Mitochondrial nitric oxide synthase

Mitochondria produce nitric oxide (NO) through a Ca(2+)-sensitive mitochondrial NO synthase (mtNOS). The NO produced by mtNOS regulates mitochondrial oxygen consumption and transmembrane potential via a reversible reaction with cytochrome c oxidase. The reaction of this NO with superoxide anion yields peroxynitrite, which irreversibly modifies susceptible targets within mitochondria and induces oxidative and/or nitrative stress. In this article, we review the current understanding of the roles of mtNOS as a crucial biochemical regulator of mitochondrial functions and attempt to reconcile apparent discrepancies in the literature on mtNOS.     

Endothelial nitric oxide synthase gene therapy for erectile dysfunction

Basic science research on erectile physiology has been devoted to investigating the pathogenesis of erectile dysfunction (ED) and has led to the conclusion that ED is predominately a disease of vascular origin. It is well recognized that the incidence of ED dramatically increases in men who suffer from diabetes mellitus, hypercholesterolemia, and cardiovascular disease. Endothelial nitric oxide synthase (eNOS) is an important factor in cardiovascular homeostasis, angiogenesis, and erectile function. Given the impact of endothelial-derived nitric oxide (NO) in vascular biology, a great deal of research over the past decade has focused on the role of NO synthesis from the endothelium in normal erectile physiology as well as in disease states. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED. Therefore, a likely target of gene therapy for the treatment of ED is eNOS. This communication reviews the role of eNOS in erectile physiology and discusses the alterations in eNOS expression in various vascular diseases of the penis. Putative gene therapy interventions to restore eNOS expression and subsequent endothelial function may represent an exciting new therapeutic strategy for the future treatment of ED.     

内源性一氧化氮合酶抑制物:一种新的内皮功能不全预测因子

血管内皮功能不全是多种心血管疾病的共同病理基础 .内源性一氧化氮合酶 (NOS)抑制物能阻止内皮细胞 (NO)合成 ,导致血管内皮功能降低 .高血压 ,动脉粥样硬化 ,糖尿病血管并发症等心血管疾病的内皮功能不全与内源性 NOS抑制物含量升高密切相关 .内源性 NOS抑制物可作为内皮功能不全的预测因子 ;阻止或抑制内源性 NOS抑制物生成可能是寻找防治心血管疾病药物的新途径 .     

线粒体靶向抗氧化剂研究进展

线粒体是细胞呼吸的主要场所,在细胞的生命周期中扮演重要角色,三羧酸循环和氧化磷酸化都是在线粒体中进行。线粒体功能障碍可导致一系列疾病,如缺血-再灌注损伤、败血症和糖尿病等。线粒体是神经退行性病变的治疗靶点,也是药物转运策略研究的引人注目的靶位。虽然线粒体所介导的疾病进程的分子机制尚未完全阐明,但氧化应激是关键的环节。开发线粒体靶向的抗氧化应激保护药物具有诱人的前景。线粒体靶向抗氧化剂是指以线粒体为作用靶位的具有抗氧化作用的药物。该文介绍了现有的线粒体靶向抗氧化剂的概念、分类及其疾病治疗研究进展。     

Correlation between brain nitric oxide synthase activity and opiate withdrawal

The opiate withdrawal induced by administration of naloxone to morphine-dependent mice correlates with an increment of calcium- dependent nitric oxide synthase (NOS) activity in the cerebellum. L-NAME, an irreversible competitive inhibitor of NOS (0.5, 5, 25, 50 mg/kg) injected sc. 45 min. prior to naloxone significantly reduced the number of escape jumps and other motor symptoms of abstinence. In addition, L-NAME also decreased NOS activity in cerebellum. L-arginine, but not D-arginine, when coadministered with L-NAME, prevented both the inhibition of NOS activity and the reduction of withdrawal symptoms induced by L-NAME in morphine-withdrawn animals. These results demonstrate a hyperactivity of the L-arginine: NO pathway in opiate withdrawal and suggests the possibility of a therapeutic use of NOS inhibitors in this state.Abbreviations EAA Excitatory amino acid - LC Locus coeruleus - L-NAME N-nitro L-arginine methyl ester - NMDA N-methyl-D-aspartate - NO Nitric oxide - NOS Nitric oxide synthase     

戊四唑癫痫大鼠脑组织一氧化氮含量和一氧化氮合酶活性变化

探讨一氧化氮在戊四唑癫病发机制中的作用。方法每天注射戊四唑建立在鼠癫痫模型,测定癫病发作后大鼠大脑皮质,海马一氧化氮和一氧化氮合酶活性变化,结果癫痫发作后海马ＮＯ含量和ＮＯＳ活性显著升高,结 戊四唑诱导的癫痫中具有致痫性。     

Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition

1. This study investigated the effects of low dose endotoxin (lipopolysaccharide, LPS) on (i) systemic haemodynamics, (ii) renal blood flow (RBF), (iii) renal cortical and medullary perfusion and (iv) renal function in the anaesthetized rat. We have also investigated the effects of nitric oxide (NO) synthase (NOS) inhibition with N^G-methyl-L-arginine (L-NMMA) on the alterations in systemic and renal haemodynamics and renal function caused by endotoxin.
2. Infusion of low dose LPS (1 mg kg⁻¹ over 30 min, n=6) caused a late fall in mean arterial blood pressure (MAP, at 5 and 6 h after LPS), but did not cause an early (at 1–4 h after LPS) hypotension. The pressor effect of noradrenaline (NA, 1 μg kg⁻¹, i.v.) was significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Infusion of L-NMMA (50 μg kg⁻¹ min⁻¹ commencing 60 min before LPS and continued throughout the experiment, n=7) abolished the delayed hypotension and significantly attenuated the vascular hyporeactivity to NA (at 2–6 h).
3. Infusion of LPS (1 mg kg⁻¹ over 30 min, n=6) caused a rapid (within 2 h) decline in renal function (measured by inulin clearance) in the absence of a significant fall in MAP or renal blood flow (RBF). L-NMMA (n=7) attenuated the impairment in renal function caused by LPS so that the inulin clearance in LPS-rats treated with L-NMMA was significantly greater than in LPS-rats treated with vehicle (control) at 3–6 h after infusion of LPS.
4. Endotoxaemia also caused a significant reduction in renal cortical, but not medullary perfusion (measured as Laser Doppler flux). Infusion of L-NMMA caused a significant further fall in cortical perfusion and a significant fall in medullary perfusion in the absence of changes in RBF.
5. Infusion of LPS resulted in a progressive increase in the plasma levels of nitrite/nitrate (an indicator of the formation of NO), so that the plasma concentration of nitrite/nitrate was significantly higher than baseline at 150 to 330 min after LPS. Infusion of L-NMMA attenuated the rise in the plasma concentration of nitrite/nitrate (at 270 and 330 min, P<0.05) caused by LPS.
6. Thus, the renal dysfunction caused by injection of low dose of endotoxin in the rat occurs in the absence of significant falls in blood pressure or total renal blood flow. Inhibition of NOS activity with L-NMMA attenuates the renal dysfunction caused by endotoxin (without improving intrarenal haemodynamics), suggesting that an overproduction of NO may contribute to the development of renal injury and dysfunction by causing direct cytotoxic effects.

     

Pesticide induced changes of nitric oxide synthase in rat brain in vitro

Organic insecticides are well known neurotoxicants. Nitric oxide (NO) is a neurotransmitter formed stoicheometrically with citrulline from L-arqinine through mediation of the enzyme nitric oxide synthase (NOS). We measured NOS activity in rat brain in vitro in the presence of selected organic insecticides such as, 10-200 microM conc carbaryl, kepone and malathion. All these three compounds inhibited NOS activity of rat brain in vitro in a concentration dependent manner. In most cases the changes observed were statistically significant. The order of potency, based on IC50 values of these insecticides, to inhibit NOS activity of rat brain, is carbaryl (105 microM) > Kepone (144 microM) > malathion (170 microM). We further demonstrated that these insecticides inhibit calmodulin (CaM)-stimulated NOS activity without affecting the basal enzyme activity. It is reported that the observed inhibition of NOS by selected insecticides may be due to interaction of these insecticides with Ca2+/CaM on which the NOS activity is well known to be dependent. This ultimately may lead to neurotoxicity of rat brain.     

Elevated serum endogenous inhibitor of nitric oxide synthase and endothelial dysfunction in aged rats

1. The purpose of the present study was to determine the relationship between endothelial dysfunction and the endogenous inhibitor of nitric oxide synthase NG,NG'-asymmetric dimethylarginine (ADMA) in aged rats. 2. Studies were performed in male adult Sprague-Dawley rats (6 months old; n = 8) and in aged rats (20 months old; n = 8). Serum levels of ADMA and L-arginine were measured by high-performance liquid chromatography and responses of endothelium-intact aortic rings to acetylcholine (ACh) were tested. Nitric oxide synthase activity in kidney tissue and serum concentrations of nitrite, a stable end-product of nitric oxide, were assayed and serum contents of malondialdehyde, derived from lipid peroxidation and serum lipid and creatinine level were determined. 3. Serum levels of ADMA increased significantly in aged rats compared with adult rats (P < 0.01), whereas serum levels of L-arginine were similar in both groups (P = NS). Accordingly, the ratio of L-arginine/ADMA in old rats was lower than that in young rats (P < 0.01). Endothelium-dependent relaxation responses to ACh in aortic rings from aged rats were impaired and these impaired responses were improved by pre-incubation of aortic rings with L-arginine. 4. Nitric oxide synthase activity in the kidney, together with serum concentration of nitrite, was significantly decreased and serum contents of malondialdehyde, cholesterol and triglycerides were increased in old compared with young rats. However, the serum creatinine level was not significantly different between adult and aged rats. 5. Endogenous ADMA may be a contributor to age-related endothelial dysfunction and increases in endogenous ADMA may be linked to lipid peroxidation in aged rats.     

Inducible nitric oxide synthase and inflammation

Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications.     

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase‐2 (MMP‐2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real‐time polymerase chain reaction, respectively. MMP‐2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol‐treated rats showed increased activity of MMP‐2. Histopathological investigation of kidneys from ethanol‐treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol‐treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS‐derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol‐treated rats might contribute to progressive renal damage.     

脑复合剂对创伤性脑损伤模型大鼠脑内NO、nNOS活性及表达的影响

目的:探讨脑复合剂治疗对创伤性脑损伤(traumatic brain injury,TBI)模型大鼠脑保护作用可能的分子机制.方法:用自由落体打击(Feeney法)建立TBI模型,分别设立假手术组、TBI模型组及中药治疗组.中药治疗组给予脑复合剂10 g· kg-1·d-1,假手术组及TBI模型组给予同等剂量的等渗盐水...     

Inhibition of neuronal nitric oxide synthase causes attenuation of cerebrovascular dysfunction in experimental heatstroke

The present study was performed to assess the prophylactic effect of 7-nitroindazole (7-NI), an inhibitor of neuronal nitric oxide synthase (nNOS), in an animal model of heatstroke. Anesthetized rats, immediately before the start of heat stress, were divided into two major groups and given the following: vehicle solution (1 mL per kg body weight) or 7-NI (5-20mg/mL per kg body weight) intraperitoneally. They were exposed to ambient temperature of 43 degrees C to induce heatstroke. Another group of rats were exposed to room temperature (24 degrees C) and used as normothermic controls. Their physiologic and biochemical parameters were continuously monitored. When the vehicle-pretreated rats underwent heat stress, their survival time values were found to be 21-25 min. Pretreatment with intraperitoneal doses of 7-NI significantly improved survival during heatstroke (55-164 min). As compared to those of normothermic controls, all vehicle-pretreated heatstroke animals displayed higher levels of core temperature, intracranial pressure, nitric oxide metabolite (NO(2)(-)), glutamate, glycerol, lactate/pyruvate ratio, neuronal damage score and nNOS expression in the hypothalamus, and tumor necrosis factor-alpha (TNF-alpha) in the serum. In contrast, all vehicle-pretreated heatstroke animals had lower levels of mean arterial pressure, cerebral perfusion pressure, cerebral blood flow, and brain PO(2). Administration of 7-NI before the start of heat exposure significantly reduced the hyperthermia, intracranial hypertension, nNOS-dependent NO(2)(-), glutamate, glycerol, lactate/pyruvate ratio, and neuronal damage score in the hypothalamus, as well as overproduction of TNF-alpha in the serum that occurred during heatstroke. The data show that reduction of nNOS-dependent NO(2)(-) with 7-NI causes attenuation of cerebrovascular dysfunction, hyperthermia, and TNF-alpha overproduction during heatstroke in the rat.     

Demonstrating the dose- and time-related effects of 7-nitroindazole on picrotoxin-induced convulsions, memory formation, brain nitric oxide synthase activity, and nitric oxide concentration in rats

In this study, the dose (50, 100, 150, and 200 mg/kg)- and time (30 and 60 min)- related effects of 7-nitroindazole (7-NI), a neuronal specific inhibitor of nitric oxide synthase (NOS) were tested on picrotoxin (5 mg/kg)-induced convulsions and memory formation in rats. The changes produced by these doses of 7-NI were determined on NOS activity and nitric oxide (NO) concentration in the brain. The effects of 7-NI were tested in animals pretreated (30 min) with L-arginine (500 and 1000 mg/kg). 7-NI, at 50 and 100 mg/kg, did not produce significant changes in NOS activity and NO concentration in the brain and memory formation. However, the convulsant action of picrotoxin was inhibited in a dose-dependent manner in these animals. A time-dependent decrease in the activity of NOS and the concentration of NO, a promotion of picrotoxin-induced convulsions, and an impairment of memory were found in animals treated with 150 and 200 mg/kg of 7-NI. The larger and not the smaller dose of L-arginine raised the concentration of NO, inhibited picrotoxin-induced convulsions and promoted memory process. Either dose of L-arginine failed to prevent 50 and 100 mg/kg of 7-NI from inhibiting convulsions. The effects of the larger doses of 7-NI (150 and 200 mg/kg) were effectively prevented by the increase of NO and not the ineffective dose of L-arginine. These results suggest that 7-NI (50 and 100 mg/kg) decreases convulsions by a nonspecific mechanism and that an inhibition of NOS by the larger doses of it (150 and 200 mg/kg) results in proconvulsant action and memory impairment. The data further show that the margin between the protective and proconvulsant doses of 7-NI is relatively narrow. These results have been taken together with the earlier reports that 7-NI produces learning impairment and fails to increase the anticonvulsant effect of traditional antiepileptic agents on experimentally induced convulsions to conclude that 7-NI can never emerge as an anticonvulsant agent for clinical use.     

慢性阻塞性肺疾病患者外周血脂肪炎症因子一氧化氮一氧化氮合成酶水平及其意义

目的分析慢性阻塞性肺疾病(COPD)患者外周血脂肪炎症因子、一氧化氮(NO)、一氧化氮合成酶(NOS)水平及其意义。方法选取我科2013年1月至2015年1月COPD稳定期患者共49例。根据是否存在肺动脉高压,分为COPD伴肺动脉高压组和COPD不伴肺动脉高压组。选取同期行体检健康志愿者22名作为对照组。对比3组外周血脂肪炎症因子、NO和NOS水平。结果 COPD伴肺动脉高压组的外周血脂肪炎症因子、NO、NOS水平显著低于COPD组不伴肺动脉高压组和对照组(P<0.05);COPD组不伴肺动脉高压组的外周血脂肪炎症因子、NO、NOS显著低于对照组(P<0.05)。COPD伴肺动脉高压组、COPD组不伴肺动脉高压组和对照组的脂肪炎症因子和NO、脂肪炎症因子和NOS、NO和NOS之间均具有相关性(P<0.05)。结论在COPD患者,特别是合并肺动脉高压的患者中,脂肪炎症因子、NO、NOS显著下降,说明三者代谢异常可能和肺循环稳态失衡密切相关。     

Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury.

1. The aim of this study was to investigate the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium-independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. 2. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg(-1)) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium-independent NOS) were evaluated 3 days after ischaemia. 3. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 4. 1400W attenuated the calcium-independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. 5. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

The activity of rat brain nitric oxide synthase following chronic antidepressant treatment

Nitric oxide synthase (NOS) is an enzyme involved in the activation of the glutamate/NMDA receptor-induced cascade of events. In this study we investigated the NOS activity in different rat brain regions after chronic electroconvulsive, imipramine and citalopram treatments. Chronic electroconvulsive treatment significantly increased the NOS activity (by 49%) in the cerebral cortex. However, chronic treatment with imipramine or citalopram did not alter the activity of NOS in all examined brain regions (cortex, hippocampus or cerebellum). The increased NOS activity after electroconvulsive but not pharmacologic (imipramine or citalopram) treatment may well reflect the differences between the adaptive changes of the NMDA receptor complex induced by these treatments.