一氧化氮合酶选择性抑制剂设计的生化基础

在体内，ＮＯ由３种一氧化氮合酶（ＮＯＳ）同工酶催化产生：内皮型ＮＯＳ（ｅｃＮＯＳ）、诱生型ＮＯＳ（ｉＮＯＳ）和脑型ＮＯＳ（ｎＮＯＳ）。选择性抑制ｉＮＯＳ有益于不同形式的休克和炎症的治疗，而对ｎＮＯＳ的抑制可保护神经不受损伤。本文简介绍了ＮＯＳ同工酰选择性抑制剂设计的生化基础和现有的选择性制剂研究概况。     

内皮一氧化氮合成障碍与高血压

198 7年 ,Ｐａｌｍｅｒ及Ｍｏｎｃａｄａ等首次证明内皮源舒张因子 (ＥＤＲＦ)是一氧化氮 (ＮＯ) ,引起世界医学界普遍关注。ＮＯ成为目前研究的热点。已证明ＮＯ参与体内许多生理和病理生理过程 ,ＮＯ对全身、肾脏血流动力学及尿钠排泄具有重要的调节作用 ,ＮＯ合成障碍参与高血压的发生和发展。本文就ＮＯ与高血压之间的关系作一综述。1　ＮＯ一般生物学特征1.1　ＮＯ的合成、代谢及生物效应　在体内ＮＯ是由Ｌ 精氨酸 (Ｌ Ａｒｇ)与氧分子经一氧化氮合酶 (ＮＯＳ)的催化下产生。ＮＯ是一种自由基气体 ,呈脂溶性 ,易通过生物膜 ,与细胞内…     

一氧化氮与内毒素性肺损伤

一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)参与机体的多种生理病理过程 ,其中ＮＯ在内毒素性肺损伤中具有很重要的作用 ,近年来对此方面研究颇多 ,但结论尚有争议 ,本文对此作一综述。1　一氧化氮一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)是新近发现的一种具有多种而复杂生物学活性的小分子物质 ,体内多种组织、细胞都能产生ＮＯ ,在肺内 ,肺的血管内皮细胞、巨噬细胞、平滑肌细胞、上皮细胞等都能产生ＮＯ ,它们均以Ｌ 精氨酸和氧为底物 ,在一氧化氮合酶 (ｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ ,ＮＯＳ)的作用下 ,生成ＮＯ和Ｌ 胍氨酸。…     

选择性神经元型一氧化氮合酶抑制剂7—硝基嘴唑的药理学研究进展

７－硝基吲唑是近年来发现的一种选择性神经元型一氧化氮合酶抑制剂，在体内它对另外两种ＮＯＳ亚型作用较弱。在分子水平上，７－ＮＩ作用于ＮＯＳ的血红素和四氢叶酸结合部位。选择性ｎＮＯＳ抑制剂有望用于临床治疗某些与ＮＯ神经毒性有关的病变。     

一氧化氮合酶抑制剂的研究进展

一氧化氮（ｎｉｔｒｉｃｏｘｉｄｅ,ＮＯ）是一种能调节细胞多种功能的信息分子,它参与心血管、外周和中枢神经以及免疫等系统生理过程和生物信号的调节。体内组织中的ＮＯ由ＮＯ合酶（Ｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ＮＯＳ）催化左旋精氨酸而合成,合成后的ＮＯ迅速跨膜扩散释放。各种调节ＮＯ释放的因素均作用于ＮＯＳ催化的化学反应过程,而体内影响该反应的ＮＯＳ在各组织的表达不同。特异性ＮＯＳ抑制剂通过调控ＮＯ的合成,对ＮＯＳ表达相关的各种疾病的预防和治疗具有重要的临床意义。本文对近年来ＮＯＳ抑制剂的研究进展作一概述。     

心血管系统 NO 释放的生理调控

心血管系统ＮＯ释放的生理调控许彦芳何瑞荣１（河北医科大学基础医学院药理教研室、１生理教研室，石家庄０５００１７）中国图书分类号Ｒ３３１．３ＮＯ参与体内心血管、中枢和外周神经以及免疫等系统生理过程的调节〔１〕。在心血管系统中，除血管内皮细胞释放ＮＯ外，...     

高血压病病人血浆一氧化氮、内皮素和过氧化物歧化酶的变化及其相关性探讨

一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)和内皮素 (ｅｎｄｏｔｈｅｌｉｎ ,ＥＴ)是血管内皮细胞产生和释放的血管舒缩因子 ,在维持血压的稳定、组织的灌流方面起着重要作用。资料表明[1] ,高血压病病人血ＮＯ浓度降低 ,舒血管功能障碍而致病。但也有人认为[2 ] ,ＥＴ的合成和释放增多是高血压病产生的重要原因。过氧化物歧化酶 (ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｕｔａｓｅ ,ＳＯＤ)是体内主要的氧自由基清除剂 ,是调节氧离子和ＮＯ水平的重要酶 ,在调节ＮＯ水平方面具有重要作用[3] 。本文对高血压病病人和健康对照组血ＮＯ、ＥＴ及ＳＯＤ进…     

Inhibitory effects of ginkgolides on nitric oxide production in neonatal rat microglia in vitro

目的：观察银杏内酯Ａ和Ｂ，ＰＡＦ拮抗剂阿帕泛（Ａｐａ）和ＮＯＳ抑制剂ＬＮＡ对新生大鼠小胶质细胞（Ｍｉ）产生ＮＯ的影响．方法：以Ｇｒｉｅｓ反应测定亚硝酸盐含量表示ＮＯ量．结果：在静息Ｍｉ，ＧＡ，ＧＢ和Ａｐａ在１－１００００ｎｍｏｌ·Ｌ－１范围对Ｍｉ产生ＮＯ没有影响，但ＬＮＡ可浓度依赖性地抑制ＮＯ产生，其ＩＣ５０（９５％可信限）值为３．４（０．８－１４９）μｍｏｌ·Ｌ－１．而在激活的Ｍｉ，ＧＡ，ＧＢ和ＬＮＡ可浓度依赖性地抑制ＮＯ产生，其ＩＣ５０（９５％可信限）值分别为５．７（１．８－１８１），１．１（０．３－４４）和０．５（０．１－２．８）μｍｏｌ·Ｌ－１，但Ａｐａ不能抑制ＮＯ产生．结论：ＧＡ和ＧＢ抑制ＬＰＳ诱导Ｍｉ产生ＮＯ．     

N-溴代-4,4-二甲基噁唑烷酮对小鼠的毒性

作者在国内首次合成了Ｎ溴代４，４二甲基噁唑烷酮（ＮＢＯ），并在小鼠体内观察了ＮＢＯ与神经性毒剂（ＶＸ、梭曼、沙林）孵温后反应液的毒性。旨在鉴定ＮＢＯ可否作为神经性毒剂的体外消毒剂。方法：首先将消毒液（ＮＢＯ）与神经性毒剂在３０℃恒温振荡器上振荡孵温３０ｍｉｎ，而后用生理盐水稀释，并用盐酸调至ｐＨ７０待用。实验分别观察了ＮＢＯ本身、ＮＢＯ与各神经性毒剂反应液对小鼠（ｉｐ）的毒性。结果指出：ＮＢＯ为１６２×１０－４ｍｏｌ／Ｌ时对小鼠ｉｐ、２４ｈ内无异常反应。以此浓度分别与不同毒剂、不同浓度…     

NOx对工人肺功能影响的调查

ＮＯｘ对工人肺功能影响的调查太原化肥厂医院（０３００２１）李晓红吴继平氮氧化物（ＮＯｘ）是氮和氧化合物的总称，包括Ｎ２Ｏ、ＮＯ、ＮＯ２、Ｎ２Ｏ３、Ｎ２Ｏ４、Ｎ２Ｏ５等，是制取、使用硝酸及一些含氮物分解时产生的有害气体，为了解ＮＯｘ对作业工人肺功能的影...     

Increased striatal dopamine efflux in vivo following inhibition of cerebral nitric oxide synthase by the novel monosodium salt of 7-nitro indazole.

The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7-nitro indazole (7-NINA). Using 7-NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7-NINA (1 mM) increased striatal dopamine efflux. The effect of 7-NINA was partially antagonized (67%) by co-perfusion with L-arginine (1 mM), the precursor of NO formation in vivo. This suggests that 7-NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo.     

In vivo nitric oxide transfer of a physiological NO carrier, dinitrosyl dithiolato iron complex, to target complex

Dinitrosyl dithiolato iron complex (DNIC) has been identified as an endogenous NO carrier, yet in vivo mechanisms of NO donation remain undefined. Transnitrosylation, in which a coordinated NO group is transferred to another metal complex, has been observed in transition-metal-nitrosyl chemistry. In this study, we used three kinds of iron dithiocarbamate complexes (Fe-DTCs) as NO acceptors to elucidate in vivo transnitrosylation of diglutathionyl dinitrosyl iron complex [DNIC-(GS)(2)]. Fe-DTCs were administered to mice after the injection of DNIC-(GS)(2) and electron paramagnetic resonance (EPR) spectra were measured both in the resected organs and in the upper abdomen of living mice. The spectral feature gradually changed from an initial DNIC-(GS)(2) signal to mononitrosyl iron dithiocarbamate one, suggesting that NO-Fe-DTC was formed through in vivo reaction of DNIC-(GS)(2) with Fe-DTC. The spectral results in in vitro and in vivo systems indicate that NO-Fe-DTCs can be formed not only by the transfer of coordinated NO-group(s) in DNIC-(GS)(2) but also by the abstraction of Fe-NO group in DNIC-(GS)(2) by free DTC ligands. Transnitrosylation proceeded more rapidly in blood than in liver and kidney; and more efficiently in kidney than in liver. Further, the ability to accept NO from DNIC was dependent on water-solubility of Fe-DTCs. Thus, in vivo transnitrosylation from DNIC to exogenous iron complex could be observed and this reaction was influenced by biological constituents and properties of iron complex. These results demonstrate that the transnitrosylation from DNIC to intrinsic NO acceptors like metalloproteins has a probable significance in in vivo NO transfer process.     

Reduced basal nitric oxide bioavailability and platelet activation in young spontaneously hypertensive rats

OBJECTIVE: To investigate the role of basal nitric oxide (NO) bioavailability for platelet activation in young spontaneously hypertensive rats before onset of hypertension. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) in platelets was used as a sensitive monitor of in vivo NO bioavailability. METHODS AND RESULTS: Whole blood samples were taken from 10-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In vivo surface-expression of P-selectin and platelet-binding of fibrinogen were assessed by flow cytometry. Platelet VASP-phosphorylation at its serine 239 (Ser239) and serine 157 (Ser157) residues was assessed using specific antibodies to determine NO bioavailability in vivo, and compared with endothelial vasomotor function. The increment in vascular tone following inhibition of NO-synthase in slightly preconstricted aortic rings was reduced indicating less NO formation under physiological stimulation (WKY 71.1+/-4.1%; SHR 57.8+/-2.4%, P<0.05). In vivo platelet VASP-phosphorylation was significantly reduced at both phosphorylation sites in SHR (mean fluorescence for Ser239: WKY: 15.2+/-0.6; SHR: 11.7+/-0.5, P<0.01; Ser157: WKY: 53.0+/-3.0; SHR: 35.0+/-3.5, P<0.05). Surface-expression of P-selectin and membrane-bound fibrinogen were significantly enhanced in SHR compared with WKY (P-selectin: WKY: 23.2+/-3.4; SHR 58.3+/-7.9, P<0.001; platelet-bound fibrinogen: WKY: 8.6+/-0.5; SHR: 13.5+/-1.1, P<0.001). In vitro preincubation of platelets with the NO donor sodium nitroprusside normalized platelet surface-expression of P-selectin in SHR. CONCLUSION: Using VASP-phosphorylation as a sensitive monitor of in vivo NO bioavailability, these data provide evidence that reduced vascular NO formation in vivo contributes to increased platelet activation in young SHR.     

Mechanisms of nitric oxide generation from nitroglycerin and endogenous sources during hypoxia in vivo

Nitroglycerin (GTN), often used in conditions of cardiovascular ischaemia, acts through the liberation of nitric oxide (NO) and the local concentration of NO in the tissue is responsible for any biological effect. However, little is known about the way in which the concentration of NO from GTN and other NO-donors is influenced by low oxygen tension in the target tissues. To evaluate the impact of changes in oxygen tension in the metabolism of NO-donors we measured exhaled NO in anaesthetized rabbits in vivo and expired NO and perfusate nitrite (NO(2)(-)) in buffer-perfused lungs in situ. The impact of acute hypoxia on NO formation from GTN, isosorbide-5-mononitrate (ISMN), dissolved authentic NO, NO(2)(-) and NO generated from endogenous NO-synthase (NOS) was studied in either model. Acute hypoxia drastically increased exhaled NO concentrations from all NO-donors studied, both in vivo and in the perfused lung. During similar conditions endogenous NO generation from NOS was strongly inhibited. The effects were most pronounced at less than 3% inspired oxygen. The mechanisms for the increased NO-formation during hypoxia seems to differ between GTN- and NO(2)(-)-derived NO. The former phenomenon is likely due to diminished breakdown of NO. In conclusion, hypoxic conditions preserve very high local NO concentrations generated from organic nitrates in vivo and we suggest that this might benefit preferential vasodilation in ischaemic tissue regions. Our findings point out the necessity to consider the influence of oxygen tension when studying the action of NO-donors.     

New methods to evaluate endothelial function: A search for a marker of nitric oxide (NO) in vivo: re-evaluation of NOx in plasma and red blood cells and a trial to detect nitrosothiols

Although plasma NOx (NO(2)(-) and NO(3)(-)) has been used as an index of nitric oxide (NO) formation in vivo, many unreasonable results appeared even after active elimination of NOx contamination from laboratory ware. For example, plasma NOx concentrations did not increase during vasodilation mediated by the NO/cGMP pathway or after organ perfusion. A possible shift of NOx from plasma to erythrocytes (RBCs) as a cause of these phenomena has been excluded, leaving the destination of NOx (after leaving plasma) unknown. Kinetic analyses have revealed that steady state NOx concentrations in plasma and whole blood did not correlate with the NOx formation rate, but rather with the NOx elimination rate. Therefore, the supposition that the NO status is directly reflected by plasma NOx concentrations appears untenable. As nitrosothiols (R-SNOs), possible carriers of NO bioactivity, have been flagged as alternative indices of NO status in vivo, efforts have been made to detect these substances. When interference by ultrafiltration was eliminated, low molecular weight R-SNOs such as nitrosocystein and nitrosogluthathione were undetectable. However, a high-molecular weight R-SNO, nitrosoalbumin, was detected in human blood. Further research is required into the significance and practical use of nitrosoalbumin as a marker of NO in vivo.     

Nitric oxide donors increase mucus gel thickness in rat stomach.

Instillation of the nitric oxide (NO) generator isosorbide dinitrate (0.1-1 mM) into the rat gastric lumen in vivo produced a dose-related increase in mucus gel thickness that was prevented by coadministration of oxyhaemoglobin (10 microM). Isosorbide dinitrate did not induce epithelial cell damage. S-Nitroso-N-acetyl-penicillamine (0.3 mM) and dibutyryl cyclic GMP (1 mM) also increased mucus thickness. These findings, along with the presence of NO synthase in the gastric mucosa, imply a role for NO in vivo in mediation of gastric mucus release.     

Brain-derived neurotrophic factor enhances histamine-induced airway responses and changes levels of exhaled nitric oxide in guinea pigs in vivo

The neurotrophin brain-derived neurotrophic factor (BDNF) occurs in elevated levels during airway inflammation, including asthma and hypoxic lung injury, and has been suggested to be associated with airway hyperresponsiveness in these conditions. The aim of the present study was to examine whether airway responses to histamine challenge and levels of exhaled nitric oxide (NO) in vivo might be altered upon BDNF treatment. Pulmonary resistance, lung compliance, insufflation pressure, and levels of exhaled NO were measured in anaesthetized guinea pigs exposed to BDNF prior to challenge with histamine and with intact or inhibited endogenous NO production. BDNF pretreatment significantly enhanced histamine-evoked increase in pulmonary resistance and insufflation pressure, as well as the decrease in lung compliance. BDNF markedly accentuated the reduction in exhaled NO following histamine challenge. In animals with inhibited endogenous NO production BDNF induced a significantly earlier histamine-evoked increase in airway responses. The present data show that BDNF can induce an augmentation of histamine-evoked airway responses and reduce levels of NO in exhaled air in vivo. Endogenous NO seems to exert a braking action on BDNF-induced enhancement of airway responses and a reduced ability to release NO may be one mechanism for increased airway response during elevated BDNF levels. Taken together this indicates that BDNF may be of importance for airway hyperresponsiveness in vivo. The interaction between BDNF and airway NO formation, and its relation to airway responses, merit further investigation.     

REGULATION OF NITRIC OXIDE PRODUCTION BY RAT ALVEOLAR MACROPHAGES IN RESPONSE TO SILICA EXPOSURE

In the present study, it was confirmed that in vivo exposure of rats to silica significantly increases nitric oxide (NO) production by bronchoalveolar lavage cells (BALC), a population of cells that includes alveolar macrophages. Possible mechanisms whereby NO production could be upregulated by rat alveolar macrophages following silica exposure were examined to determine if there is a direct effect of silica on alveolar macrophage NO production or if other factors are involved. BALC were obtained from normal male rats and cultured for 2 h. Nonadherent cells were then removed and the enriched alveolar macrophage cell populations were exposed to test agents for 18-20 h. Media nitrate and nitrite (NOx) concentrations were used to assess NO production and, in some cases, inducible NO synthase mRNA levels were indexed. In vitro exposure to silica (0.1-100 mug ml) had no significant effect on basal NO levels. Furthermore, NO generation was not additionally increased above levels induced by interferon gamma (IFN), lipopolysaccharide (LPS), or other cytokines during simultaneous incubations with silica and IFN, a 2-h pretreatment with silica followed by IFN, or preincubation with IFN, LPS, and or other cytokines before the addition of silica. To evaluate whether cell-cell interactions might be required for the induction of NO production d uring silica challenge, alveolar macrophages were cultured with splenic lymphocytes or blood-derived polymorphonuclear leukocytes. Coculture of splenic lymphocytes with alveolar macrophages resulted in media NOx levels that were greater than the additive levels from each cell type. However, the presence of silica was without additional effect on NO production by either of these cell types. Furthermore, it was found that conditioned media, derived from adherent BALC following silica treatment in vivo, could induce NO production by naive alveolar macrophages. In summary, the collective results from these experiments suggest that cell-cell communication factors, involving the interaction of pneumocytes following in vivo silica exposure, are necessary for the induction of NO by alveolar macrophages.     

Measurement by in vivo brain dialysis of nitrite and nitrate levels as indices of nitric oxide production

Nitric oxide (NO) is a free radical gas with a role in signal transduction in diverse processes. In the central nervous system, NO acts as an intercellular signaling molecule as well as a neurotoxic substance. To clarify the role of NO in the brain, we measured nitrite and nitrate levels, as indices of NO production, in the dialysate of brains in conscious rats, by using an in vivo dialysis technique. We have demonstrated that NO production in the cerebellum can be modulated by the activation of glutamate receptors and KCl stimulation. Glial cells appear to be involved in the modulation of NO production by regulating the availability of an NO precursor, L-arginine. We have also demonstrated that NO plays a role in the development of lipopolysaccharide-induced brain dysfunction and pentylenetetrazol-induced kinding. We believe that the in vivo dialysis method to measure nitrite and nitrate levels as indices of NO production is a useful tool for investigating physiological and pathophysiological roles of NO in the brain.     

Inhibition of neuronal M(2) muscarinic receptor function in the lungs by extracellular nitric oxide

1. These experiments were carried out to test whether neuronal M(2) muscarinic receptor function in the lungs is affected by nitric oxide (NO) and whether the source of the NO is epithelial or neuronal. 2. In pathogen free, anaesthetized guinea-pigs, the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction demonstrating functional neuronal M(2) muscarinic receptors. In the presence of the NO donor, 3-morpholino-sydnonimine (SIN-1), pilocarpine no longer inhibited vagally induced bronchoconstriction. In contrast, inhibiting endogenous NO with N(G)-monomethyl-L-arginine methyl ester (L-NMMA) did not affect the ability of pilocarpine to decrease vagally induced bronchoconstriction. 3. In isolated tracheas, pilocarpine inhibited contractions induced by electrical field stimulation demonstrating that neuronal M(2) muscarinic receptors function in vitro. As in the anaesthetized guinea-pigs, SIN-1 shifted the pilocarpine dose response curve to the right, demonstrating decreased neuronal M(2) receptor function. However, in vitro, L-NMMA shifted the pilocarpine dose response curve to the left, demonstrating that endogenous NO was inhibiting the ability of the M(2) receptors to decrease acetylcholine (ACh) release. 4. Both haemoglobin (Hb), which scavenges NO, and epithelial removal also shifted the pilocarpine dose response curve to the left, demonstrating that the NO inhibiting neuronal M(2) receptor function was extracellular and probably of epithelial origin. 5. In conclusion, extracellular NO appears to inhibit the ability of the M(2) receptors to decrease ACh release from the parasympathetic nerves in the lungs in vivo and in vitro in pathogen free guinea-pigs. However, while the neuronal M(2) receptors will respond to NO (from SIN-1) in vivo, there does not appear to be an endogenous source of NO since L-NMMA had no effect in vivo.