cDNA Microarray analysis of vascular gene expression after nitric oxide donor infusion in rats: Implications for nitrate tolerance mechanisms

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 μg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-μg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P<.001, analysis of variance [ANOVA]), but not after SNAP (P>.05, ANOVA) or control infusion (P> .05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the downregulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.     

Prazosin potentiates the acute hypotensive effects of nitroglycerin but does not attenuate nitrate tolerance in normal conscious rats

Sympathetic activation has been suggested as a mechanism of acute nitrate tolerance, but the available literature is not definitive. We investigated the effects of prazosin, an alpha1-adrenoceptor antagonist, on acute nitroglycerin (NTG) hemodynamics and tolerance development in normal conscious rats. The effect of prazosin bolus injection (300 microg/kg) on NTG hemodynamics was first determined after acute dosing. The extent of maximal mean arterial pressure (MAP) response and the duration of drug-induced hypotension to NTG bolus doses (5, 15, and 30 microg) were measured before and after prazosin. In separate studies, the effects of prazosin on NTG tolerance development were examined. Rats received either 10 microg/min NTG or vehicle infusion for 5 hours after predosing with prazosin (300 microg/kg). Maximal MAP response to the hourly 30-microg NTG i.v. bolus challenge dose (CD) was determined before and after prazosin, and during NTG or vehicle infusion. Our results showed that bolus doses of NTG (at 5, 15, and 30 microg) dose-dependently decreased maximal MAP by 20.8 +/- 5.8, 26.1 +/- 5.0, and 30.6 +/- 5.7 mm Hg, respectively. Prazosin caused an average of 16 mm Hg depression in MAP, and it only slightly potentiated the hypotensive effects of bolus doses of NTG both after acute dosing and during continuous infusion. Prazosin treatment prolonged the duration of NTG-induced MAP response by about 4-fold for all NTG doses examined (P < 0.01 versus corresponding dose before prazosin, ANOVA). In both prazosin-treated and untreated groups, NTG infusion significantly attenuated the MAP response of the NTG CD starting from 1 hour of infusion (P < 0.001 versus 0 hour response, ANOVA), confirming tolerance development. In the presence of NTG tolerance development, prazosin no longer enhanced the apparent duration of NTG action. The hypotensive effect produced by the 30-microg NTG CD lasted for 7 +/- 2 and 10 +/- 2 seconds for prazosin-treated and untreated groups, respectively (P > 0.05, ANOVA). Our results showed that, in both NTG-tolerant and control animals, prazosin only slightly potentiated the maximum hypotensive effects of a challenge NTG dose, but did not significantly alter the pharmacodynamics of NTG-induced hemodynamic tolerance. Thus, in our animal model, sympathetic blockade by prazosin neither prevented nor attenuated in vivo tolerance induced by NTG.     

Pharmacodynamics of in Vivo Nitroglycerin Tolerance in Normal Conscious Rats: Effects of Dose and Dosing Protocol

PURPOSE: We examined the effects of dose and dosing protocol on the pharmacodynamics of in vivo nitroglycerin (NTG) tolerance in conscious rats. Mechanism-based pharmcokinetic/pharmacodynamic (PK/PD) models were tested for their ability to describe the observed data. METHODS: Rats were infused with 1, 3, or 10 microg/min of NTG or vehicle for 10 h. Peak mean arterial pressure (MAP) response to an hourly 30 microg i.v. NTG challenge dose (CD) was measured before, during, and at 12 and 24 h after infusion. In separate experiments, the MAP effects of repeated bolus doses of NTG were compared to those after a continuous infusion, both at a total dose of 510 microg NTG. RESULTS: NTG tolerance was indicated by a decrease in peak MAP response to the CD, relative to the preinfusion peak MAP response. Tolerance toward the MAP effects of bolus CD was observed within 1 h of 10 microg/min of NTG infusion (26.8 +/- 2.8% vs. 10.6 +/- 0.4% for 0 and 1 h, respectively, p < 0.001), and the rate and extent of tolerance development increased with the infusion dose. No apparent MAP tolerance was observed when NTG was given as multiple bolus doses whereas significant MAP tolerance was observed when this dose was infused continuously. PK/PD modeling indicated that a cofactor/enzyme depletion mechanism could adequately describe the composite data. CONCLUSIONS: Our data showed that in vivo nitrate tolerance was dose- and dosing protocol-dependent. The pharmacodynamics of tolerance development are consistent with depletion of either critical enzymes or cofactors that are necessary to induce vasodilation.     

Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (-/-)) and wild-type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg(-1)s.c. 3 x daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (-/-) and (+/+) mice, the EC(50) from NTG concentration-response curve was increased by approximately 3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (-/-) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.     

Effects of obesity on the pharmacodynamics of nitroglycerin in conscious rats

Literature reports have suggested that hemodynamic response toward organic nitrates may be reduced in obese patients, but this effect has not been studied. We compared the mean arterial pressure (MAP) responses toward single doses of nitroglycerin (NTG), 0.5–50μg) in conscious Zucker obese (ZOB), Zucker lean (ZL), and Sprague-Dawley (SD) rats. NTG tolerance development in these animal groups was separately examined. Rats received 1 and 10μg/min of NTG or vehicle infusion, and the maximal MAP response to an hourly 30μg NTG IVchallenge dose (CD) was measured. Steady-state NTG plasma concentrations were measured during 10μg/min NTG infusion. The E_max and ED₅₀ values obtained were 33.9± 3.6 and 3.5±1.7μg for SD rats, 33.2±4.1 and 3.0±1.4μg for ZL rats, and 34.8±3.9 and 5.3±2.8μg for ZOB rats, respectively. No difference was found in the dose-response curves among these 3 groups (p>.05, 2-way ANOVA). Neither the dynamics of NTG tolerance development, nor the steady-state NTG plasma concentrations, were found to differ among these 3 animal groups. These results showed that ZOB rats are not more resistant to the hemodynamic effects of organic nitrates compared with their lean controls. Thus, the acute and chronic hemodynamic effects by the presence of obesity in a conscious animal model of genetic obesity.     

Nitroglycerin-induced relaxation of anorectal smooth muscle: evidence for apparent lack of tolerance development in the anaesthetized rat

1. Recent studies indicate that nitroglycerin (NTG) can produce beneficial clinical effects in healing anal fissures through the relaxation of the internal anal sphincter. The in vivo relaxation effects of NTG on the anorectal smooth muscle have not been studied and it is not known whether this tissue may also exhibit pharmacological tolerance toward NTG. 2. We have developed an in vivo procedure in the anaesthetized rat that permits continual monitoring of anorectal pressure after intravenous (i.v.) and intra-rectal application of NTG. The relaxant effects of NTG were quantified via the area-under-the-contraction-waveforms vs time curve (AUEC). 3. AUEC decreased significantly after intra-rectal bolus doses of NTG (5 - 25 microg), in a dose- and time-dependent manner. Sustained relaxation effects on anorectal pressure were also observed after continuous intra-rectal infusions of NTG. 4. Two-hours of i.v. NTG infusion led to a significant reduction in the mean arterial blood pressure (MAP) response toward a i.v. NTG (30 microg) bolus challenge. In contrast, relaxation of the anorectal pressure toward the challenge dose was not altered after NTG infusion. 5. In isolated tissues, cyclic GMP accumulation was significantly decreased after NTG pre-incubation in the rat aorta but not in the rat anorectal smooth muscle and anal sphincter. 6. These results indicate that the relaxation response toward NTG was not diminished in the anorectum under conditions that produced vascular tolerance. Thus, NTG causes significant and sustained in vivo relaxation of anorectal smooth muscle in the anaesthetized rat without evidence of tolerance development.     

Prevention of vascular nitroglycerin tolerance by inhibition of protein kinase C.

We investigated whether in vivo inhibition of protein kinase C (PKC) can prevent the development of vascular tolerance and restore the sensitivity of isolated vessels to nitroglycerin (NTG). Tolerance was induced in male Wistar rats by a constant i.v. infusion of NTG 1 mg kg-1 h-1, a dose which did not alter blood pressure. After 72 h, the aorta was removed and the sensitivity of aortic rings to NTG tested. Chronic NTG infusion resulted in a 5.5 fold decrease in NTG-sensitivity as compared with controls (vehicle), indicating the development of vascular tolerance. The simultaneous in vivo administration of the specific PKC inhibitor N-benzoyl-staurosporine (30 mg kg-1 day-1) prevented this decrease in NTG sensitivity. These results suggest a role for PKC activation in the development of vascular NTG tolerance.     

Baroreflex resetting but no vascular tolerance in response to transdermal glyceryl trinitrate in conscious rabbits.

1. We investigated whether acute (5 h) and chronic (3 days) transdermal glyceryl trinitrate (GTN) patches could cause the development of tolerance in terms of haemodynamics and vascular reactivity in the conscious rabbit. The effects of haemodynamic tolerance were assessed on arterial pressure, heart rate and the baroreflex control of heart rate, while hindquarter vascular reactivity in response to dilator and constrictor drugs and reactive hyperaemia were used to assess vascular tolerance. 2. Seven days prior to experiments, an inflatable cuff, a pulsed Doppler flow probe and an indwelling intra-aortic catheter (for i.a. agonist infusions) were implanted around the lower abdominal aorta. 3. In acute experiments, the effects of 0-5 h treatment with transdermal GTN (0 Sham), 10 or 20 mg 24 h-1) on MAP, HR and the baroreflex were examined. Chronic experiments were performed on three separated days (days 0 - before, 4 - with GTN patch and 8 - recovery). On each day, the baroreflex, reactive hyperaemic responses and hindquarter vascular dose-response curves to i.a. GTN, adenosine, acetylcholine, S-nitroso-N-acetylpenicillamine (SNAP) and methoxamine were assessed. On days 1-4, GTN was administered transdermally via a patch(es) (10 mg 24 h-1 (low dose) or 20 mg 24 h-1 (high dose); renewed every 24 h). 4. Acute treatment with 20 mg GTN 24 h-1, but not with 0 (n = 4) or 10 mg GTN 24 h-1 (n = 4), caused a significant fall in MAP (8 +/- 1 mmHg; n = 4) and resetting of the baroreflex by 5 h. Chronic GTN caused a significant fall in MAP of 8 +/- 2 and 8 +/- 2 mmHg on day 4 with low (n = 8) and high dose (n = 8), respectively, with no change in HR. There was no significant change to hindquarter vascular reactivity to i.a. infusion of GTN, nor were there any significant differences in the reactivity to i.a. adenosine, acetylcholine, SNAP or methoxamine with either low or high doses of GTN. 5. Chronic GTN treatment with low and high dose patches caused a parallel leftward shift ('resetting') of the baroreflex on day 4. By day 8, the baroreflex had still not recovered from this leftward shift 6. In the rabbit, chronic exposure to clinical nitrate patches caused haemodynamic compensation and baroreflex resetting but no evidence of vascular reactivity tolerance. Novel NO donor drugs and delivery regimens which provide intermittent dosing may prevent the development of haemodynamic resetting rather then preventing vascular tolerance, a commonly perceived difficulty in chronic nitrate therapy.     

Preservation of platelet responsiveness to nitroglycerine despite development of vascular nitrate tolerance

AIMS: Organic nitrates, via nitric oxide (NO) release, induce vasodilatation and inhibit platelet aggregation. Development of nitrate tolerance in some vascular preparations may be associated with diminished responsiveness to NO. To date it is not known to what extent vascular tolerance to organic nitrates is associated with acquired platelet hypo-responsiveness to NO. In the current study we compared the acute and chronic effects of sustained release (SR) isosorbide 5' mono-nitrate (ISMN) and transdermal nitroglycerine (TD-NTG) on blood vessels (effects on apparent arterial stiffness) and platelets (effects on responsiveness to NO donors) in patients with stable angina pectoris (SAP). METHODS: Patients (n = 34) with SAP entered a blinded randomized crossover study of ISMN (120 mg) vs. intermittent TD-NTG (15 mg 24 h(-1)). Effects of each nitrate on pulse wave reflection (augmentation index (AIx)), platelet response to adenosine di-phosphate (ADP 1 micromol l(-1)), nitroglycerine (NTG 100 micromol l(-1)) and the non-nitrate NO donor sodium nitroprusside (SNP 10 micromol l(-1)), were measured pre-dose, 4 and 8 h post dose, on three occasions: 1) at the end of a pre-nitrate phase, 2) after dosing for 7 days and 3) following 14 days of full dose therapy with either nitrate. RESULTS: Acutely, both ISMN and TD-NTG markedly reduced AIx. After 14 days, these effects were significantly attenuated (ANOVA, P = 0.018) but not abolished, indicating development of nitrate tolerance. Neither nitrate preparation affected ADP (1 micromol l(-1))-induced platelet aggregation. Platelet responsiveness to NTG (100 micromol l(-1)) and SNP (10 micromol l(-1)) was not diminished during chronic nitrate therapy, and there was no evidence of 'rebound' hyper-aggregability during 'nitrate-free' periods. CONCLUSIONS: Chronic therapy with either ISMN or TD-NTG is associated with development of vascular tolerance. Despite the induction of vascular tolerance, platelet responsiveness to NTG and SNP remains unaffected. Therefore, development of vascular tolerance is unlikely to compromise the anti-aggregatory effects of organic nitrates, or those of endogenous NO.     

Comparison of development of tolerance between nicorandil and nitroglycerin in anesthetized, open-chest dogs

Development of tolerance to nicorandil (NCR), N-(2-hydroxyethyl) nicotinamide nitrate (ester), was compared with that to nitroglycerin (NTG) in dogs. An intra-coronary arterial (i.a.) injection of NCR (30 micrograms) or NTG (3 micrograms) produced coronary vasodilation. Development of tolerance (including cross tolerance) was determined by examining whether the coronary vasodilating effect of i.a. injection of these drugs was attenuated by a 2 hr-infusion of NCR or NTG. The effect of i.a. injection of NCR was not affected by either NCR infusion (10 micrograms/kg/min, i.v.) or NTG infusion (1 or 3 micrograms/kg/min, i.v.). The effect of i.a. injection of NTG, however, was attenuated by the NTG infusion, while it was not affected by the NCR infusion. Additionally, the coronary vasodilating effect of NCR infusion (30 micrograms/kg/min, i.v.) was not attenuated by NTG infusion (3 micrograms/kg/min, i.v.). These results suggest that NCR does not produce tolerance, whereas NTG does, and that there is no cross-tolerance between NCR and NTG in terms of the coronary vasodilating effect.     

Evidences for prevention of nitroglycerin tolerance by carvedilol.

Carvedilol, a beta-blocker has shown clinically to attenuate the development of nitroglycerin (NTG) tolerance. The present study was designed to investigate the possible mechanisms whereby carvedilol could prevent NTG tolerance, particularly at the level of vascular superoxide anion (O2-) production (an important factor in nitrate tolerance) as well as modulation of certain aortic antioxidants. Rabbits were treated with NTG patch (1.5 microg/kg/min) and/or cavedilol (10 mg/kg/day) for 3 days. Relaxation of aortic segments was studied in organ chamber and rate of vascular O2- production was determined. In addition, aortic glutathione (GSH) level and superoxide dismutase (SOD) activity was also assessed. Aortic segments from NTG-treated rabbits showed a significant decrease in maximal relaxation in response to various vasodilators. Also, NTG treatment increased vascular O2- production by two-fold as compared with untreated control group. The potential source of O2- production was found to be the adventitia. In addition, treatment of rabbits with NTG induced a significant decrease in total GSH level and SOD activity by 46 and 53%, respectively, as compared with the control values. Concomitant treatment of NTG with carvedilol significantly prevented the development of NTG tolerance and normalized the rate of vascular O2- production. Moreover, carvedilol restored the normal level of aortic antioxidants mainly, total GSH and SOD.     

Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin

We investigated the involvement of calcitonin gene-related peptide (CGRP) in the vasodilatory mechanism of action of nitric oxide (NO) donors. The functional role of CGRP in NO donor-induced vasodilation of isolated rat aortic rings was determined by incubating these drugs with and without CGRP(8-37), a selective CGRP receptor antagonist. CGRP(8-37) (0.63 microM) induced rightward shifts in the vasodilatory concentration-response curves for nitroglycerin (NTG), Piloty's acid (PA), and SIN-1 (linsidomine). The EC(50) values for NTG, PA, and SIN-1 were increased by 8.3-, 5.2-, and 2.3-fold, respectively (P < 0.05). The release of CGRP from rat aorta in response to NTG and PA was measured specifically by radioimmunoassay. Thirty-minute incubations of NTG or PA with rat aorta induced 189.5 and 214.6% increases, respectively, in CGRP release when compared with the control (P < 0.05). The concentration-response curves of sodium nitroprusside (SNP), S-nitroso-acetylpenicillamine (SNAP), tetranitromethane (TNM), diethylamine NO complex (DEA-NO), and diethylenetriamine/nitric oxide adduct (DETA NONOate) were not inhibited significantly by CGRP(8-37) co-incubation (P 0.05). NO donors also were incubated with aortic strips, and NTG and PA alone induced significant formation of hydroxylamine, a NO(-) metabolite (232.4 and 364.9%, respectively, P < 0.05). These results indicate that only NTG and PA, and to a lesser extent SIN-1, stimulate the release of CGRP from the rat aorta, which subsequently contributes to the vasodilatory activity of these agents. The hydroxylamine formation suggests a possible link between NO(-) generation and CGRP release from the vascular wall.     

Effect of dosage regimen on the development of tolerance to nitroglycerin in rats

The recent introduction of several sustained delivery systems of nitroglycerin (NTG) raises the question whether the mode of drug input (e.g., sustained versus intermittent) may be a critical determinant in the development of nitrate tolerance. This hypothesis was tested in an animal model. Sixty male Sprague-Dawley rats (weighing 240-260 g) were administered a total intravenous dose of 2.5 mg NTG either as a continuous 6-h infusion (6.8 micrograms/min) or as six hourly pulse injections of 425 micrograms each. Animals were sacrificed 5 min following the termination of the infusion and 65 min following the last injection. A blood sample was taken from a central vein for plasma NTG determination, and the aorta and portal vein were isolated. Dose-response curves to NTG were determined on some of these blood vessels and on controls using an isolated tissue bath apparatus. Other blood vessels were incubated with 94 ng [14C]NTG for 60 min, and the incorporation of [14C]NTG into these tissues was determined after thin-layer chromatographic separation of NTG from its metabolites. There was no difference between the plasma NTG concentration measured at the time of sacrifice following either regimen. For the artery preparation, there was also no difference in the dose response to NTG or in the incorporation of [14C]NTG into the blood vessel from rats treated by either regimen. For the portal vein preparation, however, rats treated by continuous infusion had a similar sensitivity to NTG as controls, but there was a marked downward shift in the dose response to NTG in the veins of rats treated by the intermittent regimen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cGMP-dependent protein kinase in development of tolerance to nitroglycerine in porcine coronary arteries

BACKGROUND AND PURPOSE: The cGMP-dependent protein kinase (PKG) is a key enzyme for nitrovasodilator-induced vasodilation. The present study was to determine its role in nitrate tolerance. EXPERIMENTAL APPROACH: isolated porcine coronary arteries were incubated for 24 h with nitroglycerin (NTG) and their relaxant responses were determined. PKG activity was assayed by measuring the incorporation of (32)P into BPDEtide. PKG protein was determined by Western blotting and PKG mRNA by real-time PCR. KEY RESULTS: A 24 h incubation with NTG attenuated relaxation of coronary arteries to NTG, which was associated with decreased PKG activity. The nitrate tolerance induced with NTG at 10(-7) M was affected by a scavenger of reactive oxygen species and the tolerance induced with NTG at 10(-6) and 10(-5) M showed cross-tolerance to DETA NONOate and 8-Br-cGMP (a cell permeable cGMP analogue). PKG protein and mRNA were down-regulated by a 24 h incubation with NTG at 10(-5) M but not at 10(-7) M. Acute exposure to exogenous superoxide inhibited PKG activity stimulated by NTG at 10(-7) M but not at 10(-5) M. Superoxide had no effect on PKG activity stimulated with exogenous cGMP. CONCLUSIONS AND IMPLICATIONS: Nitrate tolerance induced by NTG at low concentrations may result from an increased production of reactive oxygen species acting on sites upstream of PKG. The tolerance induced by NTG at higher concentrations may be in part due to suppression of PKG expression resulting from sustained activation of the enzyme. These distinct mechanisms of nitrate tolerance may be of clinical significance.     

Rosuvastatin treatment protects against nitrate-induced oxidative stress

Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2 production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (-log NTG concentration evoking a half-maximal relaxation) was 6.75+/-0.06 (n=7) versus 7.75+/-0.07 (n=7) in group 3 (not exposed to NTG, P<0.05); O2 production was enhanced (10,060+/-1,205, n=7 versus 5,235+/-1,052, n=7; P<0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20+/-0.10 (n=8), P<0.05 versus group 2; O2 production was decreased (5911+/-663; n=9, P<0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2 production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.     

Recovery of vascular smooth muscle relaxation from nitroglycerin-induced tolerance following a drug-free interval. A time-course in vitro study.

Hemodynamic tolerance occurs upon continuous exposure of vascular tissues to nitroglycerin (NTG). This phenomenon is believed to be due to the depletion of the tissue sulfhydryl (SH) group, which is essential for NTG-induced increase in tissue cyclic GMP and vasorelaxation. To determine the effect of an NTG-free interval on recovery of tissue cyclic GMP accumulation and vasorelaxation following development of NTG tolerance, isolated rat aortic rings were kept in Krebs physiologic buffer at 37 degrees, precontracted with epinephrine, and exposed to NTG. The mean concentration of NTG, which relaxed the rings by 50% (EC50) upon first exposure, was 1.1 x 10(-7) M (N = 20), and vascular cyclic GMP levels after NTG increased from 21 to 46 fmol/mg (P less than 0.02). A second exposure to NTG 15 min later increased the EC50 to 1.3 x 10(-4) M and cyclic GMP levels did not change (P less than 0.001 vs first NTG exposure), indicating tolerance to NTG. However, acetylcholine-mediated relaxation of aortic rings was preserved even in NTG-tolerant rings. A second exposure of tissues to NTG separated by 30, 60, and 120 min from the first exposure progressively decreased the EC50, such that at 120 min the EC50 of NTG was 0.4 x 10(-7) M (P = NS vs first NTG exposure). Tissue cyclic GMP levels increased from 14 to 71 fmol/mg (P = NS vs first NTG exposure). These data confirm development of tolerance to the vasorelaxant effects of NTG following initial exposure. An interval of 2 hr between multiple exposures of tissues to NTG results in preservation of the smooth muscle relaxation and an increase in tissue cyclic GMP in response to NTG.     

Biochemical, hemodynamic, and vascular evidence concerning the free radical hypothesis of nitrate tolerance

Tolerance to nitroglycerin (NTG) may be due to increased superoxide anion production. Hemodynamic parameters and biochemical markers of free radical production were measured in 20 healthy male subjects at baseline, 3 h after acute transdermal NTG (0.6 mg/h), and after 5 days of continuous therapy. Transdermal NTG therapy was continued, and 2 days later all subjects received 2 g of oral vitamin C, or placebo, in a double-blind, randomized, crossover fashion. In another study of eight male subjects, forearm plethysmography was used to assess the venous responses to sublingual NTG at baseline, after 5 days of sustained transdermal NTG therapy (0.6 mg/h), and after 2 g of oral vitamin C or placebo. Systolic blood pressure decreased in response to acute transdermal NTG therapy but returned to normal after sustained NTG therapy, indicating the development of tolerance. The venous volume responses to sublingual NTG were significantly diminished after sustained therapy with transdermal NTG. Plasma lipid peroxidation products, 8-iso-PGF2 alpha, and vitamin C were unchanged by acute and sustained therapy with transdermal NTG. Vitamin C failed to restore either the hemodynamic or venous effects of NTG. These results do not support the hypothesis that nitrate therapy and tolerance is associated with increased free radical production.     

Nitrate tolerance induced by nicorandil or nitroglycerin is associated with minimal loss of nicorandil vasodilator activity.

In the current study, the vasodilator and tolerance-inducing actions of a recently developed organic nitrate vasodilator, nicorandil, were compared to nitroglycerin (NTG) in an isolated coronary artery preparation. The order of potency for relaxing U46619-constricted bovine-isolated coronary artery rings was NTG greater than isosorbide dinitrate (ISDN) greater than nicorandil. NTG was approximately 250-fold more potent than nicorandil (mean EC50 values for relaxation; 0.044 and 11.2 microM, respectively; n = 6-8). Coronary artery rings preexposed for 60 min to NTG (30 microM) were subsequently markedly less responsive to the relaxant effects of NTG (7.5-fold increase in mean EC50 value, 68.4% decrease in Emax; p less than 0.001) and ISDN (14.1-fold increase in mean EC50 value; p less than 0.001), although only marginally less responsive to nicorandil (1.75-fold increase in mean EC50 value; p less than 0.05). Thus, the coronary artery relaxant actions of nicorandil were significantly less affected by NTG-induced tolerance than were the relaxant actions of the related organic nitrate compounds, NTG and ISDN. To compare the tolerance-inducing actions of NTG and nicorandil, the relaxant actions of a series of nitric oxide (NO)-containing vasodilators were determined in control coronary artery rings and in rings preexposed for 60 min to either 30 microM NTG or 5,000 microM nicorandil. Quantitatively, similar changes in coronary artery ring responsiveness were produced by tolerance induced by NTG and nicorandil; marked attenuation of responsiveness to NTG and to the nonnitrate compound 3-morpholinosydnonimine (SIN-1), but only marginal attenuation of responsiveness to nicorandil and NO.(ABSTRACT TRUNCATED AT 250 WORDS)