Determinants of in vitro nitroglycerin tolerance induction and reversal: influence of dose regimen, nitrate-free period, and sulfhydryl supplementation

The influence of dose regimen on the induction and reversal of tolerance to nitroglycerin (NTG) is not well understood despite the current widespread clinical use of both sustained and intermittent modes of NTG administration. In an isolated coronary artery preparation both the NTG preexposure concentration and the duration of the NTG preexposure period were positive and independent determinants of the extent of NTG tolerance induction. During a "nitrate-free" or washout period, NTG tolerance was at least partially reversible. The apparent rate of NTG tolerance reversal during a "nitrate-free" period was not dependent on the absolute degree of NTG tolerance induced or on the dose regimen used to induce NTG tolerance. In this isolated vascular preparation, sulfhydryl (SH) supplementation with 1 mM N-acetylcysteine produced no significant augmentation of NTG-induced relaxations in either NTG tolerant or non tolerant tissues. N-acetylcysteine was ineffectual in attenuating the development of NTG tolerance in coronary artery preparations incubated in either Krebs bicarbonate buffer or in 10% human plasma. We conclude that in this model the NTG preexposure concentration, the duration of the NTG preexposure period, and the duration of the "nitrate-free" period are critical and independent determinants of the extent of NTG tolerance but that NTG tolerance is not significantly attenuated by SH supplementation.     

The role of lipoic acid in prevention of nitroglycerin tolerance

Besides other organic nitrates, nitroglycerin (glyceryl trinitrate; GTN) has been used to treat acute heart failure particularly due to ischemic heart disease. However, one of serious clinical problems of the GTN therapy, particularly a long-standing medication, is hemodynamic tolerance to GTN, manifested by the decreased therapeutic efficacy of the drug. The most recent studies have suggested that mitochondrial lipoate/dihydrolipoate system-dependent aldehyde dehydrogenase-2 plays a key role in nitric oxide release from GTN. The aldehyde dehydrogenase-2 performs three enzymatic activities of dehydrogenase, esterase and reductase. The reductase activity is responsible for bioactivation of organic nitrates, such as GTN yielding nitrite and dinitrate (1,2-GDN/1,3-GDN, approximately 8:1). In view of a large contribution of dihydrolipoic acid to stabilization and regeneration of thiol groups, necessary for the reductase activity of aldehyde dehydrogenase-2, we conducted studies aimed to determine whether lipoic acid administration to rats is able to prevent GTN tolerance. The studies were conducted on 4 groups of animals: control saline-treated, model GTN-tolerant, GTN + lipoic acid-treated, lipoic acid alone-administered groups. On the 9th day of experiment animals were given i.v. therapeutic dose of GTN. We measured in all animals systolic and diastolic blood pressure before injection of therapeutic dose of GTN into the cadual vein and during 20 min thereafter. Levels of nitric oxide and reactive oxygen species and activities of glutathione peroxidase and superoxide dismutase were assayed in the aorta, plasma and heart of all animals. In addition, levels of malondialdehyde, and non-protein thiols, and activities of glutathione S-transferase and gamma-glutamyl transpeptidase were evaluated in the heart and plasma. The obtained results indicate that treatment of rats with a combination of lipoic acid and GTN can efficiently counteract GTN tolerance.     

Decrease in endogenous CGRP release in nitroglycerin tolerance: role of ALDH-2

In the present study, we tested whether the decreased release of calcitonin gene-related peptide (CGRP) observed in nitroglycerin tolerance is associated with the decrease in aldehyde dehydrogenase (ALDH-2) activity. We further investigated the possible involvement of reactive oxygen species in the decrease in ALDH-2 activity. Tolerance was induced by exposure of isolated rat thoracic aortas and human umbical vein endothelial cells (HUVEC) to nitroglycerin in vitro or by pretreatment with nitroglycerin for 8 days in vivo. Pretreatment with ALDH-2 inhibitors and nitroglycerin significantly attenuated vasodilator responses to nitroglycerin concomitantly with a decrease in the release of CGRP from the isolated thoracic aorta. Nitroglycerin produced a depressor effect concomitantly with an increase in plasma concentrations of CGRP, and the effect of nitroglycerin was attenuated after pretreatment with an inhibitor of ALDH-2 or nitroglycerin for 8 days. Exposure of HUVEC to nitroglycerin for 16 h increased reactive oxygen species production and decreased ALDH-2 activity as well as cGMP production in a time-and concentration-dependent manner. Pretreatment with an ALDH-2 inhibitor also significantly decreased the cGMP production. However, tolerance to nitroglycerin in HUVEC was restored in the presence of N-acetylcysteine or captopril. The present results suggest that nitrate tolerance is, at least partially, associated with a decrease in endogenous CGRP release via a decrease in ALDH-2 activity as a result of stimulation of reactive oxygen species production.     

New insights into nitroglycerin effects and tolerance: role of calcitonin gene-related peptide

It has been shown that calcitonin gene-relate peptide plays an extensive role in cardiovascular system. CGRP is a potent vasodilator and plays an important role in mediation of nitroglycerin-induced vascular relaxation. Recently, calcitonin gene-relate peptide is emerging as a potential player in nitroglycerin tolerance. There is increasing evidence that the decreased depressor effect of nitroglycerin in tolerant states is closely related to a decrease in calcitonin gene-relate peptide release. The reduced release of calcitonin gene-relate peptide in nitroglycerin tolerance is associated with the decreased nitroglycerin biotransformation due to the mitochondrial dysfunction. Recent work has been shown that the inhibited activity of mitochondrial isoform of aldehyde dehydrogenase and the upregulation of phosphodiesterase 1A1 are the key factors that lead to the decreased nitroglycerin biotransformation in nitroglycerin tolerance, with a subsequently reduced release of calcitonin gene-relate peptide.     

Soluble guanylate cyclase activation by nitric oxide and its reversal. Involvement of sulfhydryl group oxidation and reduction

Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.     

Biochemical and pharmacological interactions between nitroglycerin and thiols. Effects of thiol structure on nitric oxide generation and tolerance reversal

Co-administration of N-acetylcysteine (NAC) with nitroglycerin (NTG) has been shown to partially reverse nitrate tolerance and to potentiate the hypotensive effect of NTG in humans. However, a high clinical dose of NAC was required for this pharmacologic interaction resulting in the production of unwanted side-effects. Therefore, sulfhydryl compounds more active than NAC need to be identified if this interaction is to be exploited clinically. We previously suggested that the effect of sulfhydryl compounds on NTG may be mediated by the formation of S-nitrosothiol or nitric oxide (NO) extracellularly to the vascular smooth muscle cell (e.g. in plasma) (Fung et al., J. Pharmacol Exp Ther 245: 524-530, 1988). In an attempt to understand the structural features which govern this thiol-catalyzed NO generation from NTG, nineteen different aliphatic and ten aromatic sulfhydryl compounds were examined with respect to their catalytic activity to generate NO from NTG in plasma. Significantly enhanced production of NO was observed with most sulfhydryl compounds examined when compared to buffer control. Among the aliphatic thiols, only mercaptosuccinic acid was more potent than NAC (2x), whereas among the aromatic thiols, both thiosalicylic acid (TSA, 10x) and TSA-methyl ester (3x) were more potent than NAC. Comparative in vitro relaxation studies were carried out using isolated (and nitrate-tolerant) rat aortic rings with NTG/TSA and NTG/NAC, in the presence of 0.5% (v/v) plasma. Under these conditions, partial reversal of NTG tolerance could be achieved with TSA, but not with NAC. These data are consistent with the view that extracellular production of NO or S-nitrosothiol serves as a tolerance-reversing mechanism of thiols on NTG. TSA appears to be a more potent sulfhydryl compound than NAC in this biochemical and pharmacologic interaction.     

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.     

血红素氧合酶-1在减轻硝酸甘油耐受中的作用研究

目的 探讨血红素氧合酶-1(HO-1)在减轻硝酸甘油耐受中的作用.方法 建立人脐静脉内皮细胞(HUVECs)的硝酸甘油耐受模型;采用HO-1诱导剂Hemin和抑制剂锌原卟啉(ZnPP )分别进行干预处理后,利用 RT-PCR以及Western blot检测HO-1的表达情况.分别采用硝酸还原酶法和荧光分光光度法检测药物干预后各组细胞NO及活性氧ROS的表达水平.结果 与对照组相比,Hemin诱导组细胞增殖水平增高(P<0.05),并且HO-1蛋白表达上调可以增加NO的含量(P<0.01),促进硝酸甘油的生物转化,该过程伴随着ROS的减少(P<0.01).结论 通过Hemin诱导HO-1过表达可以一定程度上减轻硝酸甘油的耐受,该过程与氧化应激水平的降低有关.     

Reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril: a role of calcitonin gene-related peptide

Previous studies have shown that the development of tolerance to nitroglycerin is related to a decrease in the release of endogenous calcitonin gene-related peptide (CGRP). In the present study, we explored whether endogenous CGRP is involved in reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril in rats in vivo and vitro. Tolerance was induced by exposure to nitroglycerin (4.4 x 10(-6) M) for 10 min in vitro or by pretreatment with nitroglycerin (10 mg/kg, s.c.) three times a day for 8 days in vivo. Nitroglycerin (3 x 10(-9)-10(-6) M) caused a concentration-dependent relaxation in the isolated rat thoracic aorta, an effect that was reduced by CGRP-(8-37) (3 x 10(-7) M) or capsaicin (3 x 10(-7) M). Preincubation with nitroglycerin for 10 min significantly decreased its vasodilation, which was restored in the presence of N-acetylcysteine (10(-5) M) or captopril (10(-5) M). Nitroglycerin (150 microg/kg, i.v.) produced a depressor effect and an increase in concentrations of nitric oxide and CGRP, and the effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, tolerance to nitroglycerin in vivo also was partially restored in the presence of N-acetylcysteine or captopril. The present results suggest that reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril is related to the increased release of CGRP in the rat.     

硝酸甘油耐受性机制探讨及卡维地洛的预防作用

目的　研究硝酸甘油 (NTG)连续应用耐受性的形成机制及卡维地洛对 NTG耐受性的预防作用。方法　大鼠 NTG 10 mg· kg- 1 皮下注射 ,每日 3次连用 6日引致耐受。测定耐受性形成前、后舌静脉注射 NTG对鼠尾容积的作用 ,作为硝酸甘油效应及耐受的指标 ,同时检测耐受性形成后有关组织的氧自由基 (· O2 - )、亚硝酸盐(NO2 - )、总硝酸盐 (NOX- )、超氧化物歧化酶 (SOD)活力和丙二醛 (MDA)。结果　大鼠连用 NTG后 ,再静注 NTG对鼠尾容积的增加作用明显减弱 ;主动脉和心脏组织及血浆中· O2- 和 NOX- 都明显升高 ,除血浆 NO2- 水平升高外其余组织 NO2 - 水平都明显降低 ;主动脉、肝脏和红细胞中的 MDA含量增高 ,SOD活力降低 ;卡维地洛能有效预防NTG连续应用耐受性的形成 ,完全或部分防止上述检测指标的变化。结论　 NTG连续应用期间耐受性的形成与· O2- 水平升高引起一氧化氮 (NO)氧化灭活有关 ;卡维地洛通过抗氧化、清除· O2- 等作用而有效预防或减轻 NTG连用过程中耐受性的形成。     

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)     

Loss of preconditioning in rabbits with vascular tolerance to nitroglycerin.

A preceding right ventricular overdrive pacing (VOP) of 500 b.p.m. for 5 min, markedly reduced the severity of global myocardial ischaemia produced by a subsequent 5-min VOP in conscious rabbits. This VOP-induced preconditioning developed in parallel with an increase in cardiac cyclic guanosine 3':5'-monophosphate (cyclic GMP) content. VOP-induced preconditioning was abolished when the animals had been made tolerant to the vasodilator effect of nitroglycerin (NG). In the heart of the NG-tolerant rabbits, neither VOP nor preconditioning increased cyclic GMP content. This suggests that changes by NG tolerance of cyclic GMP metabolism may account for the loss of VOP-induced preconditioning.     

Converting enzyme inhibitors and the role of the sulfhydryl group in the potentiation of exo- and endogenous nitrovasodilators.

In this study, the effect of bradykinin on coronary flow in the isolated rat heart was significantly potentiated when cysteine or the sulfhydryl-containing converting enzyme inhibitors captopril and zofenoprilat were administered simultaneously. In contrast, the effect of concomitant administration of enalaprilat only slightly increased the effect of bradykinin on coronary flow. In nitrate-tolerant hearts of rats pretreated with isosorbide dinitrate (15 mg daily), the increase in coronary flow by nitroglycerin and bradykinin was significantly less when compared to control hearts. The effect of captopril was not affected by pretreatment. The involvement of endothelium-derived relaxing factor (EDRF) in the effect of captopril was apparent from experiments with L-arginine, the precursor of EDRF, and L-NMMA, the "false" precursor of EDRF. L-Arginine increased the effect of captopril, whereas L-NMMA showed a competitive antagonism for the effect of captopril on coronary flow in the isolated rat heart. Clinically, the effect of captopril was studied in 10 patients with stable, exercise-induced angina pectoris that had been treated for 3 weeks with slow-release isosorbide dinitrate (20 mg four times daily). At day 7, a baseline exercise test was obtained. Subsequently, patients with chest pain and at least 1-mm ST-segment depression on the ECG during exercise were included. They received on day 14 and 21 either captopril (25 mg) or placebo 1 h before exercise testing in a randomized, double-blind, crossover design. Captopril significantly improved the combined score of maximal ST-segment depression, maximal workload, and time to angina when compared to placebo. No differences in the pressure-rate index at rest and during exercise were seen. These results indicate that the sulfhydryl group of certain angiotensin converting enzyme inhibitors can potentiate their effect on the endogenous nitrovasodilator EDRF. In the clinical situation, this may lead to an improved exercise performance in patients with stable angina pectoris during chronic nitrate treatment, independent of its systemic vascular effects.     

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.     

维生素C对实验兔硝酸甘油耐受性影响的研究

目的探讨维生素C对硝酸甘油耐受性的影响。方法选取30只兔龄5~6个月的健康家兔,将取出的兔胸主动脉环按随机数字表法分为3组:对照组、硝酸甘油组、硝酸甘油+维生素C组,每组10份,分别做硝酸甘油量效关系及超氧化物歧化酶(SOD)活力和丙二醛(MDA)水平测定。结果对照组的最大舒张程度为(96±7)%,硝酸甘油组的最大舒张程度为(63±7)%,硝酸甘油+维生素C组的最大舒张程度为(86±5)%,硝酸甘油组、硝酸甘油+维生素C组可以明显增强兔离体动脉环对硝酸甘油的反应性。硝酸甘油组血管组织中MDA含量明显要高于对照组和硝酸甘油+维生素C组,差异有统计学意义(P<0.01),而对照组和硝酸甘油+维生素C组的MDA含量差异无统计学意义。硝酸甘油组血管组织中SOD活力明显要低于对照组和硝酸甘油+维生素C组,差异有统计学意义(P<0.05),而对照组和硝酸甘油+维生素C组的SOD活力差异无统计学意义。结论维生素C可改善硝酸甘油的耐受性。     

Transplantation tolerance: gene expression profiles comparing allotolerance vs. allorejection

An understanding of the molecular basis of immune regulation will allow development of therapies for diseases caused by immune dysregulation and for therapeutic exploitation of the immune response in transplantation of organ grafts or stem cells. To identify critical regulatory factors in immunity, we have used a mouse model wherein infectious regulatory tolerance is inducible by CD4/CD8 blockade in recipients of vascularised heart grafts. Once established, this transplantation tolerance is robust and isolated "tolerant" spleen cells show powerful immune regulatory properties, being able to impose donor-specific allotolerance upon fully immune competent naive recipients. Here, we present a compound comparison of four gene arrays (tolerance vs. rejection, at 48 h, and at 123 h) where a relatively small number of differentially expressed genes occurred. In rejection, there was a strong progressive amplification of IFNgamma and granzyme B mRNAs. In tolerance, both ELKL motif kinase and axotrophin occurred in the group of upregulated genes. Mice lacking ELKL motif kinase develop autoimmune disease, whilst axotrophin is a newly discovered stem cell gene that has only been explored in the context of neural development. This gene expression data is the first to demonstrate a link between axotrophin and regulatory tolerance and, since axotrophin, LIF, STAT3 and c-kit each function in stem cells, we propose that common mechanisms play a central role both in developmental regulation of stem cells, and in immune regulation.     

金属硫蛋白对大鼠硝酸甘油耐药性的影响(英文)

目的:评价金属硫蛋白(metallothionein, Met)在体内是否能改善硝酸甘油耐药的发生。方法:大鼠给予硝酸甘油(nitroglycerin, Nit)贴剂治疗两天(0.05 mg·h~(-1))以产生耐药。于耐药大鼠预先给予ZnCl_2以诱导内源性Met的合成及给予外源性Met15 mg·kg~(-1)·d~(-1)连续2 d。结果:Nit ZnCl_2组大鼠肝脏、血浆Met明显高于对照组(C组)。Nit组大鼠离体主动脉环的舒张反应最低。Nit ZnCl_2组大鼠及Nit Met组大鼠对SNP的降压反应明显强于Nit组。结论:外源性Met或内源性诱导合成的Met可以改善大鼠Nit耐药的发生。