Hydrogen peroxide-induced impairment of reactivity in rat isolated aorta: potentiation by 3-amino-1,2,4-triazole.

1. In this study the impairment induced by hydrogen peroxide of vascular reactivity and the role of endogenous catalase in protection against this impairment was assessed in isolated rings of rat aorta. 2. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout depressed, in a time-dependent manner, the subsequent ability of endothelium-containing and endothelium-denuded rings to contract to phenylephrine. 3. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) to inhibit endogenous catalase had no effect by itself on subsequent phenylephrine-induced contraction. However, pretreatment with 3-amino-1,2,4-triazole did lead to a profound enhancement of the ability of hydrogen peroxide (1 mM, present for the final 30 min of the 90 min incubation, followed by washout) to depress phenylephrine-induced contraction in both endothelium-containing and endothelium-denuded rings. 4. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout inhibited, in a time-dependent manner, the subsequent ability of acetylcholine (10 nM-3 microM) to induce endothelium-dependent relaxation. Furthermore, incubation with hydrogen peroxide 1 mM (30 min, followed by washout) also inhibited relaxation induced by glyceryl trinitrate (1-100 nM) or isoprenaline (10 nM-3 microM) in endothelium-denuded rings. 5. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) had no effect by itself on relaxation induced by acetylcholine, glyceryl trinitrate or isoprenaline. In contrast, pretreatment with 3-amino-1,2,4-triazole led to profound enhancement of the ability of hydrogen peroxide (1 mM, present for final 30 min of the 90 min incubation) to block relaxation to acetylcholine, glyceryl trinitrate or isoprenaline. 6. On the basis of the actions of 3-amino-1,2,4-triazole, it is likely that endogenous catalase plays an important role in the protection of vascular reactivity of rat aorta against oxidant damage by high (1 mM) but not lower (0.1 mM) concentrations of hydrogen peroxide. The data are consistent with the promotion of non-selective damage to the vascular smooth muscle cells by hydrogen peroxide, but endothelial damage may also be sustained.

The inhibitory effect of 3-amino-1,2,4-triazole on relaxation induced by hydroxylamine and sodium azide but not hydrogen peroxide or glyceryl trinitrate in rat aorta.

1. In this study we investigated the role of catalase in relaxation induced by hydroxylamine, sodium azide, glyceryl trinitrate and hydrogen peroxide in isolated rings of rat aorta. 2. Hydrogen peroxide (1 microM-1 mM)-induced concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings. In endothelium-denuded rings, however, higher concentrations (30 microM-1 mM) of hydrogen peroxide were required to produce relaxation. The endothelium-dependent component of hydrogen peroxide-induced relaxation was abolished following pretreatment with N(O)-nitro-L-arginine methyl ester (L-NAME, 30 microM). L-NAME (30 microM) had no effect, however, on hydrogen peroxide-induced relaxation in endothelium-denuded rings. 3. Pretreatment of endothelium-denuded rings with catalase (1000 u ml-1) blocked relaxation induced by hydrogen peroxide (10 microM-1 mM). The ability of catalase to inhibit hydrogen peroxide-induced relaxation was partially blocked following incubation with 3-amino-1,2, 4-triazole (AT, 50 mM) for 30 min and completely blocked at 90 min. 4. Pretreatment of endothelium-denuded rings with methylene blue (MeB, 30 microM) inhibited relaxation induced by hydrogen peroxide (10 microM-1 mM), sodium azide (1-300 nM), hydroxylamine (1-300 nM) and glyceryl trinitrate (1-100 nM) suggesting that each acted by stimulation of soluble guanylate cyclase. 5. Pretreatment of endothelium-denuded rings with AT (1-50 mM, 90 min) to inhibit endogenous catalase blocked relaxation induced by sodium azide (1-300 nM) and hydroxylamine (1-300 nM) but had no effect on relaxation induced by hydrogen peroxide (10 microM-1 mM) or glyceryl trinitrate (1-100 nM). 6. In a cell-free system, incubation of sodium azide (10 microM-3 mM) and hydroxylamine (10 microM-30 mM) but not glyceryl trinitrate (10 microM-1 mM) with catalase (1000 u ml-1) in the presence of hydrogen peroxide (1 mM) led to production of nitrite, a major breakdown product of nitric oxide. AT (1-100 mM) inhibited, in a concentration-dependent manner, the formation of nitrite from azide in the presence of hydrogen peroxide. 7. These data suggest that metabolism by catalase plays an important role in the relaxation induced by hydroxylamine and sodium azide in isolated rings of rat aorta. Relaxation appears to be due to formation of nitric oxide and activation of soluble guanylate cyclase. In contrast, metabolism by catalase does not appear to be involved in the relaxant actions of hydrogen peroxide or glyceryl trinitrate.

Induction of nitric oxide synthase by endotoxin in rat isolated aorta but not in rat aortic smooth muscle cells grown in culture from explant.

Incubation of endothelium-denuded rings of rat aorta at 37 degrees C for 18 hours in Krebs solution led to a profound depression of the contractile actions of phenylephrine (1 nM-10 mu M). A major component of this depression of vasoconstriction was due to the relaxant actions of nitric oxide since it was reversed following inhibition of the synthesis of nitric oxide with N(G)-nitro-L-arginine methyl ester or its actions with haemoglobin (30 microM) or methylene blue (10 mu M). The depression was also reversed upon treatment with LY83583 (0.1-1 microM which generates superoxide anions, intracellularly and extracellularly, but was unaffected by hypoxanthine (100 microM)/ xanthine oxidase (16 mu/ml) which generates superoxide anion only extracellularly. The ability of polymixin B (30 microM) to inhibit the development of the depression of vasoconstriction suggests that it results from the expression of an inducible form of nitric oxide synthase, stimulated by bacterial lipopolysaccharide, contaminating the Krebs solution. In contrast to aortic rings, we found that lipopolysaccharide (10-10,000 ng/ml) alone from S. typhosa was unable to stimulate the expression of the inducible form of nitric oxide synthase in rat aortic smooth muscle cells grown in culture from explant, as assessed either by measuring the accumulation of nitrite into the culture medium during a 24 hour incubation period or by measuring the smooth muscle cyclic GMP content. Interferon-gamma (1-100 IU/ml) and interleukin-1 alpha (1-10 IU/ml) alone were, however, able to stimulate the accumulation of nitrite in a concentration-dependent manner. These inductions of nitrite accumulation were abolished following treatment with N(G)-nitro-(L)-arginine methyl ester (1 mM) and dexamethasone (1 microM). Further investigations are required to determine whether the ability of bacterial lipopolysaccharide to induce the inducible form of nitric oxide synthase in rat aortic rings, but not in rat aortic smooth muscle cells in culture, results from the presence of an endotoxin-sensitive, cytokine-secreting cell type in the vessel wall which is absent in culture, or from differences in smooth muscle phenotype in situ and in culture.

Differential sensitivity of basal and acetylcholine-stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta.

1. In this study we compared the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)-stimulated activity of NO in isolated rings of rat aorta. 2. Superoxide dismutase (SOD, 1-300 u ml-1) induced a concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings which was blocked by NG-nitro-L-arginine (L-NOARG, 30 microM), but had no effect on endothelium-denuded rings. It was likely therefore that the relaxant action of SOD resulted from protection of basally produced NO from destruction by superoxide anion, generated either within the tissue or in the oxygenated Krebs solution. 3. In contrast, a concentration of SOD (50 u ml-1) which produced almost maximal enhancement of basal NO activity, had no effect on ACh (10 nM-3 microM)-induced relaxation. 4. In the presence of catalase (3000 u ml-1) to prevent the actions of hydrogen peroxide, superoxide anion generation using hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 16 mu ml-1) produced an augmentation of PE-induced tone in endothelium-containing but not endothelium-denuded rings. This was likely to have resulted from removal of the tonic vasodilator action of basally-produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml-1), NG-monomethyl-L-arginine (L-NMMA, 30 microM) or L-NOARG (30 microM). Pyrogallol (0.1 mM) had a similar action to HX/XO, but produced an additional augmentation of tone by an endothelium-independent mechanism. 5. In contrast to their ability to destroy almost completely the basal activity of NO, HX (0.1 mM)/XO(16 mu ml-1) and pyrogallol (0.1 mM) had no effect on ACh-induced relaxation at any concentration. An increase in the concentration of HX to 1 mM or pyrogallol to 0.3 mM did, however, lead to a profound decrease in the magnitude and time course of ACh-induced relaxation at all concentrations.6. Treatment with diethyldithiocarbamate (DETCA, 0.1 mM, 1 h) to inhibit endogenous Cu-Zn SOD,augmented PE-induced tone in endothelium-containing rings and abolished the ability of HX (0.1 mM)/XO (16 mu ml-1) and L-NMMA (30 microM) to augment tone. It was likely that DETCA had led to the destruction of basal NO activity by increasing superoxide anion levels since its actions were reversed by exogenous SOD (10-300 upsilon ml-1).7. In contrast to its ability to destroy basal activity of NO completely, DETCA (0.1 mM) produced only a slight blockade of ACh-induced relaxation. However, if these tissues were subsequently treated with concentrations of HX (0.1 mM)/XO (16 mu ml-1) or pyrogallol (0.1 mM), which had no effect by themselves on ACh-induced relaxation, a profound blockade was seen and this was reversed completely with SOD (250 u ml-1).8. The data suggest that basal activity of NO is more sensitive to inactivation by superoxide anion than ACh-stimulated activity and this probably results from differential protection by endogenous Cu-ZnSOD. It is possible therefore that endogenous SOD lowers superoxide anion levels to such an extent that only small amounts of NO, such as those produced under basal conditions, are destroyed. Following generation of superoxide anion with HX/XO or pyrogallol, or inhibition of Cu-Zn SOD with DETCA,levels of the free radical will increase such that greater amounts of NO, such as those produced following stimulation with ACh, will then be destroyed.