Thiol-mediated catalysis of nitroglycerin degradation by serum proteins. Increase in metabolism was not accompanied by S-nitrosothiol production

In vitro degradation of nitroglycerin (NTG) in human plasma has been shown to be accelerated significantly by sulfhydryl compounds. The interaction between NTG and N-acetyl-1-cysteine (NAC) in human plasma produces a pharmacologically active S-nitrosothiol, which may be responsible, at least in part, for the in vivo nitrate tolerance-reversing effect of NAC. The mechanism of this thiol-mediated NTG degradation in plasma has not been identified. In this report, we examined the catalytic activity of various plasma proteins toward NAC-mediated NTG degradation and found human serum albumin (HSA) to have the highest catalytic activity among the proteins tested. However, the dinitrate metabolite distribution ratio found with HSA (favoring the 1,3- over the 1,2-isomer) was substantially different from that observed with human plasma (where equal amounts of both dinitrates were produced). In addition, the HSA-catalyzed degradation of NTG did not lead to enhanced formation of S-nitrosothiol. These findings therefore argue against a predominant role exerted by HSA in the thiol-mediated NTG metabolism in plasma. The HSA-mediated catalysis of NTG was partially blocked by pretreatment with NAC followed by a thiol-alkylating agent, suggesting that the catalytic mechanism was due, in part, to the conversion of disulfide linkages within the HSA structure to reactive sulfhydryl groups.     

Influence of N-acetylcysteine on bioactivation of nitroglycerin to nitric oxide and S-nitrosothiols in the liver and brain of mice

Three-day nitroglycerin (NTG) administration at progressively increasing doses caused a drop in the liver S-nitrosothiol (SNT) and malonyldialdehyde (MDA) concentrations below the control levels. It suggests that NTG administered in this way, exhibits antioxidant activity due to releasing the biologically active SNT and nitric oxide (NO). On the other hand, in the brain, NTG did not influence SNT concentrations, but slightly elevated NO formation. N-acetylcysteine (NAC) given jointly with NTG substantially stimulated NTG bioactivation to the biologically active NO and SNT as well in the liver as in the brain. It was accompanied by a rise in non-protein sulfhydryl thiol (NPSH) level and additional suppression of lipid peroxidation in hepatocytes. Therefore, is seems that the combined administration of NTG and thiols or other antioxidants is very much justified not only because of their influence on the vascular endothelial cells but also on such organs as the liver and brain.     

Prevention and reversal of tolerance to nitroglycerine with N-acetylcysteine

A recent study demonstrated that the sulfhydryl donor N-acetylcysteine (NAC) potentiated hemodynamic responsiveness to nitroglycerine (NTG) in patients with ischaemic heart disease. The interaction between NTG and NAC in rings of bovine coronary artery was examined. Vasodilator responses to NTG were determined after elevation of tone with the thromboxane mimetic U46619 [(15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano) prosta-5Z, 13E-dienoic acid]. NAC (1 microM-3 mM) induced no changes in tone of the preparation, but 10 microM NAC significantly potentiated responses to NTG (EC50 reduced from 0.69 +/- 0.19 microM to 0.22 +/- 0.06 microM; p less than 0.01). Increasing degrees of tolerance to NTG were produced at pH 7.4 by preincubating coronary rings with NTG in concentrations of 4.4 and 44 microM, and 0.22 mM. With 0.22 mM NTG, EC50 for subsequently administered NTG was increased to 11.0 +/- 1.8 microM (p less than 0.001 vs. control vessels). The degree of tolerance produced with this concentration of NTG was markedly attenuated by simultaneous (EC50 = 0.50 +/- 0.30 microM; p less than 0.001 vs. tolerant vessels) or subsequent (EC50 = 1.17 +/- 0.59 microM, p less than 0.001 vs. control vessels) incubation with 10 microM NAC. These data confirm that responses to NTG are modulated by sulfhydryl (or specifically cysteine) availability and suggest that in vitro tolerance to NTG is related to sulfhydryl (or cysteine) depletion. It is therefore possible that in vivo potentiation of NTG responses by NAC will be of clinical benefit in preventing or reversing loss of hemodynamic responsiveness to NTG.     

Vascular and anti-platelet actions of 1,2- and 1,3-glyceryl dinitrate.

1. The aim of this study was to investigate whether two metabolites of glyceryl trinitrate (GTN), 1,2 and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN) could account for the pharmacological effects of GTN. To this end the formation of nitric oxide (NO) from 1,2- and 1,3-GDN in the presence of bovine aortic smooth muscle cells (SMC) or endothelial cells (EC) was studied. The effects of various thiols on NO formation from these dinitrates was also evaluated. 2. 1,2-GDN or 1,3-GDN (10(-10)-10(-5) M) caused a dose-dependent relaxation of rabbit aortic strips denuded of endothelium and precontracted with phenylephrine. The dinitrates were less than one tenth as potent as GTN. 3. Incubation of 1,2-GDN or 1,3-GDN (75-2400 microM) with SMC for 30 min led to a concentration-dependent increase in nitrite (NO2-) formation but this increase was less than that produced from GTN. Likewise incubation of 1,2-GDN or 1,3-GDN with N-acetylcysteine (NAC), glutathione (GSH) or thiosalicylic acid (TSA) (all at 1 mM) for 30 min at 37 degrees C produced a concentration-dependent increase in NO2- formation. 4. Platelet aggregation induced by thrombin (40 mu ml-1) was not modified by high concentrations of 1,2-GDN or 1,3-GDN (175-700 microM). However, aggregation was inhibited when platelets were exposed to 1,2-GDN or 1,3-GDN (700 microM) in the presence of SMC (0.24-1.92 x 10(5) cells) or EC (0.8-3.2 x 10(5) cells). These effects were abrogated by co-incubation with oxyhaemoglobin (OxyHb, 10 microM) indicating that they were due to NO release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Captopril-induced reversal of nitroglycerin tolerance: role of sulfhydryl group vs. ACE-inhibitory activity.

The angiotensin-converting enzyme (ACE) inhibitor captopril has been shown to reverse vascular tolerance to nitroglycerin (NTG). Whether captopril reverses NTG tolerance by providing sulfhydryl (SH) groups or by inhibiting ACE is not clear. To examine this issue, we treated rat aortic rings with buffer, captopril (SH +, ACE inhibitory activity +), enalaprilat (SH-, ACE inhibitory activity +), or N-acetylcysteine (NAC, SH+, ACE inhibitory activity-) prior to their contraction with epinephrine and subsequent relaxation with NTG. Previous exposure of NTG-treated rings resulted in marked resistance to the vasorelaxant effect of a subsequent exposure to NTG in buffer-treated rings. Both NAC and captopril, but not enalaprilat, potentiated the vasorelaxant effects of NTG during the first exposure of vascular rings to NTG and also prevented the development of tolerance to NTG during a second exposure. Buffer-treated rings showed an inability to accumulate cyclic guanosine monophosphate (GMP) in response to a second exposure to NTG. In contrast, both NAC and captopril-pretreated rings demonstrated a persistence of cyclic GMP accumulation during the second NTG exposure. The endothelium-dependent vasodilator acetylcholine (ACh) caused relaxation of the NTG-tolerant rings and also induced cyclic GMP accumulation in these rings. In other experiments, we found that prior exposure of vascular rings to ACh did not cause resistance to the subsequent vasorelaxant effects of ACh. NAC, captopril, and enalaprilat did not modulate the effects of ACh during either the first or subsequent exposures to ACh. In addition, indomethacin did not influence the "protective" effects of NAC or captopril against NTG tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the role of Ca2+ in the toxicity of alkylating and oxidizing quinone imines in isolated hepatocytes

The cytotoxicity of acetaminophen (paracetamol) has been shown to be associated with a disruption of intracellular Ca2+ homeostasis caused by the interaction of its metabolite N-acetyl-p-benzoquinone imine (NAPQI) with hepatocyte thiols [Moore, M., et al. (1985) J. Biol. Chem. 260, 13035-13040]. Inasmuch as NAPQI can both covalently bind to thiols and oxidize thiols, we investigated the effects of two dimethylated analogues of NAPQI, one of which (2,6-dimethyl-NAPQI) primarily binds to thiols and the other of which (3,5-dimethyl-NAPQI) primarily oxidizes thiols. Of the three compounds, 2,6-dimethyl-NAPQI decreased protein thiols to the greatest extent and also inhibited hepatocyte plasma membrane Ca(2+)-ATPase to the greatest extent. The 3,5-dimethylated analogue decreased protein thiols to the least extent and inhibited the plasma membrane Ca(2+)-ATPase to a lesser extent. The cytotoxicity of all three compounds was preceded by a sustained elevation in cytosolic Ca2+ as compared to the transient rise caused by the alpha-agonist phenylephrine. Again, the 2,6-dimethyl analogue was the most potent of the three compounds. The thiol reagent dithiothreitol (DTT), which reversed the inhibition of the Ca(2+)-ATPase and the rise in cytosolic Ca2+, also protected against cytotoxicity. Agents that are known to inhibit either Ca(2+)-dependent proteases or phospholipases significantly delayed the onset of cytotoxicity caused by NAPQI and its analogues. Our results suggest that both arylation and oxidation of protein thiols may result in the elevation of cytosolic Ca2+ and in cytotoxicity and that arylation of critical thiol groups appears to be the more lethal reaction.     

N-acetylcysteine modifies the acute effects of isosorbide-5-mononitrate in angina pectoris patients evaluated by exercise testing

Nitrates are well established in the treatment of angina pectoris and the presence of sulfhydryl groups seems to be fundamental to nitrate-induced vasodilatation. The present study was performed to elucidate if large oral doses of N-acetylcysteine (NAC, 2,400 mg X 2), a donor of sulfhydryl groups, given together with a single oral dose of the long-acting nitrate, isosorbide-5-mononitrate (5-ISMN, 60 mg), would modify the nitrate effect evaluated by exercise testing before and after additional sublingual doses of nitroglycerin (NTG). Ten patients with angina pectoris and angiographically proven significant coronary artery disease were included. All patients received a baseline therapy with beta blockers. None of the patients had developed nitrate tolerance at inclusion. NAC/5-ISMN treatment significantly prolonged the total exercise time as compared with placebo/5-ISMN (7.7 +/- 2.1 min vs. 6.8 +/- 1.7 min, p less than 0.05). This increase was of such magnitude that no further effect was obtained after additional NTG doses. This study demonstrated that increased availability of sulfhydryl groups can increase the exercise capacity in angina pectoris patients treated with 5-ISMN without nitrate tolerance.     

Urinary and plasma homocysteine and cysteine levels during prolonged oral N-acetylcysteine therapy

Acute administration of N-acetylcysteine (NAC) may induce alterations in plasma and urinary levels of homocysteine (Hcy) and cysteine (Cys). We studied the effects of continuous oral NAC therapy on different Hcy and Cys plasma and urinary forms in 40 healthy subjects assigned to three groups (groups A: n = 13, no therapy; group B: n = 14, NAC 600 mg/day, and group C: n = 14, NAC 1,800 mg/day) for 1 month (T(1)). After a 1-month washout period without therapy (T(2)), all subjects were treated with oral NAC (1,800 mg/day) for 2 months and (T(3) and T(4)) reassessed monthly for plasma and urinary thiols. The treated subjects showed a significant decrease in plasma total Hcy and a slight increase in total Cys levels; the alterations of different forms of plasma thiols suggested an NAC-induced increase in disulfide forms and an increase in urinary Hcy and Cys excretion as disulfide forms. The effects appeared to be dose dependent, being more marked in subjects treated with higher dosages. This approach may be important, as an association or alternative therapy in hyperhomocysteinemic conditions of poor responses to vitamins.     

Two possible mechanisms underlying nitrate tolerance in monkey coronary arteries

1. Previous studies using isolated arteries have demonstrated cross-tolerance between nitric oxide (NO) donors such as nitroglycerin (NTG) and sodium nitroprusside (SNP). However, it remains unclear whether the vasorelaxing effect of atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase, is affected by treatment with NO donors. To investigate the cross-tolerance and interactions between NTG and ANP in coronary vasorelaxant responses, we used two models of monkey coronary arterial strips (Macaca fuscata). 2. In one model, which was induced by a 1 h treatment with 4.4 x 10(-4) mol/L NTG followed by washout of the agent for 1 h, the vasorelaxing effects of subsequent NTG were markedly attenuated, whereas those of ANP and NO were not affected. These findings suggest that the development of NTG tolerance is associated with a biotransformation process from NTG to NO. In the other model, which did not include washout after exposure to 3 x 10(-6) mol/L NTG, the vasorelaxant responses to 10(-8) mol/L ANP (31.1+/-5.4 vs 5.1+/-2.1%, respectively; P < 0.001), 10(-6) mol/L NO (61.5+/-2.4 vs 29.5+/-8.5%, respectively; P < 0.001) and 10(-8) mol/L SNP (49.4+/-6.4 vs 8.0+/-2.0%, respectively; P < 0.001) were significantly attenuated. The concentration- response curve for 8-bromo-cGMP (8-Br-cGMP) was shifted to the right, whereas responses to papaverine and forskolin were unchanged. These findings suggest that an intracellular process that occurs after the synthesis of cGMP is responsible for this interaction. 3. As a mechanism of NTG tolerance, two possible processes may be impaired: (i) biotransformation from NTG to NO; and (ii) an intracellular process that occurs after the synthesis of cGMP.     

Kinetic mechanisms for the concentration dependency of in vitro degradation of nitroglycerin and glyceryl dinitrates in human blood: metabolite inhibition or cosubstrate depletion?

The in vitro degradation of nitroglycerin (NTG) and its dinitrate metabolites in human blood and red blood cells (RBC) has been shown to exhibit apparent first-order kinetics. The decay rates of NTG and its dinitrate metabolites, however, were dependent on the initial concentration. We showed that this unusual kinetic behavior can be described mathematically by models of Michaelis-Menten kinetics combined with either competitive product inhibition or cosubstrate depletion. Experimental studies were conducted to determine the relative contribution of these two mechanisms to the observed kinetics. The effect of added thiols (the likely cosubstrates) on [14C]NTG degradation was studied separately in whole blood, reconstituted RBC, lysed RBC, and plasma. N-Acetylcysteine, L-cysteine, and D-cysteine accelerated NTG degradation in whole blood, while a similar concentration of glutathione had no effect. However, all four thiols exerted no effect on NTG kinetics in reconstituted and lysed RBC. In contrast, these thiols, as well as dithiothreitol, produced a marked increase (3-14 fold) in NTG degradation rate in plasma compared with buffer controls. Since thiol replenishment in reconstituted and lysed RBC did not abolish the concentration dependency, cosubstrate depletion due to thiols appeared unimportant as a contributor to the kinetic phenomenon. In human blood, metabolite inhibition of NTG degradation occurred along with the existence of concentration dependency. Both phenomena, however, were absent when NTG degradation was examined in rat blood. Concentration-dependent degradation in human blood was not observed for glyceryl-1-mononitrate, a compound that does not produce a nitrated metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)     

N -Acetyl-cysteine reduces homocysteine plasma levels after single intravenous administration by increasing thiols urinary excretion.

A decrease of plasma homocysteine (Hcy) may represent a therapeutic promise for reducing the impact of atherosclerosis. N -Acetyl-cysteine (NAC) is a thiol-containing compound interfering with endogenous thiols, cysteine (Cys) and Hcy, by forming with them mixed disulphides with a possibly more efficient renal clearance. The aim of this work was to assess the effect of NAC intravenous infusion on plasma levels of different forms of Hcy and particularly to verify the effect on Hcy renal excretion. We collected basal blood samples at 0.5, 1, 2, 5, 8 and 24 h after the beginning of NAC infusion (50 mg kg(-1)body wt.) and also 24-h urine samples of the day of NAC infusion and of the day before and of the day after the infusion in ten healthy subjects (mean age 73+/-15). Urinary and plasma thiols (Hcy, Cys and NAC) were assayed by HPLC. Both total plasma Hcy (approx. 69%vs basal values) and Cys (approx. 40%vs basal values) fell progressively, reaching a minimum 5 h after infusion start; total free (i.e. not bound to proteins) Hcy (2.2+/-1.8 down from 4.4+/-4.2 nmol ml(-1)) and Cys (70.4+/-39.8 down from 113. 3+/-61.2 nmol ml(-1)) decreased as well. Reduced (thiolic-free form) Hcy and Cys decreased during infusion, though not as pronounced as for the other forms. Percentagewise, out of the total plasma levels, Hcy and Cys total free form and reduced form tended to increase over infusion as well as their difference (i.e. the plasma mixed disulphide moiety), thus supporting the idea that excess NAC displaces thiols from their plasma binding sites forming mixed disulphides. Urinary total Cys and Hcy excretion significantly increased at the end of the day of NAC infusion (tenfold for Cys and fivefold for Hcy) and reduced appreciably on the following day. Also urinary excretion of the free form of Cys and Hcy increased at the end of the day of NAC infusion, although in a lower amount with respect of total amounts, meaning a reduction of percentage Cys and Hcy excreted as the free form; for none of the patients had proteinuria, the 'free' form of urine thiols has to be identified in the 'reduced' form, the difference between the total and free form reflecting the 'mixed disulphide' moiety. NAC intravenous administration induces an efficient and rapid reduction of plasma thiols, particularly of Hcy; our data support the hypothesis that NAC displaces thiols from their binding protein sites and forms, in excess of plasma NAC, mixed disulphides (NAC-Hcy) with an high renal clearance. This effect may represent the start of an alternative approach in the treatment of hyperhomocysteinaemic conditions.     

Chemical basis for the biological activity of imexon and related cyanoaziridines

Chemical aspects of mode of action of imexon and related cyanoaziridines were studied. These compounds do not alkylate DNA nor react with the epsilon-amino groups of l-lysine, despite the presence of an aziridine ring. They do react readily with biologically important sulfhydryl compounds to give products derived from either aziridine ring opening, interaction with the cyano group of cyanoaziridines, or opening of the iminopyrrolidone ring of imexon. The products from reactions of imexon and related cyanoaziridines with thiols are not as potent as their parent compounds against tumor cells. These results are consistent with biological studies that show that the mechanism of cytotoxicity involves thiol depletion followed by oxidative stress leading to apoptosis.     

Effects of N-acetylcysteine on arginase, ornithine and nitric oxide in renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion (I/R) is a complex syndrome involving several mechanisms such as renal vasoconstrictions, extensive tubular damage and glomerular injury. N-Acetylcysteine (NAC), a potent antioxidant by itself, may serve as a precursor for glutathione synthesis. The aim of this study was to investigate the possible effects of NAC on liver and kidney tissue arginase activity, ornithine and plasma nitric oxide levels during the I/R injury of kidney. METHODS: Twenty-four female Sprague-Dawley rats divided into three groups: group 1; was given saline intraperitoneally (i.p.). Saline to group 2 and NAC (300 mg kg(-1)) to group 3 were injected i.p. 30 min before induction of ischemia. Groups 2 and 3; subjected to bilateral renal ischemia (60 min) followed by reperfusion (24 h). After the reperfusion period, the rats were sacrificed and liver and kidney tissue arginase activities, ornithine and plasma nitric oxide (NO) levels were determined. RESULTS: NAC had an increasing effect on both of liver and kidney tissue arginase activities and ornithine levels while decreasing plasma NO concentration. CONCLUSION: The stimulatory effect of NAC on arginase activity may result in an inhibition of the plasma NO level. Moreover, it could be possible that one of the protective mechanisms of NAC might be through the stimulation on the both liver and kidney tissue ornithine levels.     

Increase in glucose consumption by acetylcysteine during hyperglycemic clamp. A study with healthy volunteers.

Thiols have been shown to be related to insulin secretion and to uptake of glucose into tissues. In the present study the effects of i.v. administration of acetylcysteine (N-acetylcysteine, NAC, CAS 616-91-1) on glucose consumption and plasma free thiols were studied in young healthy volunteers during hyperglycemic clamp. NAC (0.5-2.0 mg/kg) significantly increased glucose consumption. This effect was not obvious at higher doses of NAC. Plasma free NAC depended on the dose of NAC injected. The t1/2 of NAC was 11 min. NAC produced significant increases of plasma cysteine concentrations, and a slight but insignificantly increase of plasma glutathione. These data suggest that a moderate increase in plasma thiols augments glucose consumption during hyperglycemic clamp.     

Sodium nitroprusside: mechanism of NO release mediated by sulfhydryl-containing molecules

Sodium nitroprusside (SNP) is among the most widely studied nitric oxide donors, and its capability of producing NO seems to depend on its interaction with sulfhydryl-containing molecules present in vivo. The aim of this research has been the study of the mechanism of interaction between SNP and sulfhydryl-containing compounds, such as cysteine and glutathione, through detection by EPR, UV-vis, and IR spectroscopy of both the radical and nonradical species involved. An electron-transfer process can be invoked as the key step, which leads to the formation of the reduced SNP radical, the main detectable radical intermediate, and the corresponding S-nitrosothiol, the ending product of NO that can be considered the real storage and transporters of NO. When cysteine was used, a second radical species (A) is detectable: it can be accounted for by the interaction of a byproduct with unreacted cysteine.     

Hydroxyimine NO-donors; FK409 and derivatives

FK409 was discovered during a screening program for prostaglandin like compounds from microbial products by measuring inhibition of platelet aggregation and vasodilation. FK409, a semisynthetic compound derived from acidic nitrosation of microbial broth, was shown to be a potent vasodilator with a similar pharmacological profile to organic nitrates such as nitroglycerin (NTG). FK409 dilated the larger diameter coronary vascular vessels more potently than those with a smaller diameter in vitro, and was more potent than NTG in a dog angina pectoris model. Clinical development of FK409 for angina pectoris included a preliminary open efficacy study in about twenty patients with angina pectoris showing an improvement in 60-70 % of patients. Anemia proved a drug related adverse event. A new study was carried out on around 20 patients with ischemic heart disease, but in the longer term the anemia remained. It was concluded that FK409 had a comparable efficacy to organic nitrates, but an undesirable adverse effect, and development was terminated. Back-up compounds for FK409, explored improvement of the pharmacokinetic profile: an increase in duration of action and a reduction of the risk of anemia. The pharmacological action of FK409 was associated with increased cGMP levels in aortic smooth muscle; and release of NO was observed by physicochemical methods. Synthesis of chemically more stable derivatives of FK409 with slower NO release was aimed at longer pharmacokinetic profiles and a lower incidence of anemia. FR144220 and FR146881 were identified as chemically more stable compounds with a longer duration of pharmacological action.     

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)     

N-Acetylcysteine prevents but does not reverse dexamethasone-induced hypertension

1. We have shown previously that N-acetylcysteine (NAC) prevents the increase in blood pressure induced by adrenocorticotropin treatment. The present study investigated the effect of NAC on dexamethasone (Dex)-induced hypertension. 2. Male Sprague-Dawley rats were randomly divided into six groups (n = 10 in each). In a prevention study, NAC (10 g/L in the drinking water) was given for 4 days prior to and 11 days during concurrent treatment with saline (0.1 mL/rat per day) or with Dex (10 mg/rat per day). In a reversal study, daily injections of Dex or saline began 8 days before NAC and cotreatment continued for 5 days. Systolic blood pressure (SBP) was measured on alternate days using a tail-cuff system. 3. Dexamethasone significantly increased SBP from 113 +/- 4 to 139 +/- 6 mmHg (n = 10; P < 0.01). N-Acetylcysteine alone had no effect on SBP. In NAC + Dex-treated rats, SBP was significantly lower than that of Dex-treated rats (P cent < 0.01). In fully established Dex-hypertension NAC was ineffective and SBP remained high. 4. Both Dex and NAC treatments decreased bodyweight gain. N-Acetylcysteine reduced food and water consumption. Dexamethasone reduced thymus weight (P cent < 0.01) but NAC treatment did not alter this marker of glucocorticoid activity. 5. Dexamethasone tended to decrease plasma NO(x), whereas NAC restored plasma NO(x) concentrations to control levels. N-Acetylcysteine had no effect on Dex-induced increased plasma F(2)-isoprostane concentrations. 6. In conclusion, NAC partially prevented, but did not reverse, Dex-induced hypertension.     

Inhibition of human platelet aggregation by a novel S-nitrosothiol is abolished by haemoglobin and red blood cells in vitro: implications for anti-thrombotic therapy

1. S-Nitrosothiols are nitric oxide (NO) donor drugs that have been shown to inhibit platelet aggregation in platelet rich plasma (PRP) in vitro and to inhibit platelet activation in vivo. The aim of this study was to compare the platelet effects of a novel S-nitrosated glyco-amino acid, RIG200, with an established S-nitrosothiol, S-nitrosoglutathione (GSNO) in PRP, and to investigate the effects of cell-free haemoglobin and red blood cells on S-nitrosothiol-mediated inhibition of platelet aggregation. 2. The effects of GSNO and RIG200 in collagen (2.5 microg ml(-1))-induced platelet aggregation in PRP and whole blood were investigated in vitro. Both compounds were found to be powerful inhibitors of aggregation in PRP, and RIG200 was significantly more potent (IC(50)=2.0 microM for GSNO and 0.8 microM for RIG200; P=0.04). 3. Neither compound inhibited aggregation in whole blood, even at concentrations of 100 microM. Red blood cell concentrations as low as 1% of the haematocrit, and cell-free haemoglobin (> or = 2.5 microM), significantly reduced their inhibitory effects on platelets. 4. Experiments involving measurement of cyclic GMP levels, electrochemical detection of NO and electron paramagnetic resonance of haemoglobin in red blood cells, indicated that scavenging of NO generated from S-nitrosothiols by haemoglobin was responsible for the lack of effect of S-nitrosothiols on platelets in whole blood. 5. These studies suggest that scavenging of NO by haemoglobin in blood might limit the therapeutic application of S-nitrosothiols as anti-platelet agents.     

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.