Steady-state Levels Of Nitric Oxide Research Articles

Nitric Oxide Alters the Expression of γ-Globin, H-Ferritin, and Transferrin Receptor in Human K562 Cells at the Posttranscriptional Level

Cellular iron metabolism is altered during chronic inflammatory states, leading to reticuloendothelial iron sequestration and an associated anemia. To study the effects of nitric oxide (NO) on the expression of three genes involved in erythroid cell iron metabolism (gamma-globin, H-ferritin, and transferrin receptor [TfR]), we developed a series of human K562 erythroleukemic cell clones retrovirally transduced with inducible nitric oxide synthase (NOS-2) and producing different steady-state levels of NO. gamma-Globin and H-ferritin protein expression was reduced in NO-producing cells in relation to the amount of NO produced. Conversely, cell surface TfR expression increased in NO-producing clones. Both the inhibitory effects of NO on gamma-globin and H-ferritin expression and the stimulatory effect on TfR were reversed by the NOS inhibitor NG-methyl-L-arginine (NGMMA). gamma-Globin and H-ferritin mRNA levels were unaffected by NO production. In the case of TfR, NO appeared to stabilize mRNA in that the half life of TfR mRNA decreased from approximately 15 hours to less than 3 hours when NO production by NOS-transduced clones was inhibited. Thus, NO can regulate expression of these genes at the posttranscriptional level, an effect that is likely mediated by the known effect of NO on the RNA binding activity of iron regulatory protein-1 (Pantopoulos and Hentze, Proc Natl Acad Sci USA 92:1267, 1995). Furthermore, our findings suggest a mechanism for the observed relationship between NO production and the pathophysiology of the anemia of chronic disease.

Contrasting Regulation of NO and ROS in Potato Defense-Associated Metabolism in Response to Pathogens of Different Lifestyles.

Our research provides new insights into how the low and steady-state levels of nitric oxide (NO) and reactive oxygen species (ROS) in potato leaves are altered after the challenge with the hemibiotroph Phytophthora infestans or the necrotroph Botrytis cinerea, with the subsequent rapid and invader-dependent modification of defense responses with opposite effects. Mainly in the avirulent (avr) P. infestans–potato system, NO well balanced with the superoxide level was tuned with a battery of SA-dependent defense genes, leading to the establishment of the hypersensitive response (HR) successfully arresting the pathogen. Relatively high levels of S-nitrosoglutathione and S-nitrosothiols concentrated in the main vein of potato leaves indicated the mobile function of these compounds as a reservoir of NO bioactivity. In contrast, low-level production of NO and ROS during virulent (vr) P. infestans-potato interactions might be crucial in the delayed up-regulation of PR-1 and PR-3 genes and compromised resistance to the hemibiotrophic pathogen. In turn, B. cinerea triggered huge NO overproduction and governed inhibition of superoxide production by blunting NADPH oxidase. Nevertheless, a relatively high level of H2O2 was found owing to the germin-like activity in cooperation with NO-mediated HR-like cell death in potato genotypes favorable to the necrotrophic pathogen. Moreover, B. cinerea not only provoked cell death, but also modulated the host redox milieu by boosting protein nitration, which attenuated SA production but not SA-dependent defense gene expression. Finally, based on obtained data the organismal cost of having machinery for HR in plant resistance to biotrophs is also discussed, while emphasizing new efforts to identify other components of the NO/ROS cell death pathway and improve plant protection against pathogens of different lifestyles.

Folic acid enlarges the armamentarium for the treatment of coronary vasospasm

Qipshidze et al. (2010) described in the Journal of Cell Physiology that folic acid ameliorates acetylcholine-induced vasoconstriction in hyperhomocysteinemic cystathionine beta synthase heterozygote (CBS /þ) mice, and that this effect can be explained by an increase in endothelial nitric oxide (eNOS) expression and nitric oxide (NO) bioavailability in the coronary artery. The authors have investigated the role of eNOS in acetylcholine-induced vasoconstriction in CBS /þ mice by measuring total protein (by immunolabelling) and mRNA levels (by RT-PCR). However, they did not perform eNOS uncoupling assessment, which is of essential importance to evaluate the active or inactive state of eNOS. Qipshidze et al. (2010) assessed only the NO-pathway using diaminofluorescein-2-diacetat staining. However, it should be considered that compartmentalization of diaminofluorescein-2 around elastic fibers limits its potential to characterize the site of NO production at the subcellular level. Further, reaction of diaminofluorescein-2 with reagents commonly used in NO research, such as ferricyanide, N-ethylmaleimide, and tempol, may also interfere with diaminofluorescein-2 fluorescence that can be misinterpreted as NO signals. Importantly, in cell-free systems, the diaminofluorescein-2 dependent signal is enhanced in the presence of superoxide, suggesting that diaminofluorescein-2 is indeed more sensitive to the degradation product of NO, peroxynitrite, rather than to NO itself (Roychowdhury et al., 2002). This would imply that the detection of higher signals in the presence of diaminofluorescein-2 do not necessarily indicate higher levels of bioavailable NO but can rather indicate the reverse, higher levels of peroxynitrite and NO scavenging. In any case, even if diaminofluorescein-2 were specific for NO or more sensitive for NO than for peroxynitrite, higher steady state levels of NO must not necessarily indicate an effect of eNOS and higher production of NO but can also indicate reduced oxidative stress or a direct anti-oxidant effect of folate. Thus, the demonstration of recoupling of eNOS from its monomeric to its dimeric state would have been essential to justify the authors’ conclusion. In addition, UV illumination can lead to formation of high levels of nitrosating species that interferewith NO detection coming from enzymatic sources (Rodriguez et al., 2005). Therefore, assessment of NO alone by diaminofluorescein-2-diacetate cannot conclude whether or not this molecule is generated by eNOS. Other methods, such as eNOSmonomer/dimer blot, dichlorofluorescin staining, and NOS-dependent ROS production using lucigenin-enhanced chemiluminescence should also be considered. Unfortunately, the authors also did not investigate the role of folic acid-induced changes in high-energy phosphate metabolism. It has been described that folic acid, besides its well-known homocysteine-lowering and antioxidant effect, preserves the high-energy phosphate metabolism during myocardial ischemia with subsequent less myocardial necrosis, better myocardial function, and less arrhythmias (Moens et al., 2008). Title et al. (2000) have demonstrated that endothelial function, as assessed by flow-mediated dilation (FMD) in patients with coronary artery diseases, is ameliorated by reducing the level of plasma homocysteine. This is in contrast with a study ofDoshi et al. (2001), which showed no correlation between FMD improvement and plasma homocysteine level in patients with coronary artery diseases. This result is supported by Moens et al. (2007) demonstrating in patients with acute myocardial infarction that the beneficial effect of folic acid on endothelial dysfunction is independent of baseline homocysteine-level. Chambers et al. (2000) have shown that improved FMD correlates with the reduction in free (unbound)-reduced homocysteine in patients with coronary heart diseases. Unfortunately, Qipshidze et al. (2010) did not assess any of the different forms (reduced, mixed disulphides, and total) of homocysteine. It has been described that smoothmuscle constriction of the coronary artery, the so-called coronary vasospasm, is associated with increased markers of oxidative stress (Motoyama et al., 1998) and inflammation (Li et al., 2008). Interestingly, certain behavioral traits including type A personality, panic disorder, and severe anxiety, have been described to be associated with coronary artery vasospasm (Rasmussen et al., 1986; Vidovich et al., 2009). However, no data are available regarding whether these clinical situations are associated with changes in endothelial NOS. Translation of these pre-clinical data to humans, and especially to patientswith these behavioral conditions is therefore well encouraged. The data provided in the paper of Qipshidze et al. (2010) are intriguing since they open theraupetic possibilities for patient with coronary vasospasms. Nitrates and calcium channel

Nitrate Reductase- and Nitric Oxide-Dependent Activation of Sinapoylglucose:malate sinapoyltransferase in Leaves of Arabidopsis thaliana

Nitrate reductase (NR) activity is necessary for the synthesis of nitric oxide (NO), a key signaling molecule in plants. Here, we investigated the effect of NR deficiency on NO production and phenylpropanoid metabolism of Arabidopsis thaliana leaves. HPLC-mass spectrometry analysis showed that the NR double mutant (nia1 nia2) is deficient in the synthesis of sinapoylmalate (SM), the main phenylpropanoid end-product in wild-type leaves, resulting in accumulation of its precursor sinapoylglucose (SG). While real-time PCR analysis revealed no significant difference at the transcript level, sinapoylglucose:malate sinapoyltransferase (SMT) activity in leaf extracts was reduced in the mutant compared with the wild type. The low levels of SM in nia1 nia2 leaves do not result from the deficient nitrogen incorporation into amino acids, since the recovery of the amino acid content of nia1 nia2 by irrigating the plants with glutamine did not change the metabolic profile of this mutant. In contrast, an increased supply of nitrate stimulated NR activity and NO production, and enhanced SM and decreased SG levels in both genotypes. Nevertheless, sinapic acid esters in nia1 nia2 were not recovered when compared with those detected in the leaves of the wild-type plant. Mutant plants grown in medium supplemented with malate and an NO donor recovered SM to the levels of wild-type leaves. Overall, the results suggest that SMT activity is dependent on the NR-dependent steady-state levels of NO during plant development.

A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O2 over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O2 into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O2 in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.

Seed-specific elevation of non-symbiotic hemoglobin AtHb1: beneficial effects and underlying molecular networks in Arabidopsis thaliana.

BackgroundSeed metabolism is dynamically adjusted to oxygen availability. Processes underlying this auto-regulatory mechanism control the metabolic efficiency under changing environmental conditions/stress and thus, are of relevance for biotechnology. Non-symbiotic hemoglobins have been shown to be involved in scavenging of nitric oxide (NO) molecules, which play a key role in oxygen sensing/balancing in plants and animals. Steady state levels of NO are suggested to act as an integrator of energy and carbon metabolism and subsequently, influence energy-demanding growth processes in plants.ResultsWe aimed to manipulate oxygen stress perception in Arabidopsis seeds by overexpression of the non-symbiotic hemoglobin AtHb1 under the control of the seed-specific LeB4 promoter. Seeds of transgenic AtHb1 plants did not accumulate NO under transient hypoxic stress treatment, showed higher respiratory activity and energy status compared to the wild type. Global transcript profiling of seeds/siliques from wild type and transgenic plants under transient hypoxic and standard conditions using Affymetrix ATH1 chips revealed a rearrangement of transcriptional networks by AtHb1 overexpression under non-stress conditions, which included the induction of transcripts related to ABA synthesis and signaling, receptor-like kinase- and MAP kinase-mediated signaling pathways, WRKY transcription factors and ROS metabolism. Overexpression of AtHb1 shifted seed metabolism to an energy-saving mode with the most prominent alterations occurring in cell wall metabolism. In combination with metabolite and physiological measurements, these data demonstrate that AtHb1 overexpression improves oxidative stress tolerance compared to the wild type where a strong transcriptional and metabolic reconfiguration was observed in the hypoxic response.ConclusionsAtHb1 overexpression mediates a pre-adaptation to hypoxic stress. Under transient stress conditions transgenic seeds were able to keep low levels of endogenous NO and to maintain a high energy status, in contrast to wild type. Higher weight of mature transgenic seeds demonstrated the beneficial effects of seed-specific overexpression of AtHb1.

Effect of L-arginine Oral Supplementation on Response to Myocardial Infarction in Hypercholesterolemic and Hypertensive Rats

The well known metabolic functions of L-arginine have been recently increased with the discovery of its role as the substrate for the synthesis of nitric oxide (NO), which has emerged as an endogenous signaling molecule with potential therapeutic implications for cardiovascular disease. Steady-state levels of NO are derived in part from dietary sources. It has been reported that supplementation of L-arginine reduces atherosclerosis in rabbits and reduces the arterial pressure in hypertensive rats. Therefore, we investigated the effect of L-arginine supplementation using a group of induced hypercholesterolemic rats and a group of spontaneously hypertensive rats; the infarcted area in cardiac tissue of both groups was measured during the response to myocardial infarction in the ischemia-reperfusion model. Hypercholesterolemic rats supplemented with 170 mg kg(-1) of L-arginine showed a significant (P <or= 0.05) reduction in total cholesterol (25.2%) and LDL (27.8%). Spontaneously hypertensive rats supplemented with L-arginine presented a significant reduction (20.3%) in mean blood pressure (P <or= 0.05). The index infarcted area/total heart area, in both: hypercholesterolemic and hypertensive rats supplemented with L-arginine, showed a significant 36% and 29% of cardio protection (P <or= 0.05) effect, respectively. Dietary supplementation with L-arginine may represent a potentially novel nutritional strategy for the treatment of cardiovascular disease.

Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O 2 −). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O 2 − generation, besides complex III, stimulated NO degradation. Larger amounts of O 2 − were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O 2 − production were stimulated by free Ca 2+ concentration. Together, these results indicate that Ca 2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O 2 − production that favors NO degradation in potato tuber mitochondria.

Inhibition of nitric oxide synthase and farnesyltransferase change the activities of several transcription factors

Several types of cellular proteins can be modified by farnesylation and nitrosylation, of which the most significant is Ras. We used manumycin, a farnesyltransferase inhibitor, and L-NAME (Nomega-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor, for characterization of Ras-dependent downstream targets activities. Our results suggest that change of the steady-state levels of nitric oxide and inhibition of farnesylation modified the activities of several transcription factors. We have found that the inhibition of farnesylation by manumycin decreased the DNA-binding activity of nuclear factor (NF)-kappaB, did not change the DNA-binding activities of STAT, Sp1, ATF-2, and CREB, and increased the activities of c-Fos, JunD, and c-Jun. Under such conditions, phosphorylation of Akt was decreased, whereas phosphorylation of extracellular signal-regulated kinase (ERK) was increased and phosphorylation of JNK did not change. Furthermore, our results show that reduction of intracellular concentration of nitric oxides by L-NAME increases the activities of c-Fos, ATF-2 and JunD and decreases the activities of CREB, STAT, Sp1, and c-Jun. The activities of all of these transcription factors are restored to normal levels in the presence of manumycin, suggesting that simultaneous modifications of proteins by farnesylation and nitrosylation change the direction of Ras-controlled downstream pathways. Our results provide further evidence of the significance of posttranslational modifications of Ras for the specificity of transducing cascade networks and physiological outcome.

Superoxide Fluxes Limit Nitric Oxide-induced Signaling

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.

Production of nitric oxide and reactions with plant hemoglobins under hypoxic stress

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. The objectives of this study were: to examine whether NO is produced in transgenic maize suspension cell cultures and transgenic alfalfa roots exposed to hypoxic growth conditions, to determine whether there is a relationship between a class 1 hemoglobin and the amount of NO detected under hypoxic conditions, to estimate the route of formation and breakdown of NO in the tissue and to determine whether there is a relationship between NO production and aerenchyma formation. Maize suspension cell cultures and alfalfa roots, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class 1 hemoglobin. Up to 500 nmol NO/g fresh weight were detected in maize cells exposed to low oxygen tensions for 24 h. The steady state levels of NO in the different cell lines under hypoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Moreover EPR spectra showed evidence of nitrosylated heme complexes in denatured samples of hypoxically-exposed maize cells from both a wild type cell culture and a culture transformed to overexpress hemoglobin. No EPR signal characteristic of nitrosylated heme complexes was evident under aerobic conditions or in treated maize cells transformed to reduce hemoglobin expression. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO3-. Nitrate was shown to be a precursor of NO in hypoxic maize suspension cell cultures by using 15NO3- and EPR spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. There was an inverse relationship between the expression of hemoglobin and the formation of aerenchyma under hypoxic conditions. The Hb- line displayed strong evidence of aerenchyma formation under hypoxia, whereas an Hb+ line showed only slight evidence of cell breakdown characteristic of aerenchyma formation. The levels of NO expressed distal to the root tip were, at least, five fold greater than those found in the tip. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class 1 hemoglobins have a significant function in regulating NO levels.

Class-1 hemoglobins, nitrate and NO levels in anoxic maize cell-suspension cultures.

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa ( Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize ( Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)(-1) was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO(3)(-). Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using (15)NO(3)(-) and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.