Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium

Stimulation of the endothelial lining of arteries with acetylcholine results in the release of a diffusible substance that relaxes and hyperpolarizes the underlying smooth muscle. Nitric oxide (NO) has been a candidate for this substance, termed endothelium-derived relaxing factor. But there are several observations that argue against the involvement of NO in acetylcholine-induced hyperpolarization. First, exogenous NO has no effect on the membrane potential of canine mesenteric arteries. Second, although haemoglobin (believed to bind and inactivate NO (refs 11-15)) and methylene blue (which prevents the stimulation of guanylate cyclase) inhibit relaxation, neither has an effect on hyperpolarization. Finally, nitroprusside, thought to generate NO in vascular smooth muscle, relaxes rat aorta without increasing rubidium efflux. Nevertheless, nitrovasodilators, nitroprusside and nitroglycerin cause hyperpolarization in some arteries. NO might therefore be responsible for at least part of the hyperpolarization induced by acetylcholine. We now report that hyperpolarization and relaxation evoked by acetylcholine are reduced by NG-monomethyl-L-arginine, an inhibitor of NO biosynthesis from L-arginine. Thus NO derived from the endothelium can cause hyperpolarization of vascular smooth muscle, which might also contribute to relaxation by closing voltage-dependent calcium channels. Our findings raise the possibility that hyperpolarization might be a component of NO signal transduction in neurons or inflammatory cells.     

Nitric oxide in female reproductive system

Nitric oxide (NO), synthesized from L-arginine and oxygen by a family of enzymes known as nitric oxide synthase (NOS), is an effective and intercellular signal transduction molecule, and is ubiquitously present in vertebrates. To date, there are three distinct isoforms of NOS: neural NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Among them, eNOS and nNOS, also called constitutive isoforms (cNOS), require calcium for activity, and are expressed constitutively in the physiological condition. The third isoforms, iNOS, whose activity is not dependent on calcium, are produced only in response to some stimulus, including cytokines and immune stimulating factors, etc.[1].     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.     

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.     

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.     

一氧化氮参与附睾上皮细胞感染后的宿主防御

 以金黄色葡萄球菌Staphylococcus aureus感染附睾上皮细胞的体外感染模型,分别采用RT-PCR和western blot等 方法研究一氧化氮（NO）在附睾上皮细胞宿主防御中的作用。结果表明附睾上皮感染后上调iNOS RNA和蛋白水平的表达,并产生 大量的NO,采用iNOS的抑制剂L NMMA和AGE预处理后,NO水平显著降低,与空白处理组相比,抑制剂处理组的S aureus数量显著增加,这表明NO参与了附睾上皮细胞的宿主防御过程,在附睾上皮细胞的宿主防御中具有重要的作用。     

NO-independent regulatory site on soluble guanylate cyclase

Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.     

Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter

Inhibitory non-adrenergic non-cholinergic (NANC) nerves are thought to be important in the autonomic innervation of the gastrointestinal tract and other organ systems. The nature of their neurotransmitter is still debated. Speculation that nitric oxide (NO), formed from L-arginine in neuronal structures and other cells, could act as a neurotransmitter, is not yet supported by demonstration of its release upon nerve stimulation. Using a superfusion bioassay, we report the release of a vasorelaxant factor upon stimulation of the NANC nerves in the canine ileocolonic junction. Several pieces of evidence, including the selectivity of the bioassay tissues, chemical instability, inactivation by superoxide anion and haemoglobin, inhibition by NG-nitro-L-arginine (L-NNA) and potentiation by L-arginine all indicated that NO accounted for the biological activity of this transferable NANC factor.     

Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor.

Nitric oxide is an important bioregulatory molecule, being responsible, for example, for activity of endothelium-derived relaxing factor (EDRF). Acute hypertension, diabetes, ischaemia and atherosclerosis are associated with abnormalities of EDRF. Nitric oxide is thought to be a retrograde messenger in the central nervous system. The technology is not yet available for rapid detection of NO released by a single cell in the presence of oxygen and/or nitrite, so the release, distribution and reactivity of endogenous NO in biological systems cannot be analysed. Here we describe a porphyrinic microsensor that we have developed and applied to monitoring NO release in a microsystem. We selectively measured in situ the NO released from a single cell with a response time of less than 10 ms. The microsensor consists of p-type semiconducting polymeric porphyrin and a cationic exchanger (Nafion) deposited on a thermally sharpened carbon fibre with a tip diameter of approximately 0.5 microns. The microsensor, which can be operated in either the amperometric or voltammetric mode, is characterized by a linear response up to 300 microM and a detection limit of 10 nM. Nitric oxide at the level of 10(-20) mols can be detected in a single cell.     

Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.     

人参汤提取物对血管内皮细胞保护作用的研究

探讨人参汤提取物对血管内皮细胞的保护作用.采用体内、外血管内皮细胞损伤模型,测定体外血管内皮细胞破裂MTT染色后的OD值、体内血管内皮细胞损伤时血中CEC数及血清中NO的浓度.体外实验人参汤各剂量组的OD值比模型对照组降低(P<0.05)、体内实验人参汤各剂量组CEC数量、NO浓度比模型对照组都显著降低(P<0.01).实验结果表明,人参汤提取物对血管内皮细胞的损伤具有保护作用.     

Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide

Studies of cultured bovine aortic endothelial cells using quantitative chemiluminescence techniques have shown that the amount of nitric oxide released under basal conditions, or in response to either bradykinin or the calcium ionophore A23187 is insufficient to account for the vasorelaxant activities of the endothelium-derived relaxing factor (EDRF) derived from the same source. This observation contradicts previous suggestions that nitric oxide and EDRF are the same compound, but may be explained if EDRF is a compound that contains nitric oxide within its structure but is a much more potent vasodilator than nitric oxide. Such a molecule could be one of several nitrosothiols which may yield nitric oxide after a one-electron reduction. The present experiments were carried out to test the possibility that the biological activities of the endothelium-derived relaxing factor might more closely resemble those of one of these compounds, S-nitrosocysteine, than nitric oxide. Nitric oxide release from cultured bovine aortic endothelial cells was detected by chemiluminescence and bioassay experiments compared the vasodilator potencies of nitric oxide, S-nitrosocysteine, and EDRF. The results suggest that EDRF is much more likely to be a nitrosylated compound such as a nitrosothiol than authentic nitric oxide.     

Regulation between nitric oxide and MAPK signal transduction in mammals

Nitric oxide (NO) is an important biological messenger in the regulation of tissue homeostasis. It exhibits a wide range of effects during physiological and pathophysiological processes. Typical beneficial properties of NO include the regulation of vascular tone,the protection of cells against apoptosis, the modulation of immune responses, and the killing of microbial pathogens. On the other hand,NO may cause severe vasodilation and myocardial depression during bacterial sepsis or act as a cytotoxic and tissue-damaging molecule in autoimmune diseases. Mitogen-activated protein kinase (MAPK) is a family of serine/threonine protein kinases that are widely distributed in mammalian cells. MAPK cascade plays pivotal roles in gene expression, cell proliferation, differentiation, neuronal survival and programmed cell death under a variety of experimental conditions. MAPKs transduce the signal for the cellular response to extracellular stresses or stimuli. The relation between them, however, has never been reviewed. Based on our researches and other reports in the field, we review their reciprocal regulatory functions.     

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(?-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(?-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(?-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(?-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.     

Nitric oxide involved in signal transduction of Jasmonic acid-induced stomatal closure of Vicia faba L.

Nitric oxide (NO) and Jasmonic acid (JA) are two key signaling molecules involved in many and diverse biological pathways in plants. Growing evidence suggested that NO signaling interacts with JA signaling. In this work, Our experiment showed that NO exists in guard cell of Vicia faba L., and NO is involved in signal transduction of JAinduced stomata closuring: ( i ) JA enhances NO synthesis in guard cell; ( ii ) both JA and NO induced stomatal closure, and had dose response to their effects; ( iU ) there are synergetic correlation between JA and lower NO concentration in regulation of stomatal movement; (iV) JA-induced stomatal closure was largely prevented by 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO), a specific NO scavenger. An inhibitor of NO synthase (NOS) in mammalian cells, N^G-nitro-L-Arg-methyl eater (L-NAME) also inhibits plant NOS, repressing JA-induced NO generation and JA-induced stomatal closure. We presumed that NO mainly comes from NOS after JA treatment.     

下丘脑室旁核NOS／NO系统对心血管活动的调节（综述）

下丘脑室旁核（PVN）是重要的心血管活动调节中枢。PVN分布有一氧化氮（NOS）神经元,合成并释放一氧化氮（NO）。NO通过抑制各级交感神经中枢,以及影响神经内分泌活动,对心血管活动进行调节。     

Nitric oxide mediates the fungal elicitor-induced Taxol biosynthesis of Taxus chinensis suspension cells through the reactive oxygen species-dependent and -independent signal pathways

Taxol (paclitaxel, NSC-125973), a secondary me- tabolite of the Taxus species, has been recognized as one of the best anticancer drugs emerging in the last decade[1]. The production of Taxol by various Taxus spp. cells in culture has been one of the most …     

一氧化氮治疗肿瘤的研究进展

一氧化氮（NO）是一种半衰期很短的气体分子，对细胞膜具有高穿透性，能在人体内传递重要信息，并具有调节细胞的功能.NO气体分子既能维持正常细胞的生理功能和活性，又能选择性地快速耗尽肿瘤细胞的能量，诱导肿瘤细胞凋亡.研究表明：NO可以通过多种机制实现肿瘤治疗.已有一些NO供体药物表现出良好的抗肿瘤活性，精确控制NO在肿瘤部位的释放，可杀死肿瘤细胞.因此，NO气体疗法作为一种肿瘤治疗策略具有一定的应用前景.文章简述了NO的生理学特性和几种典型的NO供体，以及释放NO的生物材料在生物医学领域的应用进展.     

一氧化氮在心血管活动调控中的作用

一氧化氮是20年来医学研究的热点之一，一氧化氮在心血管活动调控和许多心血管疾病如高血压、动脉粥样硬化、器官缺血再灌注损伤的病理机制中都有重要作用．其作用即有有利的一方面，又有有害的一方面．在一氧化氮合酶作用下产生的一氧化氮对机体具有保护作用。另一方面，受细菌内毒素或细胞因子等诱导，一氧化氮合酶表达上调并产生大量一氧化氮引起细胞损伤和循环衰竭．目前有许多临床实验正在评价一氧化氮在心血管疾病中的作用．