Inhibition Of Nitric Oxide Biosynthesis Research Articles

Involvement of NO in V-ATPase Regulation in Cucumber Roots under Control and Cadmium Stress Conditions.

Nitric oxide (NO) is a signaling molecule that participates in plant adaptation to adverse environmental factors. This study aimed to clarify the role of NO in the regulation of vacuolar H+-ATPase (V-ATPase) in the roots of cucumber seedlings grown under control and Cd stress conditions. In addition, the relationship between NO and salicylic acid (SA), as well as their interrelations with hydrogen sulfide (H2S) and hydrogen peroxide (H2O2), have been verified. The effect of NO on V-ATPase was studied by analyzing two enzyme activities, the expression level of selected VHA genes and the protein level of selected VHA subunits in plants treated with a NO donor (sodium nitroprusside, SNP) and NO biosynthesis inhibitors (tungstate, WO42- and N-nitro-L-arginine methyl ester, L-NAME). Our results indicate that NO functions as a positive regulator of V-ATPase and that this regulation depends on NO generated by nitrate reductase and NOS-like activity. It was found that the mechanism of NO action is not related to changes in the gene expression or protein level of the V-ATPase subunits. The results suggest that in cucumber roots, NO signaling interacts with the SA pathway and, to a lesser extent, with two other known V-ATPase regulators, H2O2 and H2S.

Allantoin, a purine metabolite, confers saline–alkaline tolerance to sugar beet by triggering a self-amplifying feedback loop comprising jasmonic acid and nitric oxide

Saline–alkaline (SA) soil inhibits plant growth and development. A few plants have evolved strategies to combat SA stress, but the underlying mechanisms remain elusive. In the current study, we performed a combined proteome and metabolome profiling to capture the major pathways and specified metabolites involved in the sugar beet tolerance to SA stress. Protein abundance and metabolite accumulation in purine metabolism were altered to favour allantoin accumulation under SA stress. Exogenous allantoin alleviated the oxidative damage caused by SA stress, whereas inhibition of allantoin biosynthesis weakened SA tolerance. Transcriptome analysis revealed that allantoin altered the expression patterns of genes involved in jasmonic acid (JA) and nitric oxide (NO) biosynthesis to elevate their levels under SA stress. Inhibition of allantoin biosynthesis completely abolished the JA and NO accumulation. The application of JA and NO promoted the accumulation of each other, and both enhanced SA tolerance. Inhibition of JA biosynthesis completely abolished allantoin-induced SA tolerance and NO accumulation and weakened sodium nitroprusside (SNP, a donor of NO) -induced SA tolerance, whereas inhibition of NO biosynthesis only attenuated allantoin-induced SA tolerance and JA accumulation and JA-induced SA tolerance. Our results suggest that allantoin accumulation is assisted under SA stress, promoting JA accumulation and consequently NO biosynthesis. Subsequently, NO in turn assists JA biosynthesis, forming a self-amplifying feedback loop, in which JA and NO synergistically improve SA tolerance. The current study provides a novel insight into SA tolerance in sugar beet and preliminary reveals the regulatory role of allantoin in SA tolerance.

Phosphorus supplementation modulates nitric oxide biosynthesis and stabilizes the defence system to improve arsenic stress tolerance in mustard.

The interaction of mineral nutrients with metals/metalloids and signalling molecules is well known. In the present study, we investigated the effect of phosphorus (P) in mitigation of arsenic (As) stress in mustard (Brassica juncea L.). The study was conducted to investigate potential of 30 mg P·kg-1 soil P supplement (diammonium phosphate) to cope up with the adverse effects of As stress (24mg As·kg-1 soil) in mustard plants Supplementation of P influenced nitric oxide (NO) generation, which up-regulated proline metabolism, ascorbate-glutathione system and glyoxalase system and alleviated the effects of on photosynthesis and growth. Arsenic stress generated ROS and methylglyoxal content was scavenged through P-mediated NO, and reduced As translocation from roots to leaves. The involvement of NO under P-mediated alleviation of As stress was substantiated with the use of cPTIO (NO biosynthesis inhibitor) and SNP (NO inducer). The reversal of P effects on photosynthesis under As stress with the use of cPTIO emphasized the role of P-mediated NO in mitigation of As stress and protection of photosynthesis The results suggested that P reversed As-induced oxidative stress by modulation of NO formation, which regulated antioxidant machinery. Thus, P-induced regulatory interaction between NO and reversal of As-induced oxidative stress for the protection of photosynthesis may be suggested for sustainable crops.

ST Segment Yükselmeli Miyokardial Enfarktüs İle Başvuran Hastalarda Asimetrik Dimetilarjinin Düzeyinin Kisa Ve Uzun Dönem Prognoz İle İlişkisi

objecitves: ST segment elevation myocardial infarction (STEMI) is regarded as a type of acute coronary syndrome. However, the mortality of STEMI patients is substantial. Asymmetric dimethylarginine (ADMA) is the major inhibitor of nitric oxide biosynthesis in humans. Studies have shown that increased ADMA levels are associated with endothelial dysfunction and increased atherogenesis. It is aimed to show the relationship between ADMA and prognosis of ST elevation MI. Materials and methods: The study population was 45 (40 male, 5 female) patients with diagnosis of STEMI, who underwent primary percutaneous coronary intervention (PCI). Demographic data, blood tests and medical history of the patients were recorded. Ten cc of blood samples were collected right after sheat application. ADMA levels were studied by ELISA kit. Results: The follow-up period was 42.837±14.343 months. The mean age was 52.4±7.8. Only significant relation was observed between ADMA levels and heart rate (p=0.025). Other relations with demographic data, risk factors for STEMI, follow-up events such as major adverse cardiovascular events, heart failure, target vessel revascularization, reinfarction, ongoing myocardial ischemia, in-hospital events, intervention artery, type of intervention, Pre-TIMI, ejection fraction, use of tirobufan, anterior or inferior MI were not statistically significant (p>0.05). Although, a positive relation was observed between ADMA levels and chronic heart failure, it was not significant. Conclusions: Although, there was a relation with heart failure, the only significant relation was found between ADMA levels and heart rate. Further studies with greater number of participants should be performed for drawing strict conclusions.

Nitric oxide modulating ion balance in Hylotelephium erythrostictum roots subjected to NaCl stress based on the analysis of transcriptome, fluorescence, and ion fluxes

Soil salinization is one of the main stress factors that affect both growth and development of plants. Hylotelephium erythrostictum exhibits strong resistance to salt, but the underlying genetic mechanisms remain unclear. In this study, hydroponically cultured seedlings of H. erythrostictum were exposed to 200 mM NaCl. RNA-Seq was used to determine root transcriptomes at 0, 5, and 10 days, and potential candidate genes with differential expression were analyzed. Transcriptome sequencing generated 89.413 Gb of raw data, which were assembled into 111,341 unigenes, 82,081 of which were annotated. Differentially expressed genes associated to Na+ and K+ transport, Ca2+ channel, calcium binding protein, and nitric oxide (NO) biosynthesis had high expression levels in response to salt stress. An increased fluorescence intensity of NO indicated that it played an important role in the regulation of the cytosolic K+/Na+ balance in response to salt stress. Exogenous NO donor and NO biosynthesis inhibitors significantly increased and decreased the Na+ efflux, respectively, thus causing the opposite effect for K+ efflux. Moreover, under salt stress, exogenous NO donors and NO biosynthesis inhibitors enhanced and reduced Ca2+ influx, respectively. Combined with Ca2+ reagent regulation of Na+ and K+ fluxes, this study identifies how NaCl-induced NO may function as a signaling messenger that modulates the K+/Na+ balance in the cytoplasm via the Ca2+ signaling pathway. This enhances the salt resistance in H. erythrostictum roots.

Melatonin is responsible for rice resistance to rice stripe virus infection through a nitric oxide-dependent pathway

Rice stripe virus (RSV) causes one of the most important rice virus diseases of plants in East Asia. However, the molecular mechanisms controlling rice resistance to RSV infection are largely unknown. Recently, several studies presented a novel model that melatonin (MT) and nitric oxide (NO) participate in the plant-pathogen interaction in a synergetic manner. In this study, there was a difference in MT content between two rice varieties that correlated with one being susceptible and one being resistant to RSV, which suggested that MT is related to RSV resistance. In addition, a test with two NO biosynthesis inhibitors revealed that NO inhibitor were able to increase the disease incidence of RSV. A pharmacological experiment with exogenous MT and NO showed that increased MT and NO in the MT-pretreated plants led to lower disease incidences; however, only NO increased in a NO-releasing reagent [sodium nitroprusside (SNP)] pretreated plants. The expressions level of OsPR1b and OsWRKY 45 were significantly induced by MT and NO. These results suggest that rice resistance to RSV can be improved by increased MT through a NO-dependent pathway.

The involvement of NO in ABA-delayed the senescence of cut roses by maintaining water content and antioxidant enzymes activity

Abscisic acid (ABA) coordinates plant growth and development as internal signals. Nitric oxide (NO) as an essential endogenous signal molecule may prolong the vase life of cut flowers. However, the interaction of between ABA and NO during postharvest life of perishable horticultural products was not clear. Here, cut roses were used to investigate the interaction between NO and ABA during the senescence of cut flowers. The results showed that ABA increased the vase life and flower diameter of cut roses in a dose-dependent manner, with maximal biological responses at 0.5 μM. Sodium tungstate, an NO biosynthesis inhibitor, significantly suppressed the vase life and diameter enhanced by ABA, indicating that endogenous NO might be involved in ABA-regulated cut flower senescence. ABA and a NO donor sodium nitroprusside (SNP) effectively increased leaf relative water content and reduced the rate of water loss via inducing stomatal closure. ABA and NO treatments significantly increased the activity of superoxide dismutase, peroxidase and ascorbate peroxidase and the content of leaf chlorophyll. Sodium tungstate also prevented the effects of ABA mentioned above. Altogether, NO may play a crucial role in ABA-delayed the senescence of cut roses by retarding water loss, stimulating enzyme activity and maintaining leaf chlorophyll content.

Malaria in pregnancy alters l-arginine bioavailability and placental vascular development.

Reducing adverse birth outcomes due to malaria in pregnancy (MIP) is a global health priority. However, there are few safe and effective interventions. L-arginine is an essential amino acid in pregnancy and an immediate precursor in the biosynthesis of nitric oxide (NO), but there are limited data on the impact of MIP on NO biogenesis. We hypothesized that hypoarginemia contributes to the pathophysiology of MIP and that L-arginine supplementation would improve birth outcomes. In a prospective study of pregnant Malawian women, we show that MIP was associated with lower concentrations of L- arginine and higher concentrations of endogenous inhibitors of NO biosynthesis, asymmetric and symmetric dimethylarginine, which were associated with adverse birth outcomes. In a model of experimental MIP, L-arginine supplementation in dams improved birth outcomes (decreased stillbirth and increased birth weight) compared with controls. The mechanism of action was via normalized angiogenic pathways and enhanced placental vascular development, as visualized by placental microcomputerized tomography imaging. These data define a role for dysregulation of NO biosynthetic pathways in the pathogenesis of MIP and support the evaluation of interventions to enhance L-arginine bioavailability as strategies to improve birth outcomes.

Nitric Oxide Mediates 5-Aminolevulinic Acid-Induced Antioxidant Defense in Leaves of Elymus nutans Griseb. Exposed to Chilling Stress.

Nitric oxide (NO) and 5-aminolevulinic acid (ALA) are both extremely important signalling molecules employed by plants to control many aspects of physiology. In the present study, the role of NO in ALA-induced antioxidant defense in leaves of two sources of Elymus nutans Griseb. (Damxung, DX and Zhengdao, ZD) was investigated. Chilling stress enhanced electrolyte leakage, accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide radical in two E. nutans, which were substantially alleviated by exogenous ALA and NO application. Pretreatment with NO scavenger PTIO or NOS inhibitor L-NNA alone and in combination with ALA induced enhancements in electrolyte leakage and the accumulation of MDA, H2O2 and superoxide radical in leaves of DX and ZD exposed to chilling stress, indicating that the inhibition of NO biosynthesis reduced the chilling resistance of E. nutans and the ALA-enhanced chilling resistance. Further analyses showed that ALA and NO enhanced antioxidant defense and activated plasma membrane (PM) H+-ATPase and decreased the accumulation of ROS induced by chilling stress. A pronounced increase in nitric oxide synthase (NOS) activity and NO release by exogenous ALA treatment was found in chilling-resistant DX plants exposed to chilling stress, while only a little increase was observed in chilling-sensitive ZD. Furthermore, inhibition of NO accumulation by PTIO or L-NNA blocked the protective effect of exogenous ALA, while both exogenous NO treatment and inhibition of endogenous NO accumulation did not induce ALA production. These results suggested that NO might be a downstream signal mediating ALA-induced chilling resistance in E. nutans.

Nitric Oxide Plays a Central Role in Water Stress-Induced Tanshinone Production in Salvia miltiorrhiza Hairy Roots.

Nitric oxide (NO), a well-known signaling molecule plays an important role in abiotic and biotic stress-induced production of plant secondary metabolites. In this study, roles of NO in water stress-induced tanshinone production in Salvia miltiorrhiza hairy roots were investigated. The results showed that accumulations of four tanshinone compounds in S. miltiorrhiza hairy roots were significantly stimulated by sodium nitroprusside (SNP, a NO donor) at 100 μM. Effects of SNP were just partially arrested by the mevalonate (MVA) pathway inhibitor (mevinolin), but were completely inhibited by the 2-C-methyl-d-erythritol-4-phosphate pathway (MEP) inhibitor (fosmidomycin). The increase of tanshinone accumulation and the up-regulation of HMGR and DXR expression by PEG and ABA treatments were partially inhibited by an inhibitor of NO biosynthesis (Nω-nitro-L-arginine methyl ester (L-NAME)) and a NO scavenger (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)). Simultaneously, NO generation in the hairy roots was triggered by PEG and ABA, and the effects were also arrested by c-PTIO and L-NAME. These results indicated that NO signaling probably plays a central role in water stress-induced tanshinone production in S. miltiorrhiza hairy roots. SNP mainly stimulated the MEP pathway to increase tanshinone accumulation.

Different patterns of H2S/NO activity and cross-talk in the control of the coronary vascular bed under normotensive or hypertensive conditions

Hydrogen sulfide (H2S) and nitric oxide (NO) play pivotal roles in the cardiovascular system. Conflicting results have been reported about their cross-talk. This study investigated their interplays in coronary bed of normotensive (NTRs) and spontaneously hypertensive rats (SHRs).The effects of H2S- (NaHS) and NO-donors (sodium nitroprusside, SNP) on coronary flow (CF) were measured in Langendorff-perfused hearts of NTRs and SHRs, in the absence or in the presence of propargylglycine (PAG, inhibitor of H2S biosynthesis), L-NAME (inhibitor of NO biosynthesis), ODQ (inhibitor of guanylate cyclase), L-Cysteine (substrate for H2S biosynthesis) or L-Arginine (substrate for NO biosynthesis).In NTRs, NaHS and SNP increased CF; their effects were particularly evident in Angiotensin II (AngII)-contracted coronary arteries. The dilatory effects of NaHS were abolished by L-NAME and ODQ; conversely, PAG abolished the effects of SNP.In SHRs, high levels of myocardial ROS production were observed. NaHS and SNP did not reduce the oxidative stress, but produced clear increases of the basal CF. In contrast, in AngII-contracted coronary arteries of SHRs, significant hyporeactivity to NaHS and SNP was observed. In SHRs, the vasodilatory effects of NaHS were only modestly affected by L-NAME and ODQ; PAG poorly influenced the effects of SNP.Then, in NTRs, the vascular actions of H2S required NO and vice versa. By contrast, in SHRs, the H2S-induced actions scarcely depend on NO release; as well, the NO effects are largely H2S-independent. These results represent the first step for understanding pathophysiological mechanisms of NO/H2S interplays under both normotensive and hypertensive conditions.

Effects of Nitric Oxide on Neuromuscular Properties of Developing Zebrafish Embryos

Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.

High levels of arginine, citrulline and ADMA are independent predictors of cardiovascular disease

Purpose: Arginine is the sole nitrogen source for Nitric Oxide (NO) synthesis and citrulline is the major catabolic product of arginine. Arginine restores endothelial function in atherosclerotic patients, in whom there are elevated levels of Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of Nitric Oxide Synthase (NOS). In this study we investigated the relationships among plasma arginine levels, its catabolic end-product citrulline and the competitive inhibitor of NO biosynthesis (ADMA) and subclinical measures of atherosclerosis and as well incident Cardiovascular Disease (CVD). Methods: We performed a matched case-control study derived from the population-based Malmo Diet and Cancer Cardiovascular Cohort (MDC-CC), all included subjects were without history of CVD at baseline. During 12 years of follow-up, 253 individuals had a CVD event and were matched for age, sex and Framingham risk score with 253 controls. Arginine, citrulline and ADMA were profiled in baseline plasma samples using liquid chromatography-tandem mass spectrometry and relationship to incident CVD was assessed using conditional logistic regression. We further examined whether arginine, citrulline and ADMA correlated with anatomical (plaque formation) abnormalities. Results: After adjustment for traditional risk factors, inflammation (hs-CRP), diabetes status and insulin resistance (HOMA-index) at baseline, each 1SD increase of arginine (1.65, 1.21-2.26; p=0.002), citrulline (1.43, 1.13-1.80; p=0.003), and ADMA (1.34, 1.04-1.72; p=0.024), were all significantly associated with increased risk of CVD after 12 years of follow-up. Furthermore, increased levels (in quartiles) of arginine, citrulline and ADMA were cross-sectionally related with presence of more than 50% stenosis of the carotid artery; (Ptrend=0.006 for arginine, Ptrend = 0.001 for citrulline and Ptrend = 0.025 for ADMA). Conclusion: This study identifies arginine, citrulline and ADMA as independent early predictors of CVD, after taking traditional risk factors for CVD, inflammation and insulin resistance into account.

Alfacalcidol suppresses α-receptor–mediated vasoconstriction via an endothelium dependent mechanism

Vitamin D level could be related to blood pressure and cardiovascular health. However, the direct suppressor effect of vitamin D on vascular contraction has not yet been explored. Therefore, the effects of alfacalcidol (1\alpha-hydroxyvitamin D_3) and calcitriol (1\alpha,25-dihydroxyvitamin D_3) on contractions induced by phenylephrine (10^{-8} to 10^{-5} M) in mice aorta were investigated. Materials and methods: Arterial rings were suspended in isolated organ chambers filled with Krebs bicarbonate solution gassed with 95% O_2 and 5% CO_2 maintained at 37 °C. Tissue responses were recorded isometrically with a force transducer and displayed on an acquisition system. Alfacalcidol and calcitriol were applied on phenylephrine-induced active tone. Furthermore, aortic rings were preincubated with alfacalcidol and calcitriol and thereafter contracted with phenylephrine. Incubation studies were conducted by endothelium removal and in the presence of L-nitro-arginine methyl ester (L-NAME). Results: Phenylephrine-induced active tones were significantly suppressed by alfacalcidol (P < 0.05 and P < 0.01) and calcitriol (both 10^{-5} and 10^{-6} M). The pEC_{50} values of alfacalcidol and calcitriol were 7.06 ± 0.91 and 7.13 ± 0.60, respectively. Furthermore, preincubation of aortic rings with alfacalcidol (10^{-5} M) but not calcitriol (10^{-5} M) significantly inhibited phenylephrine-induced contractions (10^{-8} to 10^{-5} M, P < 0.05 and P < 0.001). The suppressive effect of alfacalcidol incubation disappeared with endothelium removal. Furthermore, alfacalcidol was unable to inhibit phenylephrine-induced contractions in the presence of L-NAME, an inhibitor of nitric oxide biosynthesis. Conclusion: In this study we report for the first time that alfacalcidol may prevent phenylephrine-induced vasoconstriction in mice aorta with an endothelium-dependent nitric oxide-mediated mechanism.

The role of nitric oxide and sulphydryls in gastric mucosal protection induced by sodium cromoglycate in rats.

The role of endogenous nitric oxide and sulphydryls in gastric protection afforded by sodium cromoglycate against ethanol-induced gastric lesions was studied in rats. Drugs were administered either intraperitoneally (i.p.) or subcutaneously (s.c.) 30, 45 or 60 min before oral administration of ethanol. Administration of cromoglycate before ethanol dose-dependently inhibited ethanol-induced gastric lesions. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, dose-dependently aggravated gastric lesions and reduced cromoglycate-induced gastric protection. The attenuating effect of L-NAME on gastric protection elicited by cromoglycate was reversible by pretreatment with L-arginine but not by D-arginine. On the other hand, ethanol-induced gastric lesions were found to be associated with a reduction of nonprotein sulphydryl content of glandular stomachs. Pretreatment with cromoglycate prevented non protein sulphydryl depletion and afforded protection. Pretreatment with N-ethylmaleimide, a sulphydryl blocker, caused dose-dependent enhancement of ethanol-induced gastric lesions and further depletion of non protein-sulphydryl. Treatment with N-ethylmaleimide before cromoglycate reduced the gastric protection that was associated with depletion of nonprotein sulphydryls. Furthermore, combined N-ethylmaleimide and L-NAME pretreatment caused a greater aggravation of ethanol-induced gastric lesions and significantly produced a higher reduction of the protective effects of cromoglycate. However, pretreatment with L-arginine only partially restored the protective effects of cromoglycate. These results suggest that the protective effects of cromoglycate may be dependent on the maintenance of a critical level of both endogenous nitric oxide and nonprotein sulphydryls in the gastric mucosa.

Effects of L-Nitro-Arginine Methyl Ester, an Inhibitor of Nitric Oxide Biosynthesis, on Intestinal Ischemia/Reperfusion Injury in Rabbits

To study whether treatment with L-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, attenuates intestinal dysfunction caused by ischemia (I) and/or reperfusion (R), rabbits were treated with L-NAME (15 mg · kg −1, intervenously) or saline olution (SS) prior to I (60 minutes) induced by occlusion of superior mesenteric artery and/or R (120 minutes). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCI using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared with a sham group, the jejunal contractions were similar in the I/R + L-NAME, but reduced in I + SS, I/R + SS, and I + L-NAME groups. The jejunal enteric nerves were damaged in the I + SS, I/R + SS, and I + L-NAME cohorts, but not among the I/R + L-NAME cohort. These results suggested that L-NAME attenuated intestinal dysfunction caused by R but not by I.

Hormonal influence on the release of endothelial nitric oxide: gender-related dimorphic sensitivity of rat aorta for noradrenaline.

Male gender shows a higher incidence of vascular disorders and this phenomenon could be explained by sexual dimorphic behaviour of vessels. Both gonadal hormones and endothelial nitric oxide (NO) are involved in the regulation of the vascular reactivity. This study aimed to evaluate a possible sexual dimorphic sensitivity of rat aorta for the catecholamine noradrenaline. To understand the role played by physiological concentrations of sex hormones, the experimental procedures were performed on isolated preparations from intact (sham-operated) and gonadectomized rats of both sexes. In parallel sets of experiments, the biosynthesis of NO was inhibited by N'-nitro-L-arginine methyl ester (L-NAME) to reveal any potential involvement of the endothelial modulator and its possible link with the endocrinous factor. In aortae from intact male and female rats, noradrenaline induced contractile effects with different potencies (mean+s.d. EC50 values 12.15 +/- 5.25 nM and 84.10 +/- 18.68 nM, respectively). Gonadectomy resulted in an increased sensitivity for noradrenaline in female vessels and a decreased sensitivity for the agonist in male vessels (EC50 values 25.64 +/- 5.04 nM and 21.70 +/ 1 1.13 nM, respectively). In aortae from intact male rats, the inhibition of NO biosynthesis resulted in a weak increase in sensitivity for noradrenaline (EC50 value 6.08 +/- 4.53 nM), whereas the increase was higher in vessels from intact female rats (EC50 value 10.38 +/- 8.40 nM). After treatment with L-NAME, aortae from gonadectomized male and female rats presented almost equivalent increases in sensitivity for the adrenergic agonist (EC50 values 6.02 +/- 3.63 nM and 9.10+/- 9.63 nM,respectively),and no significant difference in sensitivity could be recorded between intact and orchidectomized male rats, or between intact and ovariectomized female rats. It was concluded that rat aorta showed a sexual dimorphic sensitivity for noradrenaline and the female sex was more protected against the adrenergic contractile stimulus because of a higher release of endothelial NO. The gender-related difference in NO release was influenced by gonadal hormones, with the female hormones inducing an increase and the male hormones causing a reduction.

Microglia enhance manganese chloride-induced dopaminergic neurodegeneration: Role of free radical generation

Exposure to elevated levels of manganese has been shown to cause neuronal damage in the midbrain and the development of Parkinsonian symptoms. Activation of microglia and release of neurotoxic factors in particular free radicals are known to contribute to neurodegeneration. We have recently reported that manganese chloride (MnCl2) stimulates microglia to produce reactive oxygen species (ROS). The aim of this study is to determine the role of microglia in the MnCl2-induced degeneration of dopaminergic (DA) neurons that are particularly vulnerable to oxidative insult. MnCl2 (10–300 μM; 7 days) was markedly more effective in damaging DA neurons in the rat mesencephalic neuron–glia cultures than the neuron-enriched (microglia-depleted) cultures. In addition, the microglia-enhanced MnCl2 toxicity was found to be preferential to DA neurons. The microglial enhancement of DA neurotoxicity was further supported by the observation that replenishment of microglia to the neuron-enriched cultures significantly increased the susceptibility of DA neurons to the MnCl2-induced damage. Analysis of the temporal relationship between microglial activation and DA neurodegeneration revealed that MnCl2-stimulated microglial activation preceded DA neurodegeneration. Mechanistically, MnCl2 (10–300 μM) stimulated a concentration- and time-dependent robust production of ROS and moderate production of nitric oxide but no detectable release of tumor necrosis factor-alpha and interleukin-1beta. Application of free radical scavengers including superoxide dismutase/catalase, glutathione, N-acetyl cysteine and an inhibitor of nitric oxide biosynthesis significantly protected DA neurons against the MnCl2-induced degeneration. These results demonstrate that microglial activation and the production of reactive nitrogen and oxygen free radicals promote the MnCl2-induced DA neurodegeneration.

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Ectonucleotidase activities are altered in serum and platelets of l-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of Nω-Nitro- l-arginine methyl ester hydrochloride ( l-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects; thus, ADP is the most important platelet agonist and recruiting agent, while adenosine, an end product of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5′-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonucleotidase activities of serum and platelets from rats made hypertensive by oral administration of l-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days plus 7 days of l-NAME washout, in the drinking water) in comparison to normotensive control rats. l-NAME promoted a significant rise in systolic blood pressure from 112 ± 9.8 to 158 ± 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with l-NAME for 14 days when compared to control animals. In serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of l-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%, respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from l-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation.