Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

一氧化氮对水分胁迫下玉米种子萌发及幼苗生理特性的影响

以0.4mol/L的甘露醇(M)模拟水分胁迫状况,研究了外源一氧化氮(NO)供体硝普钠(SNP)对水分胁迫下玉米种子萌发、幼苗生长和生理特性的影响。结果表明:(1)水分胁迫下,玉米种子萌发和幼苗生长受到抑制,叶片丙二醛(MDA)含量、质膜相对透性、脯氨酸含量均显著增加;(2)SNP能显著提高水分胁迫下玉米种子的发芽率、发芽势、发芽指数和活力指数,增加玉米幼苗的根长、茎长、根重和整株干重,抑制水分胁迫下玉米幼苗叶片MDA含量的上升,降低叶片质膜相对透性,降低脯氨酸含量。其中以100μmol/L和200μmol/LSNP对水分胁迫的缓解效果最佳。     

Effects of nitric oxide synthase inhibitors and a substrate,L-arginine,on the cardiac function of juvenile salmonid fish

Control of cardiac function was investigated juvenile brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) using inhibitors of nitric oxide synthase (NOS), (L-NAME, NG-nitro-L-arginine and L-NMMA, NG-monomethyl-L-arginine) and a substrate of NOS (L-arginine). Salmonid alevins are excellent models for such studies since they are transparent, the beating heart is easily observed, diffusing distances are small, and they respond within a few seconds to exogenously administered chemicals. The response to inhibitors of NOS (L-NAME or L-NMMA) was tachycardia interpreted as vasoconstriction through lowered capacity for synthesis of NO. This could be reversed by addition of L-arginine and the subsequent bradycardia was explained as a vasodilation resulting from increased synthesis of NO. Blood flow into the heart is mainly via the vitelline vein and changes of flow resulting from constriction or dilation of this vessel may be probably major determinants of heart rate. The results provide evidence for the presence NOS in juvenile fish and indicate a physiological role for NO in cardiovascular control.     

一氧化氮对大鼠背根神经节神经元膜电位的作用

目的：观察一氧化氮对大鼠背根神经节神经元的作用及有关离子机制,并探讨一氧化氮在痛觉信息传递过程中的作用。方法：在分离的大鼠背根神经节标本上,应用细胞内记录技术,给予灌流一氧化氮供体硝普钠,观察硝普钠诱导的神经元膜反应。结果：大部分神经元对硝普钠敏感（79／102,77．45％）,滴加硝普钠（10～100mmol／L）后可引起浓度依赖性的超极化反应,剩余神经元没有反应。硝普钠（100mmol／L）可使神经元膜电导由（21．06±1．94）nS增加到（23．08±0．92）nS。L-NAME（非选择性一氧化氮合酶抑制剂,1mmol／L）、CdCl2（非选择性钙通道阻断剂,0．1mmol／L）、无Na^＋平衡盐液对硝普钠引起超极化反应无明显影响。四乙基碘化铵（非选择性钾通道阻断剂,10mmol／L）明显抑制硝普钠引起的超极化反应。结论：硝普钠在大鼠背根神经节神经元上可引起浓度依赖的超极化反应,且此超极化反应是钾电导介导的。     

外源一氧化氮供体对几种植物种子的萌发和幼苗生长的影响

以0、0.1、0.3、0.5、0.7、0.9mmol·L-1共6种浓度的外源一氧化氮(NO)供体硝普钠(SNP)处理豌豆、黄瓜、玉米和刺槐种子及其砂培幼苗后的结果表明:0.1～0.3mmol·L-1SNP对种子发芽势、发芽率及幼苗的根长、叶绿素含量和生物量有明显的促进作用;随着SNP浓度的增加,种子萌发和幼苗生长明显受抑制,不同植物受抑制程度的差异明显.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

WD40‐REPEAT 5a represses root meristem growth by suppressing auxin synthesis through changes of nitric oxide accumulation in Arabidopsis

Although nitric oxide (NO) is known to regulate root growth, the factor(s) modulating NO during this process have not yet been elucidated. Here, we identified Arabidopsis WD40‐REPEAT 5a (WDR5a) as a novel factor that functions in root growth by modulating NO accumulation. The wdr5a‐1 mutant accumulated less NO and produced longer roots than the wild type, whereas the WDR5a overexpression lines had the opposite phenotype. The role of NO was further supported by our observation that the NO donor sodium nitroprusside (SNP) and the NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) rescued the root meristem growth phenotypes of the wdr5a‐1 and WDR5a overexpression lines, respectively. The regulation of root growth by WDR5a was found to involve auxin because the auxin levels were similar in SNP‐treated wdr5a‐1 and wild‐type roots, but higher in untreated wdr5a‐1 roots than in wild‐type roots. In addition, the wdr5a‐1 mutant had higher production and activity levels of the auxin biosynthetic enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), in contrast to its reduced expression and activity in the WDR5a overexpression lines, and the increased root meristem growth in wdr5a‐1 was suppressed by treatment with l ‐kynurenine, which inhibits TAA1, as well as by mutating TAA1. WDR5a therefore functions in root meristem growth by maintaining NO homeostasis, and thus TAA1‐mediated auxin biosynthesis.     

Effects of exogenous nitric oxide on the antioxidant capacity of cadmium-treated soybean cell suspension

Nitric oxide (NO) is a small, ubiquitous molecule, whose physiological function in plants has recently been widely investigated. It seems that one of its pivotal properties is the antioxidant capacity, enabling plants to alleviate the effects of the oxidized stress. In this work we investigated the role of NO in soybean (Glycine max L. Cv. Navico) cell suspension treated with cadmium. Sodium nitroprusside (SNP), nitric oxide donor, markedly decreased the negative influence of Cd²⁺ on cell growth. It was also found to stimulate superoxide dismutase (SOD, EC 1.15.1.1). Using specific fluorochromes — dihydroethidine (DHE) and 2′,7′- dichlorofluorescein (DCFH-DA) it was shown that NO was very effective in reducing the level of superoxide anion (O ₂ ^·− ) and hydrogen peroxide, respectively. Furthermore, as evaluated by means of NO specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA), increased production of NO was found in Cd-treated cells. In cadmium-stressed cells SNP lowered the level of oxidized proteins. Our results suggest that the antioxidant properties of nitric oxide in Cd-treated soybean cells rely mainly on its ability to direct scavenging of ROS and stimulation of the antioxidant system.     

Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos

Aluminum (Al) is toxic to plants when solubilized into Al(3+) in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown. Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos. Exposure of H. moscheutos to Al(3+) led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP. Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

外源-氧化氮供体硝普钠对黑麦草幼苗耐碱性的影响

采用营养液砂培方法,研究外源一氧化氮(NO)供体硝普钠(SNP)对NaHCO3胁迫下黑麦草幼苗生长、活性氧代谢和渗透溶质积累的影响.结果表明:60 μmol·L-1 SNP能够缓解100 mmol·L-1NaHCO3胁迫对黑麦草幼苗生长的抑制作用,减缓NaHCO3胁迫导致的叶片O2产生速率、H2O2和丙二醛(MDA)含量的增加,提高NaHCO3胁迫下幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、质膜H+-ATP酶的活性和谷胱甘肽(GSH)、可溶性糖、可溶性蛋白质和脯氨酸的含量及K+/Na+比,降低过氧化氢酶(CAT)活性和抗坏血酸(AsA)含量,对游离氨基酸含量影响不大.上述结果表明,NO可能通过激活抗氧化系统活性、促进渗透溶质积累和改善Na+、K+平衡等缓解碱胁迫对幼苗的伤害,从而提高黑麦草的耐碱性.     

Role of nitric oxide synthase in insect cell radioresistance: an in-silico analysis

Previous studies on various insect cell lines have displayed very high radioresistance in Lepidoptera (butterflies and moths) as compared to mammals as well as other orders of Insecta including Diptera. Since NOS is known to modulate cellular radiation sensitivity, we carried out in silico analysis of Lepidopteran NOS and compared its structural and functional features including the sequence homology, predicted tertiary structure, post-translational phosphorylation and intracellular localization with the other species. Our study demonstrates that Lepidopteran NOS, while carrying significant sequence homology with mammalian nNOS, has structural/ functional features that may enhance resistance to radiation and other stress agents. A higher phosphorylation score of Lepidopteran NOS (0.885±0.02 as against 0.694±0.094 of mammalian NOS; predicted using Net Phos 2.0) was observed at many well-conserved phosphorylation sites, which may reduce NOS activation by stress agents including radiation. Further, the primarily cytoplasmic localization of Lepidopteran NOS (score 23 against 10 of mammalian NOS, derived using WoLFPSORT), aided by higher phosphorylation scores as well as sequence-driven cytoplasmic localizing signals, may significantly reduce amplification of extraneous oxidative damage. Based on these findings, we hypothesize that a primarily cytosolic and less responsive NOS could significantly contribute to radioresistance of Lepidopteran insects as well as their cultured cell lines.