response to NTG (Fig 4). pD₂ values for NTG obtained in endothelium-denuded arteries from tolerant (-6.56±0.09) and nontolerant (-6.48±0.10) rats were not significantly different (P>.05). Also, there was no significant difference between the K⁺ precontraction levels in the endothelium-denuded arteries from the tolerant and nontolerant rats (1.05±0.20 versus 1.36±0.07 g) (P>.05). These results suggest an important role of the vascular endothelium in the mechanism of development of tolerance to NTG.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this in vivo study, NTG-derived NO formation was measured in NTG-nontolerant and NTG-tolerant rats by in vivo NO spin-trapping combined with ex vivo cryogenic ESR spectroscopy. A major finding of the study is that hemodynamic and vascular tolerance to NTG may exist despite high vascular amounts of NTG-derived NO. This result does not support the concept of a reduced bioconversion of NTG to NO as a mechanism of nitrate tolerance. Rather, the present results suggest that reduced metabolic conversion of NTG to NO does not contribute to NTG tolerance in vivo.

NTG and other organic nitrates elicit vasorelaxation as a result of their metabolism to NO in vascular smooth muscle cells.¹ However, continuous exposure to NTG leads to tolerance to its hemodynamic effects. Investigations of in vivo tolerance development suggest that a reduced biological activity of NO¹⁰ and nitrate-induced neurohormonal activation (counteracting vasodilation)^{7 8 9} may participate in the development of nitrate tolerance. In vitro, however, the major hypotheses of nitrate tolerance involve a reduced metabolic conversion of NTG to NO.^{2 11 12 13} The importance of each of these mechanisms for in vivo tolerance development is currently not clear.

In this study of nitrate tolerance, development of tolerance was substantiated by a marked attenuated in vivo response to NTG, and NTG-derived NO was measured in nitrate-tolerant aortic vessels, as documented by the myograph experiments. In contrast to previous in vitro studies on bioconversion of NTG, we find that (1) NTG-derived NO in nitrate-tolerant rats increases in an NTG dose–dependent manner and (2) vascular NTG-derived NO formation is similar in tissues from nitrate-tolerant and nitrate-nontolerant rats. The same holds true for NTG-derived NO formation in heart and liver. Thus, the marked loss of hemodynamic response to NTG seems to occur without measurable changes in the bioconversion rate of NTG to NO. This is compatible with the assumption that reduced biological activity of NO, rather than reduced metabolism of NTG, contributes to in vivo development of nitrate tolerance.

Removal of the endothelium improves the NTG-mediated vasorelaxation in isolated aortic vessels of nitrate-tolerant rats in this and another recent study,¹⁰ suggesting that NO-mediated effects are modulated by a substance derived from the endothelium of nitrate-tolerant animals. Recent data from D.G. Harrison's laboratory^{10 22} suggest that in vivo nitrate tolerance is associated with an increased endothelial O₂^- production due to activation of the renin-angiotensin system. Accordingly, the endothelial O₂^- may inactivate NTG-derived NO and account for the loss of biological activity of NO. Assuming that NTG-derived NO is spin-trapped before it is exposed to O₂^- (and inactivated), our present ESR findings are in line with this mechanism of nitrate tolerance.

One explanation for the discrepancy between this in vivo and previous in vitro studies regarding the effects of nitrate tolerance on NTG-derived NO formation^{2 11 12 13} may relate to differences in the experimental setup. It is now clear that data on other aspects of nitrate tolerance (eg, thiol depletion, cross-tolerance to other nitrovasodilators, alterations in guanylyl cyclase activity and O₂^-) differ significantly depending on whether tolerance is induced in vitro or in vivo.^{10 14 15 16} Furthermore, differences in NO sampling sites, eg, in metabolically active tissue, coronary effluents, incubation media, or headspace gas may be of importance.

The amounts of NTG-derived NO trapped in vascular and organ tissues were considerably higher than those detected previously in NTG-infused rabbits.^{18 23} This finding can readily be explained by the lower dose of NTG applied in the rabbit experiments (0.5 mg·kg^-1·20 min^-1). In addition, the spin-trapping procedures were different in that a lower amount of DETC was provided to the rabbits (200 mg/kg) and exogenous nonheme iron was not used for the formation of NOFe(DETC)₂ complexes. In our previous rabbit studies, ESR signals of NO-heme were not detected, but as expected,^{24 25} a considerable part of NTG-derived NO appears in the NO-heme fraction when a higher dose of NTG is used, as in the present study. The major hemoproteins accounting for NO-heme are hemoglobin (blood), cytochrome P450/420 (liver), and myoglobin (heart). Intriguingly, in the high-dose NTG-tolerant group, 10% of the myoglobin present in the heart (400 nmol/g) was converted to NO-heme after additional NTG infusion. Whether or not oxygen handling in the myocyte is affected under this condition is not clear. However, this should be considered, because high doses of NO may show negative inotropic effects on the heart.

In summary, this is the first study to show that the in vivo bioconversion of NTG to NO is dose-dependently increased during prolonged infusion of NTG and that NTG is metabolized to NO to a similar degree in nitrate-nontolerant and nitrate-tolerant tissues. We conclude that in vivo nitrate tolerance seems not to be associated with a reduced bioconversion of NTG to NO and that the endothelium of nitrate-tolerant rats possesses an inhibitory effect on the bioactivity of NTG-derived NO.

	Selected Abbreviations and Acronyms

 

DETC = diethyldithiocarbamate ESR = electron spin resonance L-NAME = NG-nitro-L-arginine methyl ester MAP = mean arterial blood pressure NO = nitric oxide NTG = nitroglycerin O2- = superoxide anion

	Acknowledgments

 
This study was supported by grants from the Danish Heart Foundation, the NOVO Foundation, the Christa and Viggo Peterson Foundation, and the Land Hessen (grant ATG 91) to Dr Mulsch.

Received February 15, 1996; revision received May 15, 1996; accepted May 15, 1996.

	References

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