In vivo protein tyrosine nitration in S. cerevisiae: Identification of tyrosine-nitrated proteins in mitochondria

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Δyhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Δyhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Δyhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Δyhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Nitric oxide synthase activity and the level of nitrates/nitrites in brain regions during spontaneous morphine withdrawal in rats

Activity of nitric oxide synthase (NOS) and concentrations of nitrate/nitrites (NO _x ^? ) were measured in brain regions of rats during spontaneous morphine withdrawal, which was modeled in male Wistar rats. The animals were injected with the increasing intraperitoneal doses (10–100 mg/kg, twice a day) of morphine hydrochloride for 6 days. Thirty six hours after the last injection the severity of the spontaneous morphine withdrawal syndrome was determined by specific autonomic and locomotor indices The withdrawal was accompanied by the increase of both NOS activity and NO _x ^? levels in the midbrain and hippocampus, the decrease of these parameters in striatum and hypothalamus, and lack of changes in cerebral cortex and brain stem. In cerebellum NOS activity decreased whereas NO _x ^? concentrations remained unchanged. In the cerebral cortex, striatum, midbrain, and cerebellum activity of NOS and NO _x ^? concentrations correlated with the withdrawal syndrome severity and also with the specific signs of abstinence.     

Metal-catalyzed protein tyrosine nitration in biological systems

Abstract

Protein tyrosine nitration is an oxidative postranslational modification that can affect protein structure and function. It is mediated in vivo by the production of nitric oxide-derived reactive nitrogen species (RNS), including peroxynitrite (ONOO^?) and nitrogen dioxide (^?NO₂). Redox-active transition metals such as iron (Fe), copper (Cu), and manganese (Mn) can actively participate in the processes of tyrosine nitration in biological systems, as they catalyze the production of both reactive oxygen species and RNS, enhance nitration yields and provide site-specificity to this process. Early after the discovery that protein tyrosine nitration can occur under biologically relevant conditions, it was shown that some low molecular weight transition-metal centers and metalloproteins could promote peroxynitrite-dependent nitration. Later studies showed that nitration could be achieved by peroxynitrite-independent routes as well, depending on the transition metal-catalyzed oxidation of nitrite (NO₂^?) to ^?NO₂ in the presence of hydrogen peroxide. Processes like these can be achieved either by hemeperoxidase-dependent reactions or by ferrous and cuprous ions through Fenton-type chemistry. Besides the in vitro evidence, there are now several in vivo studies that support the close relationship between transition metal levels and protein tyrosine nitration. So, the contribution of transition metals to the levels of tyrosine nitrated proteins observed under basal conditions and, specially, in disease states related with high levels of these metal ions, seems to be quite clear. Altogether, current evidence unambiguously supports a central role of transition metals in determining the extent and selectivity of protein tyrosine nitration mediated both by peroxynitrite-dependent and independent mechanisms.     

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5 s), therefore direct NO quantification is challenging. An indirect method – based on quantifying conversion of an l-Arg- to l-Cit-derivative by ¹H NMR spectroscopy – is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a–j and 11a–c were better or equivalent substrates for the eNOS enzyme (NO₂^? production = 19–46 μM) than native l-Arg (NO₂^? production = 25 μM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by ¹H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide‐dependent mechanism

Protein tyrosine (Tyr) nitration is a post‐translational modification yielding 3‐nitrotyrosine (NO₂–Tyr). Formation of NO₂–Tyr is generally considered as a marker of nitro‐oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O₂ transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO₂–Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO₂–Tyr25 and NO₂–Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and by alternative means to nitrate reductase, probably via a ˙NO synthase‐like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires  + H₂O₂ and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to . Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO₂–Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles

S-Nitrosoglutathione (GSNO) is a nitrosothiol which plays a major role in the metabolism of NO in higher plants mediating signaling processes. Protein tyrosine nitration (NO₂–Tyr) is a post-translational modification which contributes to protein regulation. The subcellular localization of GSNO, S-nitrosoglutathione reductase (GSNOR), an enzyme which catalyzes its decomposition and protein tyrosine nitration was studied in pea (Pisum sativum L.) leaf plants with the aid of the electron microscopy immunogold-labeling technique. Our findings show that GSNO, GSNOR and nitrated proteins are present in the different subcellular compartments of leaf cells which include chloroplasts, cytosol, mitochondria, and peroxisomes. Given that pea peroxisomes are one of the cell compartments where nitric oxide (NO) has been thoroughly studied, our results provide additional insights into the metabolism of NO in this organelle where NO and GSNO could function as signal molecules in cross talk between the different cell compartments.     

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

Background and Aims Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper.Methods The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO₂-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis.Key Results Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit.Conclusions The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H₂O₂, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.     

Isolation and Some Properties of an Aminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Evolution of nitrogen oxides (NO_(x), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO_(x) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO_(x). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and ¹⁵N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO_(x) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO_(x). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate (¹⁵NO₃⁻) reduction during the assay.     

Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1

Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the ‘Trp triad.’ Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH°) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.     

Functional consequences of actin nitration: in vitro and in disease states

To link the phenomena of inflammatory-induced increases in protein nitrotyrosine (NO₂Tyr) derivatives to protein dysfunction and consequent pathological conditions, the evaluation of discrete NO₂Tyr modifications on specific proteins must be undertaken. Mass spectrometric (MS) proteomics-based strategies allow for the identification of all individual proteins that are nitrated by separating tissue homogenates using 2D gel electrophoresis, detecting the nitrated proteins using an anti-NO₂Tyr antibody, and then identifying the peptides generated during an in-gel proteolytic digest using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) MS. Actin, one of the most abundant proteins in eukaryotic cells, constitutes 5% or more of cell protein and serves with other cytoskeletal proteins as a critical target for nitration-induced functional impairment. Herein, examples of actin nitration detected under physiological conditions in various models of human disease or in clinically derived tissues are given and the impact that this post-translational protein modification can have on cell and organ function is discussed.     

15N-CIDNP investigations during tryptophan, N-acetyl-L-tryptophan, and melatonin nitration with reactive nitrogen species

Tryptophan and melatonin are nitrated by peroxynitrite; tryptophan residues in proteins are susceptible to attack by reactive nitrogen species. Nitrated tryptophan might therefore be used as a biomarker for the involvement of reactive species derived from nitrogen oxide in a variety of pathophysiological conditions. The radical character of the tryptophan (Trp) and N-acetyl-L-tryptophan (N-AcTrp) nitration with peroxynitrite is shown using (15)N-CIDNP. During the decay of peroxynitrite-(15)N in the presence of Trp at pH 5 in the probe of a (15)N-NMR spectrometer, the (15)N-NMR signals of various nitrated tryptophans ((15)NO(2)-Trp) show emission (E). The effects are built up in radical pairs [Trp( radical), 15NO2 ](F) formed by diffusive encounters of radicals 15NO2 and Trp( radical) generated during decay of peroxynitrite-(15)N in the presence of Trp. Similar (15)N-CIDNP effects are observed during reaction of Trp and/or N-AcTrp using the nitrating systems H(15)NO(3), H(15)NO(4) and H(2)O(2)/15NO2 /HRP, which are also built up in radical pairs [Trp, 15NO2 ](F). During nitration of melatonin (Mel) with peroxynitrite-(15)N and H(15)NO(4), the (15)N-NMR signal of 4-nitromelatonin (4-(15)NO(2)-Mel) shows emission arising from radical pairs [Mel, 15NO2 ](F) which are formed in an analogous manner.     

Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells

Nitro-arachidonic acid (NO₂-AA) is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II) produces an increase in reactive oxygen species (ROS) production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO₂-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO₂-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells). Treatment of HK-2 cells with ANG II increases the production of superoxide (O₂^●-), nitric oxide (^●NO), inducible nitric oxide synthase (NOS2) expression, peroxynitrite (ONOO^-) and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO₂-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO^-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO₂-AA. Whereas the activity of succinate dehydrogenase (SDH) and ATP synthase (ATPase) were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO₂-AA. Moreover, NO₂-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO₂-AA, identifying this compound as a promising pharmacological tool to prevent ANG II–induced renal disease.     

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO₃⁻ uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO₃⁻ leaves) or 40 mm NO₃⁻ solution (high-NO₃⁻ leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO₃⁻-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO₃⁻ and high-NO₃⁻ leaves, indicating that NO₃⁻ had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO₃⁻ compared with low NO₃⁻ leaves showed evidence that NO₃⁻ enhanced the protein kinase activity. These results support the concept that NO₃⁻, or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).     

Aluminum overload increases oxidative stress in four functional brain areas of neonatal rats

Background

Higher aluminum (Al) content in infant formula and its effects on neonatal brain development are a cause for concern. This study aimed to evaluate the distribution and concentration of Al in neonatal rat brain following Al treatment, and oxidative stress in brain tissues induced by Al overload.

Methods

Postnatal day 3 (PND 3) rat pups (n =46) received intraperitoneal injection of aluminum chloride (AlCl₃), at dosages of 0, 7, and 35 mg/kg body wt (control, low Al (LA), and high Al (HA), respectively), over 14 d.

Results

Aluminum concentrations were significantly higher in the hippocampus (751.0 ± 225.8 ng/g v.s. 294.9 ± 180.8 ng/g; p < 0.05), diencephalon (79.6 ± 20.7 ng/g v.s. 20.4 ± 9.6 ng/g; p < 0.05), and cerebellum (144.8 ± 36.2 ng/g v.s. 83.1 ± 15.2 ng/g; p < 0.05) in the HA group compared to the control. The hippocampus, diencephalon, cerebellum, and brain stem of HA animals displayed significantly higher levels of lipid peroxidative products (TBARS) than the same regions in the controls. However, the average superoxide dismutase (SOD) activities in the cerebral cortex, hippocampus, cerebellum, and brain stem were lower in the HA group compared to the control. The HA animals demonstrated increased catalase activity in the diencephalon, and increased glutathione peroxidase (GPx) activity in the cerebral cortex, hippocampus, cerebellum, and brain stem, compared to controls.

Conclusion

Aluminum overload increases oxidative stress (H₂O₂) in the hippocampus, diencephalon, cerebellum, and brain stem in neonatal rats.     

Activation of Glutamate Receptors Stimulates the Formation of Nitrite in Synaptosomes from Rat Cerebellum

Abstract: Synaptosomes from rat cerebellum were used to investigate the involvement of different glutamate receptor subtypes in the control of the synthesis of nitric oxide (NO), measured as its breakdown product nitrite (NO₂^-). Synaptosomes incubated in the presence of NAD|PH and l -arginine produced measurable levels of NO₂^-, which were reduced by addition of Nω-nitro-l -arginine methyl ester, an inhibitor of nitric oxide synthase. The selective ionotropic glutamate receptor agonist N-methyl-d -aspartate (NMDA) induced a pronounced increase in NO₂^-formation, which was prevented by Nω-nitro-l -arginine methyl ester and by the specific NMDA receptor antagonist Dl -2-amino-5-phosphonovaleric acid (AP-5). The NMDA-induced increase in NO₂^-formation was blocked by chelation of extracellular Ca²⁺ with EGTA. Both l -glutamate and the selective agonist for the metabotropic glutamate receptors (β)-1-aminocyclopentane-trans-1,3-dicarboxylic acid raised NO₂^-production, which retumed to control levels after addition of Nω-nitro-l -arginine methyl ester. The selective glutamate ionotropic receptor agonist (R,S)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid did not cause any change in NO₂ formation. The stimulatory effect of l -glutamate was blocked by the metabotropic glutamate receptor antagonist Dl -2-amino-4-phosphonobutyric acid but was unaffected by the selective NMDA receptor blocker AP-5. Removal of extracellular Ca²⁺ by EGTA did not affect the action of l -glutamate; whereas W-7, an inhibitor of calmodulin, and dantrolene, a compound that blocks the mobilization of Ca²⁺ from intracellular stores, abolished the effect of l -glutamate on NO₂^-formation. It is suggested that stimulation of ionotropic NMDA receptors activates NO metabolism by causing an influx of Ca²⁺ from the extracellular space, whereas activation of metabotropic receptors by l -glutamate provokes a mobilization of Ca²⁺ from intracellular stores, which stimulates nitric oxide synthase activity by forning Ca²⁺/calmodulin complexes.