João Laranjinha
Research interests
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InterestsNeurobiology, Neurobiochemistry, Neurodegenerative Diseases, Neurodegeneration
Publications
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6.08Impact points
Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress.
Free radical biology & medicine. 03/2012; 52(6):1093-100.
Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, includin... [more] Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12dyn/cm(2)) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H(2)O(2)). Physiological concentrations of H(2)O(2) (flux of 0.1nM/min and 15μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H(2)O(2)- and LSS-induced eNOS phosphorylation and reduced (•)NO levels. We propose a model in which LSS promotes the formation of signaling levels of H(2)O(2), which in turn activate p38α MAPK and then stimulate eNOS, leading to increased (•)NO generation and protection of endothelial function.
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Modulation of cellular respiration by endogenously produced nitric oxide in rat hippocampal slices.
Methods in molecular biology (Clifton, N.J.). 01/2012; 810:73-88.
Nitric oxide (•NO) is a ubiquitous signaling molecule that participates in neuromolecular phenomena associated with memory formation as well as in excitotoxicity. In the hippocampus, neuronal •NO production is coupled to the activation of the NMDA-type of glutamate receptor. More recently, Cytochrom... [more] Nitric oxide (•NO) is a ubiquitous signaling molecule that participates in neuromolecular phenomena associated with memory formation as well as in excitotoxicity. In the hippocampus, neuronal •NO production is coupled to the activation of the NMDA-type of glutamate receptor. More recently, Cytochrome c oxidase has emerged as a novel target for •NO, which competes with O 2 for binding to this mitochondrial complex. This reaction establishes •NO not only as a regulator of cellular metabolism but possibly also as a regulator of mitochondrial production of reactive oxygen species which participate in cellular signaling. A major gap in the understanding of •NO bioactivity, namely, in the hippocampus, has been the lack of knowledge of its concentration dynamics. Here, we present a detailed description of the simultaneous recording of •NO and O2 concentration dynamics in rat hippocampal slices. Carbon fi ber microelectrodes are fabricated and applied for real-time measurements of both gases in a system close to in vivo models. This approach allows for a better understanding of the current paradigm by which an intricate interplay between •NO and O 2 regulates cellular respiration.
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6.08Impact points
Intragastric nitration by dietary nitrite: implications for modulation of protein and lipid signaling.
Free radical biology & medicine. 11/2011; 52(3):693-8.
Inorganic nitrite, derived from the reduction of nitrate in saliva, has recently emerged as a protagonist in nitric oxide ((•)NO) biology as it can be univalently reduced to (•)NO, in the healthy human stomach. Important physiological implications have been attributed to nitrite-derived (•)NO in the... [more] Inorganic nitrite, derived from the reduction of nitrate in saliva, has recently emerged as a protagonist in nitric oxide ((•)NO) biology as it can be univalently reduced to (•)NO, in the healthy human stomach. Important physiological implications have been attributed to nitrite-derived (•)NO in the gastrointestinal tract, namely modulation of host defense, blood flow, mucus formation and motility. At acidic pH, nitrite generates different nitrogen oxides depending on the local microenvironment (redox status, gastric content, pH, inflammatory conditions), including (•)NO, nitrogen dioxide ((•)NO(2)), dinitrogen trioxide (N(2)O(3)), and peroxynitrite. Thus, the gastric environment is a significant source of nitrating and nitrosating agents, especially in individuals consuming a nitrate/nitrite-rich diet on a daily basis. Both, the gastric lumen and mucosa contain putative targets for nitration, not only proteins and lipids from ingested aliments but also endogenous proteins secreted by the oxyntic glands. The physiological and functional consequences of nitration of gastric mediators will impact on local processes including food digestion and ulcerogenesis. Additionally, gastric nitration products (such as nitrated lipids) may be absorbed and affect systemic pathways. Thus, dietary ingestion of nitrate will have direct consequences for endogenous protein nitration, as indicated by our preliminary data.
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3.57Impact points
Nitric oxide and DOPAC-induced cell death: from GSH depletion to mitochondrial energy crisis.
Molecular and cellular neurosciences. 06/2011; 48(1):94-103.
The molecular mechanisms inherent to cell death associated with Parkinson's disease are not clearly understood. Diverse pathways, sequence of events and models have been explored in several studies. Recently, we have proposed an integrative mechanism, encompassing the interaction of nitric oxide... [more] The molecular mechanisms inherent to cell death associated with Parkinson's disease are not clearly understood. Diverse pathways, sequence of events and models have been explored in several studies. Recently, we have proposed an integrative mechanism, encompassing the interaction of nitric oxide (•NO) and a major dopamine metabolite, dihydroxyphenylacetic (DOPAC), leading to a synergistic mitochondrial dysfunction and cell death that may be operative in PD. In this study, we have studied the sequence of events underlying the mechanisms of cell death in PC12 cells exposed to •NO and DOPAC in terms of: a) free radical production; b) modulation by glutathione (GSH); c) energetic status and d) outer membrane mitochondria permeability. Using Electron Paramagnetic Resonance (EPR) it is shown the early production of oxygen free radicals followed by a depletion of GSH reflected by an increase of GSSG/GSH ratio in the cells treated with the mixture of •NO/DOPAC, as compared with the cells individually exposed to each of the stimulus. Glutathione ethyl ester (GSH-EE) and N-acetylcysteine (NAC) may rescue cells from death, increasing GSH content and preventing ATP loss in cells treated with the mixture DOPAC/•NO but failed to exert similar effects in the cells challenged only with •NO. The depletion of GSH is accompanied by a decreased activity of mitochondrial complex I. At a later stage, the concerted action of DOPAC and •NO include a rise in the ratio Bax/Bcl-2, an observation not evident when cells were exposed only to •NO. The results support a free radical-induced pathway leading to cell death involving the concerted action of DOPAC and •NO and the critical role of GSH in maintaining a functional mitochondria.
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3.54Impact points
Evidence for a pathway that facilitates nitric oxide diffusion in the brain.
Neurochemistry international. 06/2011; 59(1):90-6.
Nitric oxide (()NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized ()NO diffusion in the rat brain in vivo. By direct sub-second measurement of ()NO, ... [more] Nitric oxide (()NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized ()NO diffusion in the rat brain in vivo. By direct sub-second measurement of ()NO, we determined the diffusion coefficient of ()NO in the rat brain cortex. The value of 2.2×10(-5)cm(2)/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate ()NO free diffusion). These results reinforce the view of ()NO as a fast diffusing messenger but, noticeably, the data indicates that neither ()NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between ()NO free diffusion coefficient and that obtained in the brain. Overall, the results support that ()NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates ()NO diffusion, such as cell membranes and other hydrophobic structures.
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8.20Impact points
Brain nitric oxide inactivation is governed by the vasculature.
Antioxidants & redox signaling. 03/2011; 14(6):1011-21.
The mechanisms underlying nitric oxide ((•)NO) synthesis and inactivation in the brain are essential determinants of (•)NO neuroactivity. Although (•)NO production is well characterized, the pathways of inactivation in vivo remain largely unknown. Here, we characterize the kinetics and the major mec... [more] The mechanisms underlying nitric oxide ((•)NO) synthesis and inactivation in the brain are essential determinants of (•)NO neuroactivity. Although (•)NO production is well characterized, the pathways of inactivation in vivo remain largely unknown. Here, we characterize the kinetics and the major mechanism of (•)NO inactivation in the rat brain cortex and hippocampus in vivo by measuring locally applied (•)NO with carbon-fiber microelectrodes (CFMs) and ceramic-based microelectrode arrays (MEAs). An apparent first-order clearance was observed in both brain regions, with decay rate constants (k) of (•)NO signals of 0.67 to 0.84 per second, significantly higher than the k obtained in agarose gel (0.099 per second), used as a (•)NO diffusion-control medium. (•)NO half-life in vivo, estimated by mathematical modeling, was 0.42 to 0.75 s. Experiments using MEAs support that the (•)NO diffusion radius is heterogeneous and related to local metabolic activity and vascular density. After global ischemia, k decreased to control values of diffusion in gel, but during anoxia, k decreased only 21%. Additionally, k in brain slices was threefold to fivefold lower than that in vivo, and hemorrhagic shock induced a 53% decrease in k. Overall, the results support that (•)NO scavenging by circulating erythrocytes constitutes the major (•)NO-inactivation pathway in the brain.
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6.08Impact points
Dynamic and interacting profiles of *NO and O2 in rat hippocampal slices.
Free radical biology & medicine. 04/2010; 48(8):1044-50.
Nitric oxide (*NO) is a ubiquitous signaling molecule that participates in the neuromolecular phenomena associated with memory formation. In the hippocampus, neuronal *NO production is coupled to the activation of the NMDA-type of glutamate receptor. Although *NO-mediated signaling has been associat... [more] Nitric oxide (*NO) is a ubiquitous signaling molecule that participates in the neuromolecular phenomena associated with memory formation. In the hippocampus, neuronal *NO production is coupled to the activation of the NMDA-type of glutamate receptor. Although *NO-mediated signaling has been associated with soluble guanylate cyclase activation, cytochrome oxidase is also a target for this gaseous free radical, for which *NO competes with O(2). Here we show, for the first time in a model preserving tissue cytoarchitecture (rat hippocampal slices) and at a physiological O(2) concentration, that endogenous NMDA-evoked *NO production inhibits tissue O(2) consumption for submicromolar concentrations. The simultaneous real-time recordings reveal a direct correlation between the profiles of *NO and O(2) in the CA1 subregion of the hippocampal slice. These results, obtained in a system close to in vivo models, strongly support the current paradigm for O(2) and *NO interplay in the regulation of cellular respiration.
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2.51Impact points
Diffusion of nitric oxide through the gastric wall upon reduction of nitrite by red wine: physiological impact.
Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 04/2010; 22(3):235-41.
In this work we showed that nitric oxide produced via red wine- and ascorbate-dependent reduction of nitrite diffuses through the rat stomach, inducing smooth muscle relaxation. The studies encompassed ex vivo and in vivo models of diffusion. Regarding the former, luminal *NO generated from a mixtur... [more] In this work we showed that nitric oxide produced via red wine- and ascorbate-dependent reduction of nitrite diffuses through the rat stomach, inducing smooth muscle relaxation. The studies encompassed ex vivo and in vivo models of diffusion. Regarding the former, luminal *NO generated from a mixture of physiologic nitrite and ascorbate or wine diffuses across the stomach wall, being 8-20% of that produced in the mucosal side detected at high microM range (>100 microM) in the serosal side. In order to evaluate whether cellular dysfunction was associated with *NO diffusion at the microM range, the gastric tissue exposed to *NO was evaluated in terms of carbachol-induced muscle contraction in fundal strips and mitochondrial respiration and showed to remain functional and metabolically active. Moreover, pre-contracted gastric strips were shown to relax 86.5+/-5.5% (control) and 75.0+/-4.0% (nitrite/ascorbate-exposed tissue) when challenged with acidified nitrite. The studies in the living animal support the diffusion of luminal *NO to the gastric vasculature as, following addition of nitrite/ascorbate to rat stomach in vivo, *NO was not detected in the serosal environment but concentrations as high as 31 microM of *NO were detected outside the stomach after cardiac arrest. Collectively, the results establish a link between the consumption of nitrite and dietary reductants (e.g., wine polyphenols) and stomach muscle relaxation via the local chemical generation of *NO.
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3.91Impact points
In vivo modulation of nitric oxide concentration dynamics upon glutamatergic neuronal activation in the hippocampus.
Hippocampus. 02/2010; 21(6):622-30.
Nitric oxide ((•)NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time ... [more] Nitric oxide ((•)NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time dynamics of this molecule require the use of rapid analytical methods that can more accurately describe its signaling in vivo. This is critical for understanding how the kinetics of (•)NO-dependent signaling pathways is translated into physiological or pathological functions. In these studies, we used (•)NO selective microelectrodes coupled with rapid electrochemical recording techniques to characterize for the first time the concentration dynamics of (•)NO endogenously produced in hippocampus in vivo following activation of ionotropic glutamate receptors. Both L-glutamate (1-100 mM) and N-methyl-D-aspartate (NMDA; 0.01-5 mM) produced transient, dose-dependent increases in extracellular (•)NO concentration. The production of (•)NO in the hippocampus by glutamate was decreased by the nNOS inhibitor 7-NI. Intraperitoneal administration of the NMDA receptor blocker, MK-801, and the inhibitor of α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor, NBQX, applied locally greatly attenuated glutamate-evoked overflow of (•)NO. Thus, (•)NO overflow elicited by activation of glutamate receptors appeared to result from an integrated activation of ionotropic glutamate receptors, both of the NMDA and AMPA receptors subtypes. Additionally, distinct concentration dynamics was observed in the trisynaptic loop with stronger and longer lasting effects of glutamate activation on (•)NO overflow seen in the CA1 region as compared with the dentate gyrus. Overall, the results provide a quantitative and temporal basis for a better understanding of (•)NO activity in the rat hippocampus.
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3.24Impact points
Dietary polyphenols generate nitric oxide from nitrite in the stomach and induce smooth muscle relaxation.
Toxicology. 09/2009;
Nitrite, considered a biological waste and toxic product, is being regarded as an important physiological molecule in nitric oxide (NO) biochemistry. Because the interaction of dietary phenolic compounds and nitrite would be kinetically (due to the high concentrations achieved) and thermodynamically... [more] Nitrite, considered a biological waste and toxic product, is being regarded as an important physiological molecule in nitric oxide (NO) biochemistry. Because the interaction of dietary phenolic compounds and nitrite would be kinetically (due to the high concentrations achieved) and thermodynamically (on basis of the redox potentials) feasible in the stomach, we have studied the potential reduction of nitrite by polyphenols present in several dietary sources. By measuring the time courses of NO production in simulated gastric juice (pH 2), the efficiency of the compounds studied is as follows: B5 dimer<catechin<epicatechin<chlorogenic acid<B2 dimer<oleuropein<B8 dimer<quercetin<Epicatechin-3-O- gallate. The initial rates of NO production fall in a narrow range (c.a. 1-5muMs(-1)) but the distinct kinetics of the decay of NO signals suggest that competition reactions for NO are operative. The proof of concept that, in the presence of nitrite, phenol-containing dietary products induce a strong increase of NO in the stomach was established in an in vivo experiment with healthy volunteers consuming lettuce, onions, apples, wine, tea, berries and cherries. Moreover, selected mixtures of oleuropein and catechin with low nitrite (1muM) were shown to induce muscle relaxation of stomach strips in a structure-dependent way. Data presented here brings strong support to the concept that polyphenols consumed in a variety of dietary products, under gastric conditions, reduce nitrite to NO that, in turn, may exert a biological impact as a local relaxant.
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3.91Impact points
Stimulation of NMDA and AMPA glutamate receptors elicits distinct concentration dynamics of nitric oxide in rat hippocampal slices.
Hippocampus. 01/2009;
Nitric oxide ((*)NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of (*)NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is... [more] Nitric oxide ((*)NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of (*)NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N-methyl-D-aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of (*)NO. Using a (*)NO-selective microsensor inserted in the diffusional spread of (*)NO in the CA1 region of rat hippocampal slices, we measured its real-time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration-dependent (*)NO production but encompassing distinct kinetics for lag phases and slower rates of (*)NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D-(-)-2-amino-5-phosphonopentanoic acid, AP5) and AMPA (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX) receptors. When using glutamate as a stimulus, (*)NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca(2+) rises to levels to induce a peak of (*)NO from the background. Whereas extracellular Ca(2+) was required for the (*)NO signals, Philanthotoxin-4,3,3 (PhTX-4,3,3) a toxin used to target Ca(2+)-permeable AMPA receptors, attenuated (*)NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca(2+)-permeable AMPA receptors in the Ca(2+) activation of neuronal NOS is suggested. (c) 2008 Wiley-Liss, Inc.
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5.43Impact points
A comparative study of carbon fiber-based microelectrodes for the measurement of nitric oxide in brain tissue.
Biosensors & bioelectronics. 12/2008; 24(4):704-9.
The measurement of Nitric oxide (NO) in real-time has been a major concern due to the involvement of this ubiquitous free radical modulator in several physiological and pathological pathways in tissues. Here we performed a study aiming at evaluating different types of carbon fibers, namely Textron, ... [more] The measurement of Nitric oxide (NO) in real-time has been a major concern due to the involvement of this ubiquitous free radical modulator in several physiological and pathological pathways in tissues. Here we performed a study aiming at evaluating different types of carbon fibers, namely Textron, Amoco, Courtaulds and carbon nanotubes (University of Kentucky) covered with Nafion/o-phenylenediamine (o-PD) for NO measurement in terms of sensitivity, LOD, response time and selectivity against major potential interferents in the brain (ascorbate, nitrite and dopamine). The results indicate that, as compared with the other carbon fibers and nanotubes, Textron carbon fiber microelectrodes coated with two layers of Nafion and o-PD exhibited better characteristics for NO measurement as they are highly selective against ascorbate (>30,000:1), nitrite (>2000:1) and dopamine (>80:1). These coated Textron microelectrodes showed an average sensitivity of 341+/-120pA/microM and a detection limit of 16+/-11nM. The better performance of the Textron fibers is likely related to a stronger adhesion or more uniform coating of the Nafion and o-PD polymers to the fiber surface. In addition, the background current of the Textron carbon fibers is low, contributing to the excellent signal-to-noise for detection of NO.
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6.08Impact points
The potent vasodilator ethyl nitrite is formed upon reaction of nitrite and ethanol under gastric conditions.
Free radical biology & medicine. 09/2008; 45(4):404-12.
By acting as a bioreactor, affording chemical and mechanical conditions for the reaction between dietary components, the stomach may be a source of new bioactive molecules. Using gas chromatography-mass spectrometry we here demonstrate that, under acidic gastric conditions, ethyl nitrite is formed i... [more] By acting as a bioreactor, affording chemical and mechanical conditions for the reaction between dietary components, the stomach may be a source of new bioactive molecules. Using gas chromatography-mass spectrometry we here demonstrate that, under acidic gastric conditions, ethyl nitrite is formed in microM concentrations from the reaction of red wine or distilled alcoholic drinks with physiological amounts of nitrite. Rat femoral artery rings and gastric fundus strips dose-dependently relaxed upon exposure to nitrite:ethanol mixtures. In contrast, when administered separately in the same dose ranges, nitrite evoked only minor vasorelaxation while ethanol actually caused a slight vasoconstriction. Mechanistically, the relaxation effect was assigned to generation of nitric oxide ((*)NO) as supported by direct demonstration of (*)NO release from ethyl nitrite and the absence of relaxation in the presence of the soluble guanylyl cyclase inhibitor, ODQ. In conclusion, these results suggest that ethanol in alcoholic drinks interacts with salivary-derived nitrite in the acidic stomach leading to the production of the potent smooth muscle relaxant ethyl nitrite. These findings reveal an alternative chemical reaction pathway for dietary nitrate and nitrite with possible impact on gastric physiology and pathophysiology.
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4.00Impact points
Glutamate induces release of glutathione from cultured rat astrocytes--a possible neuroprotective mechanism?
Journal of neurochemistry. 06/2008; 105(4):1144-52.
Glutamate is the major excitatory amino acid of the mammalian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regulation of extracellular glutamate levels via glutamat... [more] Glutamate is the major excitatory amino acid of the mammalian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regulation of extracellular glutamate levels via glutamate transporters and metabolic and antioxidant support. In this study, we report that cultures of rat astrocytes incubated with high extracellular glutamate (5 mM) exhibit a twofold increase in the extracellular concentration of the tripeptide antioxidant glutathione (GSH) over 4 h. Incubation with glutamate did not result in an increased release of lactate dehydrogenase, indicating that the rise in GSH was not because of membrane damage and leakage of intracellular pools. Glutamate-induced increase in extracellular GSH was also independent of de novo GSH synthesis, activation of NMDA and non-NMDA glutamate receptors or inhibition of extracellular GSH breakdown. Dose-response curves indicate that GSH release from rat astrocytes is significantly stimulated even at 0.1 mM glutamate. The ability of astrocytes to increase GSH release in the presence of extracellular glutamate could be an important neuroprotective mechanism enabling neurones to maintain levels of the key antioxidant, GSH, under conditions of glutamate toxicity.
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2.47Impact points
LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion.
Journal of agricultural and food chemistry. 06/2008; 56(10):3798-804.
Dietary phenolic compounds may act as antioxidants in vitro, but because of structural modifications during absorption, its role based on concentrations high enough to afford an antioxidant protection needs to be re-evaluated. We have explored the hypothesis that red wine procyanidins interact with ... [more] Dietary phenolic compounds may act as antioxidants in vitro, but because of structural modifications during absorption, its role based on concentrations high enough to afford an antioxidant protection needs to be re-evaluated. We have explored the hypothesis that red wine procyanidins interact with low density lipoproteins (LDL) and that, at this location, the phenolic compounds efficiently protect LDL from oxidation and maintain LDL alpha-tocopherol at a high steady state concentration by recycling it back from the alpha-tocopheroxyl radical. To this end, human plasma was supplemented with wine procyanidins and isolated LDL were challenged with a constant flux of peroxyl radicals. As compared with LDL from plasma-free procyanidins, those LDL better resisted lipid oxidation and exhibited longer lag-phases of alpha-tocopherol consumption. The procyanidins, depending on their structure, were able to reduce the UV-induced alpha-tocopherol radical in a micellar system, as evidenced by electron paramagnetic ressonance. Mechanistically, the protection of LDL was interpreted in terms of quenching of peroxyl radicals and the recycling of alpha-tocopherol by the procyanidins bound to the lipoproteins. These results support the notion that, in human plasma, the procyanidins, via binding to LDL, may act as efficient local antioxidants.
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4.29Impact points
Diphenyl diselenide, a simple glutathione peroxidase mimetic, inhibits human LDL oxidation in vitro.
Atherosclerosis. 04/2008;
Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe)(2), a simple organoseleno compound, against copper (Cu(2+)) and peroxyl radical-induced human L... [more] Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe)(2), a simple organoseleno compound, against copper (Cu(2+)) and peroxyl radical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe)(2) caused a dose-dependent inhibition of Cu(2+)-induced lipid peroxidation, which was correlated to thiol consumption. (PhSe)(2) increased lipid peroxidation lag phase and decreased lipid peroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) levels. Consistent with these observations, (PhSe)(2) showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe)(2) also displayed a dose-dependent protective effect against Cu(2+)-induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe)(2) toward the lipid moieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected against Cu(2+)-induced oxidation. The results presented herein are the first to show that (i) (PhSe)(2) inhibits lipid peroxidation in human isolated LDL in vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moieties of human LDL. Taken together, such data render (PhSe)(2) a promising molecule for pharmacological studies with respect to the atherogenic process.
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1.90Impact points
In vivo real-time measurement of nitric oxide in anesthetized rat brain.
Methods in enzymology. 02/2008; 441:351-67.
During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of... [more] During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of .NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the .NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-d-aspartate-subtype glutamate receptors. Moreover, the kinetics of .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.
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6.08Impact points
Red wine-dependent reduction of nitrite to nitric oxide in the stomach.
Free radical biology & medicine. 12/2007; 43(9):1233-42.
Nitrite may be a source for nitric oxide (*NO), particularly in highly acidic environments, such as the stomach. Diet products contribute also with reductants that dramatically increase the production of *NO from nitrite. Red wine has been attributed health promoting properties largely on basis of t... [more] Nitrite may be a source for nitric oxide (*NO), particularly in highly acidic environments, such as the stomach. Diet products contribute also with reductants that dramatically increase the production of *NO from nitrite. Red wine has been attributed health promoting properties largely on basis of the reductive antioxidant properties of its polyphenolic fraction. We show in vitro that wine, wine anthocyanin fraction and wine catechol (caffeic acid) dose- and pH-dependently promote the formation of *NO when mixed with nitrite, as measured electrochemically. The production of *NO promoted by wine from nitrite was substantiated in vivo in healthy volunteers by measuring *NO in the air expelled from the stomach, following consumption of wine, as measured by chemiluminescence. Mechanistically, the reaction involves the univalent reduction of nitrite, as suggested by the formation of *NO and by the appearance of EPR spectra assigned to wine phenolic radicals. Ascorbic and caffeic acids cooperate in the reduction of nitrite to *NO. Moreover, reduction of nitrite is critically dependent on the phenolic structure and nitro-derivatives of phenols are also formed, as suggested by caffeic acid UV spectral modifications. The reduction of nitrite may reveal previously unrecognized physiologic effects of red wine in connection with *NO bioactivity.
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6.14Impact points
NO-mediated apoptosis in yeast.
Journal of cell science. 10/2007; 120(Pt 18):3279-88.
Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogena... [more] Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.
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5.98Impact points
Nitric oxide signaling is disrupted in the yeast model for Batten disease.
Molecular biology of the cell. 08/2007; 18(7):2755-67.
The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-D... [more] The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease.
Following (10)
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Afaf Kamal-Eldin
United Arab Emirates University -
Jan Frank
Universität Hohenheim -
Ricardo M Santos
Center for Neuroscience and Cell Biology -
Ana Ledo
University of Coimbra -
Marcos Bürger
Universidade de Brasília
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