Norepinephrine Increases Cyclic GMP Levels in Cerebellar Cells from Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.     

Regulation of Drosophila Visual System Development by Nitric Oxide and Cyclic GMP

Photoreceptors undergoing target selection in the optic lobeof Drosophila express a nitric oxide sensitive soluble guanylatecyclase (sGC). At the same time, cells in the target regionof the optic lobe express nitric oxide synthase (NOS). Pharmacologicalinhibition of NOS, NO or sGC leads to disruption of the retinalprojection pattern in vitro, and the extension of individualretinal axons beyond their appropriate targets. The disruptiveeffects of NOS inhibition in vitro are prevented by adding acGMP analog. Mutations in the sGC alpha subunit gene, Gc1, reducesGC expression and attenuate NO-sensitive retinal cGMP productionin the visual system. Although the retinal projection patternis undisturbed in Gc1 mutants, they lack positive phototaxisas adults, suggesting inappropriate connections exist betweenthe photoreceptors and optic lobe interneurons in these flies.Preliminary results show that heat-shock expression of wild-typeGc1 during metamorphosis can restore positive phototaxis insevere Gc1 mutants. These in vivo results support the in vitrofindings that NOS and sGC activity are required to promote theappropriate retinal innervation of the optic lobe.     

Two Pathways of Cyclic GMP Production Through Glutamate Receptor-Mediated Nitric Oxide Synthesis

The selective agonists for the metabotropic glutamate receptor and the ionotropic non-N-methyl-D-aspartate (NMDA) glutamate receptor, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), respectively, increased the cyclic GMP (cGMP) content in cerebellar slices prepared from adult rats. The ACPD-induced rise in cGMP level was blocked by compounds known to antagonize metabotropic glutamate receptors, such as DL-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyric acid, but not by ionotropic glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the AMPA-induced rise in cGMP level was suppressed by CNQX. Both rises in cGMP level involved nitric oxide synthase (NOS), because NG-methyl-L-arginine (NMLA), an inhibitor of NOS, blocked both cGMP level rises, and excess L-arginine reversed the effect of NMLA. After lithium chloride treatment, which could exhaust phosphatidylinositol phosphates, ACPD no longer increased cGMP levels, whereas AMPA was still effective. In a calcium-free medium, ACPD still induced a rise in cGMP level, whereas AMPA did not. When the molecular layer was isolated to determine the cGMP content separately from that in the rest of the cerebellar cortex, it was found that ACPD raised the cGMP level mainly in the molecular layer, whereas AMPA raised it in both sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of In Vivo Striatal Transmitter Release by Nitric Oxide and Cyclic GMP

Abstract: The effects of nitric oxide (NO) and cyclic GMP on in vivo transmitter release in the rat striatum were investigated using microdialysis sampling in urethane-anaesthetised animals. The NO release-inducing substances S -nitrosoacetylpenicillamine (SNAP), S -nitrosoglutathione (SNOG), and sodium nitroprusside (SNP) increased extracellular concentrations of aspartate (Asp), glutamate (Glu), γ-aminobutyric acid (GABA), taurine (Tau), acetylcholine (ACh), and serotonin (5-HT). Dopamine (DA) concentrations were decreased by SNAP but were increased by SNOG and SNP. An NO scavenger, haemoglobin, blocked or reduced the effects of SNAP on transmitter release. However, the control carrier compounds for SNAP, SNOG, and SNP (penicillamine, glutathione, and potassium ferricyanide, respectively, which do not induce release of NO) also increased GABA, Tau, DA, and 5-HT concentrations. When NO gas was given directly by dissolving it in degassed Ringer's solution, DA concentrations decreased significantly, and those of Asp, Glu, GABA, Tau, ACh, and 5-HT increased. These effects of NO gas were all inhibited by coadministration of haemoglobin and for GABA, Tau, ACh, and DA showed some calcium dependency. The cyclic GMP agonists 8-bromo-cyclic GMP and dibutryl-cyclic GMP stimulated dose-dependent increases in Asp, Glu, GABA, Tau, ACh, DA, and 5-HT concentrations. Increased striatal transmitter release in response to NO may therefore be mediated by its stimulatory action on cyclic GMP formation. NO inhibition of DA release may be mediated indirectly through its stimulation of local cholinergic and GABAergic neurones.     

Inhibition of Nitric Oxide Synthase Blocks N-Methyl-D-Aspartate-, Quisqualate-, Kainate-, Harmaline-, and Pentylenetetrazole-Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.     

Nitric Oxide Synthase in Spontaneously Hypertensive Rats

Since its discovery by Furchgott and Zawadzki in 1980 [18], endothelium-derived relaxing factor (EDRF) has been shown to play a central role in the cardiovascular system [10]. The endothelial product is chemically equivalent to nitric oxide (NO) [23, 40] or a biochemical congener thereof [48]. Fifteen years ago, this small, simple and highly toxic molecule was known as a lengthy list of environmental pollutants found in unsavory haunts such as smoke and smog, and even as destroyer of ozone, suspected carcinogen, and precursor of acid rain. In addition, NO seems an unlikely biological jack of all trades for most of the body's functions are regulated by extraordinarily large and complex proteins and compounds. But over the past decade, diverse lines of evidence have converged to show that this sometime poison is a fundamental player in the everyday business of the human body.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Induction of Nitric Oxide Synthase in Glial Cells

Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOS-like activity that was L-arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS-like activity. C6 cells treated with LPS also showed Ca(2+)-independent and L-arginine-dependent NOS-like activity. N18 cells demonstrated constitutive Ca(2+)-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells.     

Nitric Oxide Synthase Activity in the Molluscan CNS

Abstract: Putative nitric oxide synthase (NOS) activity was assayed in molluscan CNS through histochemical localization of NADPH-diaphorase and through measurement of l -arginine/ l -citrulline conversion. Several hundreds of NADPH-dependent diaphorase-positive neurons stained consistently darkly in the nervous system of the predatory opisthobranch Pleurobranchaea californica , whereas stained neurons were relatively sparse and/or light in the other opisthobranchs ( Philine, Aplysia, Tritonia, Flabellina, Cadlina, Armina, Coriphella , and Doriopsilla sp.) and cephalopods ( Sepia and Rossia sp.). l -Arginine/ l -citrulline conversion was β-NADPH dependent, insensitive to removal of Ca²⁺, inhibited by the calmodulin blocker trifluoperazine, and inhibited by the competitive NOS inhibitor N -nitro- l -arginine methyl ester ( l -NAME) but not d -NAME. Inhibitors of arginase [ l -valine and (+)- S -2-amino-5-iodoacetamidopentanoic acid)] did not affect l -citrulline production in the CNS. NOS activity was largely associated with the particulate fraction and appeared to be a novel, constitutive Ca²⁺-independent isoform. Enzymatic conversion of l -arginine/ l -citrulline in Pleurobranchaea and Aplysia CNS was 4.0 and 9.8%, respectively, of that of rat cerebellum. l -Citrulline formation in gill and muscle of Pleurobranchaea was not significant. The localization of relatively high NOS activity in neuron somata in the CNS of Pleurobranchaea is markedly different from the other opisthobranchs, all of which are grazers. Potentially, this is related to the animal's opportunistic predatory lifestyle.     

一氧化氮合酶的若干研究进展

一氧化氮合酶(NOS)是一氧化氮(NO)生物学与医学研究的重要内容.近年来,对NOS酶本质及其生化与分子生物学特性甚至某些分子遗传学方面的认识都在迅速发展和深化.研究表明,干预NOS-NO途径的某些环节,如酶激活、NO合成、释放与转运甚至有关酶的编码基因及其表达,将为某些临床问题的解决提供新的思路和手段．     

Nitric Oxide Synthase in Bovine Superior Cervical Ganglion

Abstract: We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by N ^G-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca²⁺/calmodulin dependent and required NADPH and tetrahydrobiopterin as co-factors. Superoxide dismutase protected and oxyhemo-globin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohisto chemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-β-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.     

Endothelial Nitric Oxide Synthase Single Nucleotide Polymorphism and Left Ventricular Function in Early Chronic Kidney Disease

Background

Chronic kidney disease (CKD) is associated with accelerated cardiovascular disease and heart failure. Endothelial nitric oxide synthase (eNOS) Glu298Asp single nucleotide polymorphism (SNP) genotype has been associated with a worse phenotype amongst patients with established heart failure and in patients with progression of their renal disease. The association of a cardiac functional difference in non-dialysis CKD patients with no known previous heart failure, and eNOS gene variant is investigated.

Methods

140 non-dialysis CKD patients, who had cardiac magnetic resonance (CMR) imaging and tissue doppler echocardiography as part of two clinical trials, were genotyped for eNOS Glu298Asp SNP retrospectively.

Results

The median estimated glomerular filtration rate (eGFR) was 50mls/min and left ventricular ejection fraction (LVEF) was 74% with no overt diastolic dysfunction in this cohort. There were significant differences in LVEF across eNOS genotypes with GG genotype being associated with a worse LVEF compared to other genotypes (LVEF: GG 71%, TG 76%, TT 73%, p = 0.006). After multivariate analysis, (adjusting for age, eGFR, baseline mean arterial pressure, contemporary CMR heart rate, total cholesterol, high sensitive C-reactive protein, body mass index and gender) GG genotype was associated with a worse LVEF, and increased LV end-diastolic and systolic index (p = 0.004, 0.049 and 0.009 respectively).

Conclusions

eNOS Glu298Asp rs1799983 polymorphism in CKD patients is associated with relevant sub-clinical cardiac remodelling as detected by CMR. This gene variant may therefore represent an important genetic biomarker, and possibly highlight pathways for intervention, in these patients who are at particular risk of worsening cardiac disease as their renal dysfunction progresses.     

Characterisation of Nitric Oxide Synthase in Three Cnidarian-Dinoflagellate Symbioses

Background

Nitric oxide synthase (NOS) is an enzyme catalysing the conversion of L-arginine to L-citrulline and nitric oxide (NO), the latter being an essential messenger molecule for a range of biological processes. Whilst its role in higher vertebrates is well understood little is known about the role of this enzyme in early metazoan groups. For instance, NOS-mediated signalling has been associated with Cnidaria-algal symbioses, however controversy remains about the contribution of enzyme activities by the individual partners of these mutualistic relationships.

Methodology/Principal Findings

Using a modified citrulline assay we successfully measured NOS activity in three cnidarian-algal symbioses: the sea anemone Aiptasia pallida, the hard coral Acropora millepora, and the soft coral Lobophytum pauciflorum, so demonstrating a wide distribution of this enzyme in the phylum Cnidaria. Further biochemical (citrulline assay) and histochemical (NADPH-diaphorase) investigations of NOS in the host tissue of L. pauciflorum revealed the cytosolic and calcium dependent nature of this enzyme and its in situ localisation within the coral''s gastrodermal tissue, the innermost layer of the body wall bearing the symbiotic algae. Interestingly, enzyme activity could not be detected in symbionts freshly isolated from the cnidarians, or in cultured algal symbionts.

Conclusions/Significance

These results suggest that NOS-mediated NO release may be host-derived, a finding that has the potential to further refine our understanding of signalling events in cnidarian-algal symbioses.     

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.     

诱导型一氧化氮合酶与疾病

炎症是众多疾病如自体免疫紊乱、神经退行性病变、心血管疾病和癌症发展的病理机制,诱导型一氧化氮合酶在炎症过程中被诱导表达,产生过量的一氧化氮,引发炎症级联反应,进而导致以上多种疾病发生。抑制诱导型一氧化氮合酶表达在体内体外实验及临床使用中均体现抗炎效果和症状改善。本文综述了诱导型一氧化氮合酶在炎症过程中诱导表达及与各类重大疾病联系的最新进展,并展望了诱导型一氧化氮合酶抑制剂作为抗炎治疗策略的前景。     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

大鼠脑组织中一氧化氮合酶测定

在含有一氧化氮合酶(NOS)底物左族精氨酸(L-Arg), 辅助因子还原性辅酶Ⅱ(NADPH)、四氢生物蝶呤(BH₄)、黄素单核苷酸(FMN), 黄素腺嘌呤二核苷酸(FAD)以及Ca²⁺、钙调蛋白等溶液中加入大鼠脑组织匀浆离心上清液, 组成酶反应体系. 37℃温育80min, 应用N-(1-萘基)-乙二胺、对氨基苯磺酸的重氮、偶氮反应测定酶反应体系中一定时间内NO代谢产物NO^-₂浓度变化, 建立一种简便的NOS活性测定方法. 反应体系最佳pH为7.4, K_m=0.1mmol/L, 体系内NO^-₂生成量与加入样品量之间有良好线性关系(r=0.998). 此方法简单、方便、重复性好, 批内CV为3.69%, 批间CV为5.16%. 10只健康大鼠脑组织中NOS活性为(39.61±7.64)nmol/(min·g).