Sulfhydryl Donor Research Articles

Anti‐angiogenic treatment of human cancer: Pitfalls and promises

Anti‐angiogenic treatment of human cancer: Pitfalls and promises

More on Interference of N-Acetylcysteine in Measurement of Acetaminophen

Acetaminophen (ACET) is a common analgesic and antipyretic drug that in excessive concentrations may cause acute liver damage and acute renal failure (1). Although the normal elimination of ACET seems to involve conjugation of a toxic alkylating metabolite with liver glutathione to yield a detoxified conjugation product, excessive doses of ACET deplete liver glutathione stores, causing accumulation of the toxic metabolite N -acetyl-imidoquinone (1)(2)(3). Acetylcysteine (NAC, N -acetyl-3-mercaptoalanine) is the drug of choice for the treatment of an ACET overdose. Although the mechanism responsible for the ability of NAC to serve as an effective antidote in vivo has not been fully elucidated, a major route of detoxification seems to depend on the ability of NAC to serve as a potent sulfhydryl donor that restores depleted …

Effects of N-acetylcysteine on nitroglycerin-induced relaxation and protein phosphorylation of porcine coronary arteries.

We investigated the effects of the sulfhydryl-donor, N-acetylcysteine (NAC), on nitroglycerin (NTG)-induced relaxation of the vascular smooth muscle. Addition of histamine to isolated porcine coronary arteries induced an initial rapid contraction followed by a gradual decrease in tonic contraction. NTG applied to the coronary artery strips before histamine caused relaxation of the histamine-induced rapid (3 min) and tonic (48 min) contraction. The inhibition of the tonic contraction by NTG was less at 48 min than at 3 min. Application of NAC (NTG-NAC) enhanced the relaxing effects of NTG on the histamine-induced tonic contraction rather than the acute contraction. In phosphorylation studies, changes in the phosphorylation of an intermediate filament, desmin, were parallel with changes in contraction in NTG-treated and NTG-NAC samples at 48 min. These phosphorylation changes of desmin at 48 min, which might be responsible for tonic phase contraction, were more extensive than those of myosin light chain (MLC) phosphorylation at 3 min, which might be responsible for acute contraction. These results suggest that treatment with the sulfhydryl donor, NAC, inhibited the phosphorylation of desmin associated with the enhancement of NTG-induced relaxation, which might be related to the mechanisms of recovery from NTG tolerance by sulfhydryl groups.

Potentiation by isoniazid of relaxation induced by nitrovasodilators in rat aorta.

The influence of isoniazid (ISO) preincubation on the relaxant effects of a group of nitrovasodilators was examined in norepinephrine-contracted rat aortic rings with and without endothelium. ISO displaced to the left the dose-response curves to all nitrovasodilators and increased the negative logarithm of their median effective concentration (EC50) values. Potentiation was minimal with sodium nitrite and increased progressively with sodium nitroprusside, nitroglycerin (NTG), and isosorbide dinitrate. The phenomenon occurred in rings with and without endothelium and was not seen with the nitric oxide-releasing agent acetylcholine or with the nonspecific vasodilator hydralazine. These results confirm previous observations of potentiation of NTG vasorelaxation by ISO and extend them to other nitrovasodilators. Potentiation is attributed to the previously postulated inhibition by ISO of pyridoxal-requiring enzymes involved in the breakdown of homocysteine, leading to its intracellular accumulation. Increased availability of this sulfhydryl donor would lead in turn to enhanced bioactivation and vasorelaxant effects of nitrovasodilators. The different degrees of potentiation observed would be related to the sulfhydryl requirement of each compound for bioactivation. Alternatively, enhanced bioactivation of the nitrovasodilators could be due to induction by ISO of P-450 enzymes involved in this process.

More on Interference of N-Acetylcysteine in Measurement of Acetaminophen

Acetaminophen (ACET) is a common analgesic and antipyretic drug that in excessive concentrations may cause acute liver damage and acute renal failure (1). Although the normal elimination of ACET seems to involve conjugation of a toxic alkylating metabolite with liver glutathione to yield a detoxified conjugation product, excessive doses of ACET deplete liver glutathione stores, causing accumulation of the toxic metabolite N -acetyl-imidoquinone (1)(2)(3). Acetylcysteine (NAC, N -acetyl-3-mercaptoalanine) is the drug of choice for the treatment of an ACET overdose. Although the mechanism responsible for the ability of NAC to serve as an effective antidote in vivo has not been fully elucidated, a major route of detoxification seems to depend on the ability of NAC to serve as a potent sulfhydryl donor that restores depleted …

Polarographic detection of nitric oxide released from cardiovascular compounds in aqueous solutions

In order to detect the concentration of nitric oxide, known to be one of the biologically active principles of certain cardiovascular compounds, a highly selective polarographic/amperometric device was used. The nitric oxide-releasing properties of sodium nitroprusside, nitroglycerine, nicorandil, and the molsidomine metabolite, 3-morpholinosydnonimine, were compared in the following cell-free experimental solutions in vitro: in Krebs-Henseleit solution with and without a sulfhydryl donor, l-cysteine, in an acidic, reducing medium, and in Krebs-Henseleit solution with superoxide dismutase enzyme. Sodium nitroprusside released similar concentrations of nitric oxide in Krebs-Henseleit solution and in the acidic, reducing medium. l-Cysteine inhibited the release of nitric oxide at physiological pH. In the presence of nitroglycerine, nitric oxide signals were detected in the acidic, reducing environment and in l-cysteine-rich Krebs-Henseleit solution but not in the absence of the sulfhydryl donor. Amperometric signals could not be detected after adding nicorandil in all the experimental conditions used. 3-Morpholinosydnonimine released nitric oxide only in the presence of the superoxide dismutase enzyme. Our results suggest that the polarographic electrode is able to detect the release of nitric oxide from sodium nitroprusside, nitroglycerine, and 3-morpholinosydnonimine in the absence of biological material. The present observations support the importance of the chemical environment during the detection of nitric oxide from donor compounds in the common in vitro bathing systems.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin.

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Nitrate tolerance, rebound, and their clinical relevance in stable angina pectoris, unstable angina, and heart failure

Vascular tolerance develops rapidly in isolated vascular strips exposed to millimolar concentrations of nitroglycerin. Several mechanisms, including depletion of sulfhydryl groups, reduced biotransformation of nitrates to NO or nitrosothiols, oxygen free radical injury, and downregulation of a membrane-bound enzyme or a nitrate receptor, have been proposed, but the exact mechanism responsible for in-vitro tolerance remains unknown. In-vivo tolerance of the beneficial effects of nitrates on hemodynamics, myocardial ischemia, and exercise performance develops rapidly. It has been suggested, but remains to be proven, that development of venous tolerance and not arterial tolerance is responsible for the attenuation of nitrate effects during long-term nitrate therapy. Several mechanisms, including neurohormonal activation, depletion of sulfhdryl groups, and the shift of fluid from the extravascular to intravascular compartment have been implicated. However, the use of agents to counteract these mechanisms (ACE inhibitors, sulfhydryl donors, diuretics) has produced conflicting results. Thus, at present the mechanism responsible for in vivo tolerance to nitrates remains unknown. Both in vitro and in vivo vascular tolerance to nitrates can be prevented or minimized by providing nitrate-free or low-nitrate intervals. However, during nitrate-free periods, rebound phenomena (rest angina in patients with ischemic heart disease or a deterioration in exercise performance prior to the renewal of the morning dose in patients with stable angina) remain a clinical concern. When treating patients with stable angina pectoris, it must be recognized that none of the nitrate preparations or formulations can provide round-the-clock antianginal or antiischemic prophylaxis. In these patients, beneficial antianginal and antiischemic effects of nitrates for 10-14 hours during the daytime can be maintained by using formulations and dosing regimens that avoid or minimize the development of tolerance (standard formulation of isosorbide-5-mononitrate, 20 mg in the morning and 7 hours later; slow-release formulation of isosorbide-5-mononitrate, 120-240 mg once a day; or nitroglycerin patch delivering 0.6 nitroglycerin per hour for 10-12 hours each day). Only the patch on and off treatment is associated with nitrate rebound. Although intermittent nitrate therapy is not associated with the development of tolerance, this strategy cannot be recommended for treating unstable angina because rebound angina during nitrate-free periods complicates clinical decision making. In the acute phase of unstable angina, continuous treatment with intravenous nitroglycerin is recommended because it permits rapid up- or down-titration. Tolerance towards antianginal and antiischemic effects does develop in a substantial number of patients with 24 hours, but this can be overridden by dose escalation and restoration of the therapeutic effectiveness of nitroglycerin. Tolerance towards the beneficial effects of nitrates on hemodynamics and on exercise performance also develops rapidly during continuous or long-term nitrate therapy, and for these reasons nitrates are not used as first-line therapy to treat chronic heart failure. In combination with hydralazine, high-dose isosorbide dinitrate (30-40 mg four times a day) improves survival, but this combination therapy is inferior to ACE inhibitors.

Sulfur Mustard Induces Apoptosis and Necrosis in Endothelial Cells

Sulfur Mustard (SM) is a vesicant or blistering chemical warfare agent, for which there still is no effective therapy. Endothelial cells are one of the major cellular targets for SM. The mechanism of endothelial cell death during SM injury is poorly understood. We studied the effect of exposure of endothelial cells to 0–1000 μMSM over the time course of 2–24 hr to determine the role of apoptotic and necrotic patterns of cell death in endothelial injury induced by SM. SM concentrations ≤250 μMinduced exclusively apoptosis which was observed after 5 hr in 30% of endothelial cells. Exposure to SM concentrations ≥500 μMcaused apoptosis and necrosis to the same extent in 60–85% of all cells after 5 to 6 hr. Necrosis was accompanied by a significant (∼50%) depletion of intracellular ATP, while in apoptotic cells ATP remained at the level similar to healthy cells. Interestingly, disruption of the long actin filament stress fibers and rounding of cells preceded other features of apoptosis—DNA fragmentation, membrane budding, and apoptotic body formation. In apoptotic cells, microfilaments formed constricted perinuclear bands, which were not observed in necrotic cells. Pretreatment with 50 mMN-acetyl-L-cysteine (NAC), a sulfhydryl donor and antioxidant, nearly eliminated the apoptotic features of cell death but did not prevent necrosis in response to SM. NAC pretreatment alone induced reorganization of actin filaments into an enhanced network of long stress fibers instead of a dominant cortical band of actin. NAC pretreatment prevented loss of cell adherence and cell rounding following exposure to 250 μMSM. The effect of NAC on cytoskeletal organization and its ability to eliminate SM-induced apoptosis suggests that actin filament organization may be an important element in cellular susceptibility to apoptotic stimuli.

Effects of Adriamycin on the Balance of Glomerular Protein Metabolism: Studies in Isolated Glomeruli of the Atlantic Hagfish, <i>Myxine glutinos</i><i>a</i>

Alterations in the glomerular metabolic balance such as an increase protein synthesis and/or diminished proteolytic activities with a concomitant enrichment of protein into the glomerulus have previously been associated with various renal diseases. The present study was aimed to closer define which pathomechanisms initially cause alterations in glomerular protein metabolism by using the anthracycline antibiotic Adriamycin (ADR) as a model compound to experimentally induce a glomerular lesion. As an alternative multicellular system, isolated and in vitro-incubated glomeruli of the Atlantic hagfish Myxine glutinosa were applied. ADR pretreatment caused a significant increase in glomerular protein synthesis (control (c): 55.1 ± 4.7 DPM/h/µg protein, ADR: 90.7 ± 7.5) accompanied by an elevated glomerular protein content. The sulfhydryl donor N-acetylcysteine (NAC) was applied as a free radical scavenger and showed no protective properties on the increase in protein synthesis (ADR + NAC: 86.2 ± 11.5 DPM/h/µg protein). Further, ADR caused a significant inhibition of RNA synthesis (c: 12.3 ± 1.2 DPM/h/µg protein, ADR: 6.0 ± 0.7). The proteolytic activity was diminished in glomerular homogenates of ADR-treated animals (c: 4.91 ± 1.49 U/h/mg protein, ADR: 1.71 ± 1.02). Based on these results, a radical-mediated effect of ADR on protein synthesis could possibly be ruled out. It is suggested instead that the increased protein synthesis is due to a net-effect of protein synthesis and proteolytic degradation occurring at the same time. The low translation rate caused by the lack of RNA could be disguised by the diminished degradation of proteins. This leads over time to a net increase of protein synthesis and to an enrichment of total protein in the glomerulus, subsequently causing functional alterations of the glomerular filtration barrier. A decreased glomerular proteolytic activity is therefore regarded to be the most crucial patho-mechanism responsible for disturbances in the glomerular protein balance during the development of ADR glomerulopathy in M. glutinosa.

Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock patients: Results from a prospective, randomized, double-blind study

Deactivation of endothelium-derived relaxing factor due to an increased oxygen radical load during sepsis may contribute to an impairment in microcirculatory blood flow. We investigated whether treatment with the sulfhydryl donor and oxygen radical scavenger, N-acetylcysteine, would improve whole-body oxygen consumption (VO2), gastric intramucosal pH, and veno-arterial CO2 gradient (veno-arterial PCO2) during septic shock. Prospective, randomized, double-blind study conducted over 2 yrs. Septic shock patients admitted to the intensive care unit. Fifty-eight patients requiring hemodynamic monitoring (radial and pulmonary artery catheters) due to septic shock, were included in this study. All patients were examined within 72 hrs after the onset of sepsis. They were optimally resuscitated by conventional means with volume and inotropic agents, and exhibited stable clinical conditions (hemodynamic values, body temperature, hemoglobin, FIO2). A gastric tonometer was inserted to measure the gastric intramucosal pH. Subjects randomly received either 150 mg/kg of intravenous N-acetylcysteine or placebo over a 15-min period, then a continuous infusion of 12.5 mg/hr of N-acetylcysteine or placebo over approximately 90 mins. Infusion measurements were begun 60 mins after the beginning of infusion and lasted approximately 30 mins. The infusion was then discontinued and 2 hrs later the final measurements were taken. Basic patient characteristics (age, sex, Acute Physiology and Chronic Health Evaluation [APACHE] II scores, Multiple Organ Failure scores) did not differ significantly, nor did pre- and 2-hr postinfusion measurements differ between any of the groups. Thirteen (45%) patients responded (i.e., showed an increase in VO2 > 10%, reaching a mean of 19%) to the N-acetylcysteine infusion. The N-acetylcysteine responders also showed an increase in gastric intramucosal pH, a decrease in veno-arterial PCO2, an increase in oxygen delivery, cardiac index, stroke index, and left ventricular stroke work index, as well as a significant decrease in systemic vascular resistance in comparison to baseline. The N-acetylcysteine nonresponders, as well as the patients in the placebo group, did not show any significant changes in any of these variables. The N-acetylcysteine responders had a higher survival rate (69%) than the non-responders (19%) and were studied earlier after onset of sepsis (37 hrs) than the nonresponders (61 hrs). The only significant difference between the entire N-acetylcysteine group (which included responders plus nonresponders) and the placebo group was an increased VO2 in the entire N-acetylcysteine group during infusion measurements. N-acetylcysteine provided a transient improvement in tissue oxygenation in about half of the septic shock patients, as indicated by an increase in VO2 and gastric intramucosal pH and a decrease in veno-arterial PCO2. The higher survival rate in the N-acetylcysteine responders and the fact that half of the patients receiving N-acetylcysteine did not respond, suggests that, in some patients, sepsis irreversibly damages the microvasculature to the extent that N-acetylcysteine has no effect. If analyzed by intention to treat, the N-acetylcysteine did not produce effects that were significantly different from the placebo. Whether the N-acetylcysteine challenge was merely diagnostic or whether N-acetylcysteine can be effective in the treatment of sepsis deserves further investigation.

A modified product inhibition model describes the nonlinear pharmacokinetics of nicorandil in rats.

Nicorandil, a vasodilator which acts through both cyclic GMP accumulation and K+ channel opening, has been used in the treatment of various cardiovascular diseases. We have examined the pharmacokinetics of nicorandil in the rat as a function of dose, as both i.v. boluses (9 doses, 0.75-12 mg, n = 1.4 per dose), and as a 5-hr infusion followed by a 5-hr washout (6 doses, 10-500 micrograms/kg/min, n = 3 per dose). Plasma nicorandil concentrations were determined by HPLC. Nicorandil plasma concentrations increased disproportionately with dose, but nicorandil elimination obeyed apparent monoexponential kinetics, and the apparent half-life (t1/2) increased with dose. In addition, the approach to apparent steady-state during the infusion phase was not overtly sensitive to the drastic changes in t1/2 observed. Pharmacokinetic modelling with several nonlinear models, viz: Michaelis-Menten with parallel first-order, cosubstrate depletion and competitive product inhibition, were carried out. Addition of the sulfhydryl donor, N-acetyl-L-cysteine, did not change the pharmacokinetics of nicorandil, providing experimental indication that a cosubstrate depletion model might not be applicable. To described the unique pharmacokinetics, a modified product inhibition model was developed. This new model includes the classic competitive product inhibition equation, describing both parent and product kinetics, and it incorporates, in addition, separate first-order elimination rate constants for both nicorandil and the inhibition metabolite. Experimental evidence showed that N-(2-hydroxyethyl) nicotinamide, the major metabolite of nicorandil in rats, and nicotinamide (niacinamide) itself, indeed inhibited nicorandil elimination.

Sulfhydryl group donors potentiate the hypotensive effect of acetylcholine in rats.

Nitric oxide mediates the vasodilator and hypotensive responses of acetylcholine infusion. It has been reported that nitric oxide could be protected from free radical destruction by forming an S-nitrosothiol compound. Furthermore, sulfhydryl donors such as N-acetylcysteine or thiosalicylic acid enhance nitric oxide production from nitroglycerin. Consequently, the hypotensive effect of intravenous acetylcholine infusion might be potentiated during the simultaneous administration of sulfhydryl donors. The objective of the present study was to test in Okamoto spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (1) whether the hypotensive effect of acetylcholine (10 micrograms/kg per minute) was affected by the simultaneous administration of N-acetylcysteine (10 micrograms/kg per minute) or thiosalicylic acid (10 micrograms/kg per minute), and (2) whether NG-nitro-L-arginine-methyl ester (100 micrograms/kg per minute) administration was able to reverse the changes induced by acetylcholine plus N-acetylcysteine or acetylcholine plus thiosalicylic acid. The administration of acetylcholine reduced (P < .05) mean arterial pressure in WKY rats (13 +/- 2%) and SHR (14 +/- 2%) without affecting urine flow rate, urinary sodium excretion, and glomerular filtration rate. In the presence of N-acetylcysteine, the acetylcholine-induced reduction in mean arterial pressure was potentiated (P < .05) in WKY rats (24 +/- 4%) and SHR (20 +/- 2%). These changes in mean arterial pressure were accompanied by significant reductions in urine flow rate and urinary sodium excretion in WKY rats, as well as in glomerular filtration rate in SHR.2

Enhancement of renal and hepatic glutathione metabolism by dimethylthiourea

Dimethylthiourea (DMTU), a free radical scavenger, was administered to rats to study its effect on renal and hepatic glutathione metabolism, since it is a potential sulfhydryl donor. Six hours following DMTU, renal GSH content was significantly ( P < 0.05) increased (10%), and was increased further after 24 h (28%) ( P < 0.001). Hepatic GSH content was also significantly ( P < 0.001) elevated at 6 and 24 h (5 and 33%, respectively). Seven days of daily DMTU therapy significantly ( P < 0.001) increased renal and hepatic GSH content by 36 and 54%, respectively, which was associated with a significant ( P < 0.001) increase in the renal activities of glutathione peroxidase (GP) by 38%, glutathione transferase (GT) by 92%, and glutathione reductase (GR) by 19% ( P < 0.05). Significantly increased activities of hepatic GP by 84% ( P < 0.01) and GT by 101% ( P < 0.001) also occurred in DMTU-treated rats after 7 days of continuous therapy. From these data, we conclude that DMTU stimulates renal and hepatic GSH metabolism, which may be important in mediating DMTU's protective effect against free radical-induced tissue injury.

Acute effects of nitroglycerin depend on both plasma and intracellular sulfhydryl compound levels in vivo. Effect of agents with different sulfhydryl-modulating properties.

Changes in sulfhydryl (SH) compound availability may alter the hemodynamic effect of nitroglycerin (NTG). Data on the relation between NTG effect and thiol levels are, however, limited to in vitro experiments. The present study investigates how intracellular and extracellular changes in SH group concentrations (cysteine and glutathione [GSH]) affect the responsiveness to NTG in vivo. GSH and cysteine levels in plasma, vena cava, and aorta were measured after administration of N-acetylserine (placebo, n = 6), N-acetylcysteine (NAC, extracellular and intracellular SH donor, n = 6), oxothiazolidine (OXO, intracellular SH donor, n = 6), buthionine sulfoximine (BSO, intracellular GSH-depleting agent, n = 6), BSO+NAC (n = 6), and BSO+OXO (n = 6) in chronically catheterized conscious rats. In addition, the effect of 2.5 mg NTG/kg i.v. on mean arterial pressure (MAP) was determined before and after the same treatment. NAC (5 mmol/kg i.v. for 2 hours) significantly (p < 0.05) increased extracellular cysteine and GSH levels and potentiated the hypotensive effect of NTG (from 26 +/- 3 to 31 +/- 4 mm Hg [mean +/- SEM], p < 0.05). OXO (5 mmol.kg-1 x hr-1 i.v. for 2 hours) significantly increased intracellular cysteine and GSH levels but had no effect on NTG responsiveness (p > 0.05). BSO (1 g i.p. three times within 24 hours) significantly decreased intracellular GSH levels (p < 0.05) and attenuated the effect of NTG (from 28 +/- 3 to 16 +/- 2 mm Hg). The results suggest that the acute hypotensive effect of NTG in vivo is: 1) increased by high extracellular GSH and/or cysteine levels (NAC), 2) decreased by low intracellular GSH levels (BSO), and 3) unaffected by high intracellular levels of cysteine and GSH (OXO).

Positive inotropic effect of acetylcysteine in cardiomyopathic Syrian hamsters.

Several laboratories have provided indirect evidence that the myocardium of the cardiomyopathic Syrian hamster (CMH) is chronically ischemic on the basis of microvascular spasm. We previously reported evidence supporting a defect in the ryanodine-sensitive sarcoplasmic reticulum calcium release channel (SRCRC) in CMH. A relation between alterations in SRCRC and chronic ischemia has not yet been explored. A potential mechanism could be the effects of changes in redox state on thiol groups. Thiol reagents have previously been shown to regulate calcium release from SRCRC. Accordingly, we studied the inotropic effects of the sulfhydryl donors, acetylcysteine (AC), cysteine, and cystine in CMH. AC was a positive inotrope in isolated papillary muscles prepared from CMH, but not F1B controls (F1B) (p < 0.01). No significant differences were noted in inotropic responses to cysteine or cystine. AC blunted the response of CMH > F1B control papillary muscle preparations to stimulation frequency (p < 0.01). The actual tension generated (in mg/mm2) by CMH was no longer different than F1B with addition of AC (10(-3) M), ryanodine (10(-8) M), or verapamil (5 x 10(-7) M). These findings are consistent with a defect in SRCRC in CMH. This defect may be primary or may provide a novel mechanism for hibernating myocardium owing to chronic ischemia.

Biochemical mechanism of organic nitrate action

Increasing evidence suggests that organic nitrate action derives from their metabolic conversion to nitric oxide (NO) in the vascular smooth muscle cell. The primary catalytic activity of this process appears to reside at the cellular plasma membrane. There is no concrete evidence to indicate that NO formation is preceded by the production of inorganic nitrite ion or that the NO produced needs to form S-nitrosothiols before it can activate guanylate cyclase to produce cyclic guanosine 3′,5′-monophosphate (cGMP). Although sulfhydryl donors can partially reverse nitroglycerin-induced tolerance in patients, this phenomenon (by itself) is not sufficient to implicate intracellular sulfhydryl depletion as an operating mechanism of clinical nitrate tolerance. This is because sulfhydryl donors can react with nitroglycerin extracellularly to form S-nitrosothiols, and nonsulfhydryl compounds, such as enalapril and hydralazine, can prevent the development of in vivo nitrate tolerance. In addition to the cellular biochemical reactions, organic nitrates also produce systemic biochemical effects through altering neurohormonal status. These systemic effects may contribute significantly to the development of nitrate tolerance in therapeutic situations.

Nitrate tolerance. How to prevent it or minimize its effect.

Continuous therapy with nitrates rapidly produces tolerance along with loss or diminution of circulatory, antianginal, and anti-ischemic effects. Development of tolerance can be avoided by various approaches. In patients with stable angina, intermittent use of nitrates with long nitrate-free intervals, use of transdermal nitroglycerin during the day or oral isosorbide dinitrate or isosorbide-5-mononitrate twice daily in the morning and early afternoon, and intermittent use of nitrates in combination with another class of antianginal agent are appropriate. In patients with unstable angina, continuous therapy with intravenous nitroglycerin is recommended during the acute phase of angina. Despite development of partial tolerance, oral isosorbide dinitrate, 30 to 60 mg four times a day, plus hydralazine may be useful for patients with congestive heart failure who cannot tolerate angiotensin-converting enzyme inhibitors. Concomitant use of sulfhydryl donors or angiotensin-converting enzyme inhibitors, agents that might theoretically prevent nitrate tolerance, is not recommended. Data on these agents are conflicting, and added costs and adverse effects are likely to preclude their use in the future.

Preventive administration of intravenous N-acetylcysteine and development of tolerance to isosorbide dinitrate in patients with angina pectoris.

Development of tolerance to organic nitrates may be related to depletion of sulfhydryl groups in vascular smooth muscle. N-Acetylcysteine (NAC), a sulfhydryl donor, has been reported to potentiate the effect of nitroglycerin and reverse tolerance in humans. However, its ability to prevent or delay the development of nitrate tolerance in patients with angina pectoris has not been established. Ten patients with stable angina pectoris were treated with intravenous isosorbide dinitrate (ISDN; 5 mg/hr) combined with NAC (2 g i.v. over 15 minutes followed by 5 mg/kg/hr) or matching placebo for 30 hours in a double-blind, randomized, crossover study with a washout interval of 8 days. Bicycle exercise tests were performed before and at 1 1/2, 8, 20, 24, and 30 hours after start of treatment. After 24 hours of infusion, exercise parameters were not significantly different from pretreatment values (p greater than 0.05) during ISDN plus placebo, indicating development of tolerance to ISDN. In contrast, time to onset of angina, time to 1-mm ST segment depression, and total amount of ST segment depression were still significantly improved after 24-hour infusion of ISDN plus NAC (p less than 0.05). In addition, compared with placebo, a significant difference (p less than 0.05) in favor of NAC was observed regarding time to angina (507 +/- 63 versus 445 +/- 69 seconds, mean +/- SEM), time to 1-mm ST segment depression (435 +/- 43 versus 407 +/- 45 seconds), and total ST segment depression (1.8 +/- 0.9 versus 3.1 +/- 0.4 mm). These results suggest that infusion of high doses of NAC in combination with ISDN for 30 hours affects and partially prevents the development of tolerance to antianginal effects normally observed during infusion with ISDN.

Management of myocardial ischemia: Role of intermittent nitrate therapy

Tolerance to long-term nitrate therapy has now been documented in a large number of clinical studies. These trials have demonstrated attenuation of effect over time but a return of responsiveness with the start of nitrate-free intervals. Intermittent therapy prevents attenuation or tolerance when used as the initial treatment regimen. This strategy is appropriate for use with isosorbide dinitrate, as well as intravenous or transdermal nitroglycerin. Transmucosal nitroglycerin has not been associated with tolerance; the lack of such an effect may be a result of rapid increases and decreases in drug levels. In the future, sulfhydryl donors may also prove useful in prevention or reversal of nitrate tolerance.