Inhibition of nitrergic neurotransmission in the bovine retractor penis muscle by an oxidant stress: effects of superoxide dismutase mimetics

1. A number of superoxide dismutase (SOD) mimetics were examined both biochemically for their ability to inhibit the superoxide-catalyzed reduction of cytochrome c and nitro blue tetrazolium, and functionally for their ability to mimic authentic Cu/Zn SOD in restoring nitrergic neurotransmission in bovine retractor penis (BRP) muscle following its inhibition by oxidant stress.
2. The SOD mimetics investigated were CuSO₄, MnCl₂, CuDIPS (copper [II] [diisopropylsalicylate]₂), MnTBAP (manganese [III] tetrakis 4-benzoic acid porphyrin), MnTMPyP (manganese [III] tetrakis 1-methyl-4-pyridyl porphyrin pentachloride), tiron (4,5-dihydroxy-1,3-benzene disulphonic acid), PTIYO (4-phenyl,2,2,5,5,-tetramethyl-3-imidazolin-1-yloxy-3-oxide) and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl).
3. The rank order of potency in inhibiting the reduction of cytochrome c was: CuSO₄⩾MnCl₂⩾CuDIPS⩾MnTMPyP>MnTBAP>tempol⩾tiron>PTIYO.
4. The requirement for EDTA (0.1 mM) prevented assessment of the activity of CuSO₄, MnCl₂ and CuDIPS in the assay involving inhibition of reduction of nitro blue tetrazolium. However, the rank order of potency for those agents which could be examined (MnTMPyP>MnTBAP>tiron⩾tempol>PTIYO) was essentially similar to that seen in the cytochrome c assay.
5. Inhibition of endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA, 3 mM, 120 min) in BRP muscle strips, followed by addition of the superoxide anion generator, LY 83583 (1 μM), resulted in almost complete abolition of nitrergic relaxation (4 Hz, 10 s).
6. Authentic Cu/Zn SOD (1–300 u ml⁻¹), CuSO₄ (0.1–300 μM), MnCl₂ (0.1–100 μM) and MnTMPyP (10–300 μM) each restored nitrergic transmission by around 50%. However, CuDIPS (0.1–30 μM), MnTBAP (0.1–100 μM), tempol (10 μM–3 mM), PTIYO (1–300 μM) and tiron (10 μM–10 mM) all failed to restore nitrergic transmission.
7. The ability of MnTMPyP to restore nitrergic neurotransmission may therefore provide a lead in the development of SOD mimetics as therapeutic agents in the treatment of neuropathies associated with oxidant stress.

     

Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta

1. In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion.
2. Ascorbate produced concentration-dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1–300 μM, reached a maximum of 45.3±2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 μM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid.
3. Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM–10 μM) on endothelium-containing rings or adenosine (0.1 μM–3 mM) on endothelium-denuded rings.
4. An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml⁻¹). This reduced maximal acetylcholine-induced relaxation from 96.7±1.3% to 42.4±3.5% (P<0.001). Treatment with ascorbate (30 μM–3 mM) reversed this blockade in a concentration-dependent manner.
5. Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Quinacrine increases endothelial nitric oxide release: role of superoxide anion

The effect of acute quinacrine treatment on agonist-induced nitric oxide (NO) release was investigated in cultured human endothelial cells using electrochemical monitoring of the in situ NO concentration. Quinacrine dose-dependently increased NO release with an apparent EC50 of 0.2 microM and a maximal effect at 1 microM. Quinacrine did not modify the dependence of NO release on extracellular L-arginine. Acceleration or deceleration of O2- dismutation, which altered NO release in control cells, did not modify it in quinacrine-treated cells. Quinacrine did not modify NO amperometric signal or reaction with O2- produced by xanthine oxidation. In the presence of quinacrine, agonist-induced NO release became Mg2+ -independent and could not be attributed to an inhibition of phospholipase A2 activity. Quinacrine made NO release insensitive to Cu2+ chelation. The present study demonstrates that acute treatment by low quinacrine concentrations increases endothelial NO release, possibly through an inhibition of O2- production.     

Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta

1. The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated.
2. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.3 and 0.1 μM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium.
3. High concentrations of catechin (in the range of 10⁻¹ g l⁻¹) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium.
4. The NO synthase inhibitor, N^ω-nitro-L-arginine-methyl-ester (L-NAME, 300 μM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds.
5. In contrast to superoxide dismutase (SOD, 100 u ml⁻¹), neither RWPC nor leucocyanidol affected the concentration-response curve for the NO donor, SIN-1 (3-morpholino-sydnonimine) which also produces superoxide anion (O₂⁻).
6. In aortic rings with endothelium, RWPC (10⁻² g l⁻¹) produced a 7 fold increase in the basal production of guanosine 3′ : 5′-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (300 μM).
7. Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe²⁺-diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (300 μM).
8. These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O₂⁻. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.

     

不同脂质载体对超氧化物歧化酶(SOD)渗入小白鼠红细胞能力的观察

本文观察了单纯注射超氧化物歧化酶和脂质体及脂肪乳携带超氧化物歧化酶渗入小白鼠红细胞的能力。发现脂质体及脂肪乳携带超氧化物歧化酶渗入小白鼠红细胞的能力比单纯注射超氧化物歧化酶大(P<0.05～0.01)。该结果为解决超氧化物歧化酶渗入细胞发挥治疗作用提供了简易方法和实验根据。     

Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

Aim: The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relaxation against superoxide anion inhibition in the mouse duodenum. Methods: The effects of exogenous SOD, N , N '-bis(salicylidene) ethylenediamine chloride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues. Results: Diethyldithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electrical field stimulation (EFS). SOD and ascorbate partially prevented the inhibitory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by EUK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exogenous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8. Conclusion: These results suggest that the nitrergic neurotransmission in the mouse duodenum is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endogenous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.     

Arsenic alters monocyte superoxide anion and nitric oxide production in environmentally exposed children

Arsenic (As) exposure has been associated with alterations in the immune system, studies in experimental models and adults have shown that these effects involve macrophage function; however, limited information is available on what type of effects could be induced in children. The aim of this study was to evaluate effects of As exposure, through the association of inorganic As (iAs) and its metabolites [monomethylated arsenic (MMA) and dimethylated arsenic (DMA)] with basal levels of nitric oxide (NO⁻) and superoxide anion (O₂⁻), in peripheral blood mononuclear cells (PBMC) and monocytes, and NO⁻ and O₂⁻ produced by activated monocytes. Hence, a cross-sectional study was conducted in 87 children (6-10 years old) who had been environmentally exposed to As through drinking water. Levels of urinary As species (iAs, MMA and DMA) were determined by hydride generation atomic absorption spectrometry, total As (tAs) represents the sum of iAs and its species; tAs urine levels ranged from 12.3 to 1411 μg/g creatinine. Using multiple linear regression models, iAs presented a positive and statistical association with basal NO⁻ in PBMC (β = 0.0048, p = 0.049) and monocytes (β = 0.0044, p = 0.044), while basal O₂⁻ had a significant positive association with DMA (β = 0.0025, p = 0.046). In activated monocytes, O₂⁻ showed a statistical and positive association with iAs (β = 0.0108, p = 0.023), MMA (β = 0.0066, p = 0.022), DMA (β = 0.0018, p = 0.015), and tAs (β = 0.0013, p = 0.015). We conclude that As exposure in the studied children was positively associated with basal levels of NO⁻ and O₂⁻ in PBMC and monocytes, suggesting that As induces oxidative stress in circulating blood cells. Additionally, this study showed a positive association of O₂⁻ production with iAs and its metabolites in stimulated monocytes, supporting previous data that suggests that these cells, and particularly the O₂⁻ activation pathway, are relevant targets for As toxicity.     

Effects of superoxide dismutase mimetics on the activity of nitric oxide in rat aorta.

A number of structurally distinct superoxide dismutase (SOD) mimetics were examined to determine if they shared the ability of authentic Cu/Zn SOD to produce endothelium-dependent relaxation of rings of rat aorta by protecting basal nitric oxide from destruction by endogenously produced superoxide anion. MnCl2 (10 nM-100 microM), CuSO4 (100 nM-1 mM) and CuDIPS (Cu [II]-[diisopropylsalicylate]2; 100 nM-30 microM) each mimicked the ability of Cu/Zn SOD (0.1-300 u ml(-1)) to produce relaxation of phenylephrine-precontracted aortic rings in a manner inhibited by endothelial removal or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM). In contrast, MnTMPyP (Mn [III] tetrakis [1-methyl-4-pyridyl] porphyrin; 10 nM-30 microM) augmented phenylephrine-induced contraction and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction rather than protection of basal nitric oxide activity. Pretreatment with Cu/Zn SOD (250 u ml(-1)) blocked this augmentation suggesting that it arose paradoxically through destruction of nitric oxide by superoxide anion. The spin trap agents tiron (100 nM-1 mM), tempol (100 nM-1 mM) and PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide; 100 nM-300 microM) all failed to promote endothelium-dependent relaxation. In fact, the last two augmented phenylephrine-induced tone and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction of basal nitric oxide activity. This destruction was unaffected by pretreatment with Cu/Zn SOD (250 u ml(-1)) and probably reflected the direct ability of tempol and PTIYO to destroy nitric oxide. Thus, the ideal SOD mimetic for protection of nitric oxide activity in conditions of oxidant stress still awaits development.     

Increased renal vascular resistance in zinc-deficient rats: role of nitric oxide and superoxide

1. Zinc deficiency (ZD) induces many kinds of pathological states. However, the effects of ZD on haemodynamics remain unclear. In the present study, we measured mean blood pressure (BP) and renal blood flow (RBF) under anaesthesia and calculated renal vascular resistance (RVR) from these parameters in rats maintained on a ZD diet (0.5 p.p.m. zinc) for 4 weeks. 2. Zinc deficiency did not change mean BP, but significantly reduced RBF and increased RVR (each P < 0.01). In addition, these effects of ZD were reversible. 3. Because Cu/Zn superoxide dismutase (SOD) is a zinc-containing enzyme and superoxide is a potent scavenger of nitric oxide (NO), a vasodilator, we hypothesized that one of the mechanisms by which ZD increases RVR is by decreasing NO bioavailability by the enhanced formation of superoxide due to low Cu/Zn SOD activity. To test this hypothesis, we observed the roles of NO and superoxide in the mechanism, after having confirmed the low activity of Cu/Zn SOD in the kidneys of ZD rats. 4. Administration of the SOD mimetic tempol (5 mg/kg per min) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that superoxide was responsible for the mechanism. Low doses of the NO donor sodium nitroprusside (SNP; 2.0 micro g/kg per min, continuous) decreased RVR to a significantly smaller extent in ZD rats compared with control, whereas a high dose of SNP (0.75 mg/kg, bolus) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that the mechanism includes an inhibition of NO activity in ZD, which is most likely to be a scavenging of NO by the activated superoxide. 5. In summary, ZD may increase RVR. The mechanism probably includes changes in NO and superoxide activities.     

Endogenous nitric oxide attenuates beta-adrenoceptor-mediated relaxation in rat aorta

1. Divergent evidence suggests that the intracellular signalling pathways for beta-adrenoceptor-mediated vascular relaxation involves either cAMP/protein kinase (PK) A or endothelial nitric oxide (NO) release and subsequent activation of cGMP/PKG. The present study identifies the relative roles of NO and cAMP, as well as dependence on the endothelium for beta-adrenoceptor-mediated relaxation of rat isolated aortas. 2. Cumulative concentration-response curves to isoprenaline (0.01-3 micromol/L) in phenylephrine (0.1 micromol/L)-preconstricted endothelium-intact and -denuded aortas were constructed. Isoprenaline-mediated relaxation was partially reduced by endothelium removal and the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (0.1 mmol/L), but not by the cAMP antagonist (Rp)-cyclic adenosine-3',5'-monophosphorothioate (Rp-cAMPS; 0.5 mmol/L). 3. In contrast, in endothelium-denuded aortas, the isoprenaline-mediated relaxation was inhibited by Rp-cAMPS and this inhibition was lost in the presence of the NO donor sodium nitroprusside (1 nmol/L). This effect was not due to phosphodiesterase (PDE) activity because the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (1 micromol/L) failed to affect the isoprenaline vasorelaxant response. 4. The K(+) channel blocker tetraethylammonium (TEA; 1 mmol/L) attenuated isoprenaline-induced relaxation in endothelium-denuded aorta, but its effect was non-additive with Rp-cAMPS, suggesting that the K(+) channel component may involve cAMP. In endothelium-intact aortas, TEA but not Rp-cAMPS reduced isoprenaline relaxation, suggesting an additional non-cAMP component. 5. These findings suggest that beta-adrenoceptors induce vascular smooth muscle relaxation by acting through the NO-cGMP pathway and, when that is disrupted by endothelium removal or the presence of an NO synthase inhibitor, the cAMP pathway in smooth muscles is used. The lack of cAMP participation in endothelium-intact vessels may be because NO suppresses or overrides the cAMP effect.     

Developmental changes in endothelium-dependent vasodilation and the influence of superoxide anions in perinatal rabbit pulmonary arteries

1. ACh-induced vasodilation was investigated in pulmonary arteries from 8 and 2 day pre-term foetal, neonatal (0–12 h and 4 day old) and adult rabbits. The effects of superoxide anion generation [with hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 15 mu ml⁻¹)], endogenous superoxide dismutase (SOD) inhibition [with the Cu-Zn SOD inhibitor triethylenetetramine (TETA, 1 mM)], endogenous superoxide anion scavenging [by superoxide dismutase (SOD, 50 u ml⁻¹)] and inhibition of endothelial nitric oxide synthase (eNOS) [with, N^ω-nitro-L-arginine methylester (L-NAME, 0.1 mM)], on basal and ACh-induced NO activity were studied by examining phenylephrine-induced contraction and ACh-induced vasodilation respectively.
2. L-NAME and endothelium removal abolished all ACh-induced vasodilation and 1 μM sodium nitroprusside fully dilated all vessels. ACh-induced vasodilation was absent in the 8 day pre-term foetus and 0–12 h neonate but present at all other ages. L-NAME itself contracted 2 day pre-term foetal vessels. At 0–12 h, SOD, but not the phosphodiesterase 5 inhibitor zaprinast (1 μM), uncovered ACh-induced vasodilation. At this age SOD reduced phenylephrine-induced contraction which was not influenced by TETA, L-NAME or HX/XO, and L-NAME itself did not cause contraction. This suggests both ACh-induced and basal NO activity are compromise in these vessels by endogenous superoxide anion production and deficiencies in endogenous SOD activity.
3. In 4 day vessels, but not adult vessels, L-NAME, TETA and HX/XO augmented contractions to phenylephrine, and L-NAME itself induced vasoconstriction, suggesting that basal NO and SOD activities were present by 4 days but were not evident in the adult. ACh-induced NO activity, and the influence of endogenous SOD on this, were present in the adult (and 4 day) vessels as superoxide generation with HX/XO significantly reduced ACh-induced vasodilation and this effect was inhibited by SOD and augmented by TETA.
4. Increased oxygen tensions >500 mmHg attenuated ACh-induced vasodilation in the foetal but not neonatal rabbits. Raising the oxygen tension from ∼20 to ∼120 mmHg revealed ACh-induced vasodilation in the 8 day pre-term vessels.
5. In summary, superoxide anion accumulation combined with deficiencies in SOD activity may transiently compromise basal and ACh-induced NO activity at birth. Experimental oxygen tensions markedly influence ACh-induced vasodilation in foetal rabbit pulmonary arteries.

     

内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节

目的　以内皮细胞产生NO的关键酶———eNOS(内皮一氧化氮合酶 )为研究目标 ,探讨外源性内皮源性超极化因子EDHF(EETs)对内皮细胞合成NO的影响。方法　在原代培养 3～ 4代以内的牛主动脉内皮细胞中 ,分别加入不同浓度 (5 0～ 2 0 0nmol·L-1)的 8,9 EET、11,12 EET、14 ,15 EET ,作用 1h后用不同的方法收获细胞。用WesternBlot以及NorthernBlot方法检测EETs对eNOS基因表达的影响 ;同时通过检测L [3 H] 精氨酸转化为L [3 H] 瓜氨酸的量研究EETs对NOS活性的影响。结果　显示 8,9 EET、11,12 EET、14 ,15 EET均呈浓度依赖性地增加eNOS蛋白质的表达 ,并提高eNOSmRNA表达水平以及NOS酶活性。结论　外源性EDHF对eNOS基因表达是一种正反馈调节作用 ,从而能够促进内皮细胞NO的产生 ,通过药物调节内皮表氧化酶进而促进eNOS基因表达可作为防治心血管疾病的新策略     

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.     

Vascular non-endothelial nitric oxide induced by swimming exercise stress in rats

1. Herein, we report the effects of acute or chronic forced swimming on vascular responsiveness to angiotensin (Ang) II. 2. The possible involvement of locally produced substances, such as nitric oxide (NO) and prostanoids, in these effects were studied in rat thoracic aorta and superior mesenteric arteries. 3. Chronic, but not acute, swimming reduced the efficacy (maximal effect; Emax) of AngII in thoracic aorta and mesenteric arteries, either with intact or denuded endothelium. 4. The efficacy of AngII was reduced in the presence of indomethacin in mesenteric arteries, but not in the aorta, from either control or chronically stressed rats. 5. Treatment with NG-monomethyl-l-arginine reversed the effect of chronic stress on the response to AngII, suggesting that chronic stress may increase non-endothelial NO activity in both the aorta and mesenteric arteries. 6. The effects of acute and chronic stress on vascular reactivity were selective for AngII because no changes were observed on the effects of phenylephrine.     

依达拉奉对LPC所致兔胸主动脉内皮功能的影响及机制

目的探讨依达拉奉(edaravone,Eda)对溶血磷脂酰胆碱(lysophosphatidylcholine,LPC)所致家兔血管内皮损伤的影响及机制。方法家兔胸主动脉环分别与LPC(5 mg.L-1)和Eda(25～100μmol.L-1)单独孵育或共孵育,分别检测乙酰胆碱诱导的内皮依赖性舒张反应和硝普钠诱导的非内皮依赖性舒张反应,血管组织中一氧化氮(nitric oxide,NO)和丙二醛(malonaldehyde,MDA)含量以及超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果 5 mg.L-1LPC孵育血管环30 min,明显抑制了乙酰胆碱诱导的内皮依赖性舒张反应,但没有影响硝普钠诱导的非内皮依赖性舒张反应,降低了血管组织中NO含量和SOD活性而增加了MDA含量。25～100μmol.L-1Eda分别孵育血管环15min,再与5 mg.L-1LPC共同孵育30 min,明显改善LPC所致的血管舒张功能的损害,升高了血管组织中NO含量和SOD活性而降低了MDA含量。结论 Eda对LPC所致的血管内皮依赖性舒张功能的损伤具有明显的保护作用,该效应可能与其抗氧化作用有关。     

Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000×g rat cerebellar supernatant preparation in a concentration-dependent manner (IC₅₀ value=12.9 μM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a K_i value of 2.57 μM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl₂/calmodulin, indicating that the presence of CaCl₂/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl₂/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.     

Nitric oxide, adenosine deaminase, xanthine oxidase and superoxide dismutase in patients with panic disorder: alterations by antidepressant treatment

OBJECTIVE: In the present study, we aimed to investigate whether nitric oxide (NO) levels and activities of xanthine oxidase (XO), superoxide dismutase (SOD), and adenosine deaminase (ADA) are associated with Panic disorder (PD) as well as impact of psychopharmacological treatments on NO, SOD, ADA, and XO levels in PD. METHOD: In this study, 32 patients and 20 healthy controls were included. The serum levels of NO, XO, SOD, and ADA were measured in the patients and controls. The patients were treated with antidepressant. RESULTS: ADA and XO levels of the patients were significantly higher than the controls. SOD levels of the patients were significantly lower than the controls but the difference was not statistically significant. Although NO levels of the patients were higher than the controls, the difference was not statistically significant. There was no correlation between PAS and the parameters studied (SOD, ADA, XO, and NO) of the patients. After 8 weeks of antidepressant treatment, ADA and SOD activities were increased whereas NO and XO levels decreased significantly. CONCLUSION: ADA, XO activity may have a pathophysiological role in PD, and prognosis of PD. Activity of these enzymes may be used to monitor effects of the antidepressant treatment.     

化疗对急性白血病外周血NO、SOD、OFR及MDA影响的探讨

目的 :探讨化疗对急性白血病 (AL)外周血一氧化氮 (NO)、超氧化物歧化酶 (SOD)、氧自由基 (OFR)及丙二醛 (MDA)的影响。方法 :对 4 6例 AL患者的外周血 ,采用比色法检测其 NO、SOD、OFR和 MDA的水平变化 ,并与正常人进行比较。结果 :AL患者外周血 NO水平与正常对照差异无显著性 (P>0 .0 5 ) ,而 SOD、OFR水平明显低于正常对照组 (P<0 .0 1) ,MDA水平化疗前明显高于正常对照组 (P<0 .0 1) ,化疗药物对 AL 外周血中各个时期的影响差异无显著性 (P>0 .0 5 )。结论 :NO在急性白血病发病过程中的影响还需进一步商榷 ,SOD和 OFR低水平状态及 MDA水平升高提示参与了急性白血病的发病过程 ,化疗药物对患者外周血中的 NO、SOD、OFR和 MDA水平的变化影响不大。     

Sequential induction of heme oxygenase-1 and manganese superoxide dismutase protects cultured astrocytes against nitric oxide

Nitric oxide (NO) is a widely recognized mediator of physiological and pathophysiological signal transmission. In an attempt to better understand the molecular actions of NO in astrocytes, stress protein expression in response to NO donor sodium nitroprusside was investigated. Heme oxygenase-1 (HO-1) has been identified as an inducer of manganese superoxide dismutase (MnSOD), playing a cytoprotective role under the condition of nitrosative stress. We present evidence that the sequential induction of HO-1 and MnSOD protects astrocytes from NO toxicity: (1) both HO-1 and MnSOD expression were induced by NO; (2) NO-mediated increase in MnSOD activity was partly abolished by HO-1 inhibitor Zn(II) protoporphyrin IX (ZnPP); (3) pretreatment of astrocytes with a nontoxic dose of NO protected the cells against the later treatment with a toxic dose of NO; (4) inhibition of HO-1 by ZnPP sensitized astrocytes to the nontoxic dose of NO resulting in a marked cytotoxicity; and (5) adenovirus-mediated overexpression of MnSOD protected astrocytes from the NO toxicity. The molecular action of NO in astrocytes appears to be dose-dependent. While a high dose of NO exerts cytotoxicity leading to the tissue damage in the central nervous system, a low dose of NO may act as an important signaling molecule in astrocytes with concurrent induction of cytoprotective proteins such as HO-1 and MnSOD.