Nitric oxide (NO) is synthesized in vivo by a group of nitric oxide synthases, exhibiting a wide ... more Nitric oxide (NO) is synthesized in vivo by a group of nitric oxide synthases, exhibiting a wide array of biological actions. Much less attention has been devoted to the non enzymatic production of NO. However, it has been shown that in acid/reducing environments nitrite may be reduced to NO. Examples include ischemia-reperfusion situations and the oral and stomach cavities. Due to their reducing capacity, phenolic antioxidants found in human diet may potentially yield NO from nitrite. In this work we have studied the ability of phenolic acids (mainly caffeic and p-coumaric acids) to reduce nitrite to NO, as a function of pH. NO production was measured electrochemically and phenol oxidation measured by UV and electron Paramagnetic Resonance (EPR) spectroscopies. At the concentrations relevant in vivo, caffeic acid efficiently reduces nitrite to nitric oxide (rising up to microM range), the effect being larger at pH 1.5 (stomach pH) as compared with pH 5.5 (a value that can be found in ischemia- reperfusion conditions). A structurally related compound, p-coumaric acid, failed to induce the production of NO. The reduction of nitrite to NO was substantiated by measuring the o- semiquinone radical of caffeic acid by EPR. In view of the possible interaction in vivo, the reduction of nitrite to NO by caffeic acid may acquire physiopathological relevance in both, ischemia-reperfusion conditions and in the stomach compartment. Introduction NO can be produced in vivo by a non enzymatic pathway involving nitrite reduction, given that an acid/reducing environment is present. This environment occurs in ischemia-reperfusion were pH drops to 5.5 (Zweier et al. 1995) and in stomach compartment at pH 1.5. Nitrites and nitrates are ingested in the diet. Nitrate is absorbed in the upper small intestine (Mowat and McColl 2001) and c.a. 25 % of ingested nitrate undergoes enterosalivary recirculation (Bartholomew and Hill 1984) coming back to the mouth and then converted to nitrite in saliva by action of nitrate reductase expressed by microorganisms (Spiegelhalder et al. 1976).Caffeic acid is a catechol compound precursor of flavonoids with potent antioxidant activity (for review see Laranjinha 2001) and is found naturally in a wide variety of foods as part of the human diet. Therefore, caffeic acid and nitrite are likely to be found simultaneously in the diet, oral cavity, stomach and plasma, suggesting that a mutual interaction may occur. In this work we have studied the ability of caffeic to reduce nitrite to NO, as a function of pH. Results were compared with those obtained with p-coumaric acid, a molecule structurally related to caffeic acid but with much lower reducing properties.
Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell functio... more Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell function and communication. A role for H2O2 as an intercellular signaling molecule and neuromodulator in the brain has become increasingly apparent, with evidence showing this biological oxidant to regulate neuronal polarity, connectivity, synaptic transmission and tuning of neuronal networks. This notion is supported by its ability to diffuse in the extracellular space, from source of production to target. It is, thus, crucial to understand extracellular H2O2 concentration dynamics in the living brain and the factors which shape its diffusion pattern and half-life. To address this issue, we have used a novel microsensor to measure H2O2 concentration dynamics in the brain extracellular matrix both in an ex vivo model using rodent brain slices and in vivo. We found that exogenously applied H2O2 is removed from the extracellular space with an average half-life of t1/2 = 2.2 s in vivo. We determined the in vivo effective diffusion coefficient of H2O2 to be D* = 2.5 × 10−5 cm2 s−1. This allows it to diffuse over 100 μm in the extracellular space within its half-life. Considering this, we can tentatively place H2O2 within the class of volume neurotransmitters, connecting all cell types within the complex network of brain tissue, regardless of whether they are physically connected. These quantitative details of H2O2 diffusion and half-life in the brain allow us to interpret the physiology of the redox signal and lay the pavement to then address dysregulation in redox homeostasis associated with disease processes.
Nitric Oxide (NO) is a messenger involved in various physiologic processes in the brain, such as ... more Nitric Oxide (NO) is a messenger involved in various physiologic processes in the brain, such as modulation of neurotransmitter release, long term potentiation and neurovascular coupling [1]. Unlike most signaling molecules, NO diffusion is not restricted by cell membranes, which means that its concentration dynamics cannot be controlled by vesicle storage/exocytose or intracellular uptake processes [2]. Instead of being released from vesicles like classical neurotransmitters, NO is enzymatically synthesized, mainly in synaptic terminals containing NMDA receptors physically coupled to the neuronal isoform of NO synthase (nNOS). At this location NO production is triggered by activation of nNOS, via calcium influx into the post-synaptic terminal through N-Methyl-DAspartate (NMDA) receptors activated by glutamate [1].
The brain is highly rich in lipids, which accounts for roughly 50% of its dry weight. The brain l... more The brain is highly rich in lipids, which accounts for roughly 50% of its dry weight. The brain lipidome, generally characterized over half a century ago, is comprised of thousands of biochemical structures expressed differentially as a function of brain region, structure, cell type and subcellular compartment. Lipids play diverse structural and functional roles in the brain, not only due to their chemical diversity but also due to the unique hydrophobic environment that they create. This lipophilic milieu promotes interactions involving reactive oxygen and nitrogen species that may not occur, at least at a similar extent, in aqueous environments.In the present chapter, we have focused on 3 distinct types of bioactive lipids and the roles played in brain physiology and pathology: nitrated fatty acids, cholesterol and endocannabinoids. These lipids are diverse in origin and bioactivity: (1) nitrated fatty acids result from biochemical modification of dietary fatty acids by nutrients ...
The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical sc... more The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical sciences since it was promoted from atmospheric air pollutant to biological ubiquitous signaling molecule. Its unique physical chemical properties expand beyond its radical nature to include fast diffusion in aqueous and lipid environments and selective reactivity in a biological setting determined by bioavailability and reaction rate constants with biomolecules. In the brain, •NO is recognized as a key player in numerous physiological processes ranging from neurotransmission/neuromodulation to neurovascular coupling and immune response. Furthermore, changes in its bioactivity are central to the molecular pathways associated with brain aging and neurodegeneration. The understanding of •NO bioactivity in the brain, however, requires the knowledge of its concentration dynamics with high spatial and temporal resolution upon stimulation of its synthesis. Here we revise the understanding of the role of neuronal-derived •NO in brain physiology, aging and degeneration, focused on changes in the extracellular concentration dynamics of this free radical and the regulation of bioenergetic metabolism and neurovascular coupling.
The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...
Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in b... 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 Nω-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...
The brain has high energetic and metabolic demands, which requires a precise communication betwee... more The brain has high energetic and metabolic demands, which requires a precise communication between neuronal cells and the blood vessels. Neurovascular coupling (NYC) allows the allocation of resources to areas of higher energetic requirement with temporal and regional precision. One key mediator of NYC is nitric oxide ( • NO) produced in activated neurons by nNOS. Enzymatic production of • NO can be impaired in conditions like hypoxia, ageing and neurodegenerative diseases, impacting NYC. We hypothesize that low-level changes in basal • NO levels can result from modulation of circulating nitrite levels via dietary intervention with nitrate-rich foods, improving cerebrovascular function and NYC in aging and degeneration or stroke. In this work, we used gas phase chemiluminescence to quantify nitrite reduction between pH 6.0–7.4 in the presence of ascorbate, the more abundant reducing agent found in the brain extracellular space and released from neurons upon glutamatergic stimulation. Results support nitrite reduction to • NO by ascorbate as a function of pH. In vivo studies to assess the impact of nitrite-driven • NO in the functionality of NYC were implemented and preliminary data support a contribution of this mechanism to the maintenance of NYC.
Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in b... 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.
Oxidative Stress, Inflammation and Angiogenesis in the Metabolic Syndrome, 2009
ABSTRACT Free radicals-mediated oxidation of biomolecules and oxidative stress were implausible n... more ABSTRACT Free radicals-mediated oxidation of biomolecules and oxidative stress were implausible notions just a few decades ago. The discovery of superoxide dismutase in the late 1960’s triggered intense research that ultimately led to the description of the production of free radicals and oxidants by mitochondria and by several metabolic pathways in mammalian cells. In view of the threats imposed by free radicals and oxidants, life in an aerobic environment required antioxidant strategies to prevent and repair potential oxidative damage to vital cell components. As a corollary of these discoveries Helmut Sies formulated the concept of oxidative stress, emphasizing the balance in the dynamic equilibrium between oxidants and antioxidants. Later, it was recognized that free radicals and oxidants are not only noxious cellular stressors but also play an essential role in cellular signalling and redox regulation of metabolic process. In particular, it has been appreciated the involvement of free radical and oxidants in discreet redox pathways, suggesting that specific mechanisms have evolved for free radicals and oxidants signalling. This and other observations, arguing against a global imbalance between oxidants and antioxidants, led to an update of the notion of oxidative stress in order to emphasize discreet and compartmentalized cellular redox circuits. The updated concept of oxidative stress may thus help project novel therapeutic approaches selectively directed to targets and disease conditions.
Nitric oxide (NO) is synthesized in vivo by a group of nitric oxide synthases, exhibiting a wide ... more Nitric oxide (NO) is synthesized in vivo by a group of nitric oxide synthases, exhibiting a wide array of biological actions. Much less attention has been devoted to the non enzymatic production of NO. However, it has been shown that in acid/reducing environments nitrite may be reduced to NO. Examples include ischemia-reperfusion situations and the oral and stomach cavities. Due to their reducing capacity, phenolic antioxidants found in human diet may potentially yield NO from nitrite. In this work we have studied the ability of phenolic acids (mainly caffeic and p-coumaric acids) to reduce nitrite to NO, as a function of pH. NO production was measured electrochemically and phenol oxidation measured by UV and electron Paramagnetic Resonance (EPR) spectroscopies. At the concentrations relevant in vivo, caffeic acid efficiently reduces nitrite to nitric oxide (rising up to microM range), the effect being larger at pH 1.5 (stomach pH) as compared with pH 5.5 (a value that can be found in ischemia- reperfusion conditions). A structurally related compound, p-coumaric acid, failed to induce the production of NO. The reduction of nitrite to NO was substantiated by measuring the o- semiquinone radical of caffeic acid by EPR. In view of the possible interaction in vivo, the reduction of nitrite to NO by caffeic acid may acquire physiopathological relevance in both, ischemia-reperfusion conditions and in the stomach compartment. Introduction NO can be produced in vivo by a non enzymatic pathway involving nitrite reduction, given that an acid/reducing environment is present. This environment occurs in ischemia-reperfusion were pH drops to 5.5 (Zweier et al. 1995) and in stomach compartment at pH 1.5. Nitrites and nitrates are ingested in the diet. Nitrate is absorbed in the upper small intestine (Mowat and McColl 2001) and c.a. 25 % of ingested nitrate undergoes enterosalivary recirculation (Bartholomew and Hill 1984) coming back to the mouth and then converted to nitrite in saliva by action of nitrate reductase expressed by microorganisms (Spiegelhalder et al. 1976).Caffeic acid is a catechol compound precursor of flavonoids with potent antioxidant activity (for review see Laranjinha 2001) and is found naturally in a wide variety of foods as part of the human diet. Therefore, caffeic acid and nitrite are likely to be found simultaneously in the diet, oral cavity, stomach and plasma, suggesting that a mutual interaction may occur. In this work we have studied the ability of caffeic to reduce nitrite to NO, as a function of pH. Results were compared with those obtained with p-coumaric acid, a molecule structurally related to caffeic acid but with much lower reducing properties.
Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell functio... more Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell function and communication. A role for H2O2 as an intercellular signaling molecule and neuromodulator in the brain has become increasingly apparent, with evidence showing this biological oxidant to regulate neuronal polarity, connectivity, synaptic transmission and tuning of neuronal networks. This notion is supported by its ability to diffuse in the extracellular space, from source of production to target. It is, thus, crucial to understand extracellular H2O2 concentration dynamics in the living brain and the factors which shape its diffusion pattern and half-life. To address this issue, we have used a novel microsensor to measure H2O2 concentration dynamics in the brain extracellular matrix both in an ex vivo model using rodent brain slices and in vivo. We found that exogenously applied H2O2 is removed from the extracellular space with an average half-life of t1/2 = 2.2 s in vivo. We determined the in vivo effective diffusion coefficient of H2O2 to be D* = 2.5 × 10−5 cm2 s−1. This allows it to diffuse over 100 μm in the extracellular space within its half-life. Considering this, we can tentatively place H2O2 within the class of volume neurotransmitters, connecting all cell types within the complex network of brain tissue, regardless of whether they are physically connected. These quantitative details of H2O2 diffusion and half-life in the brain allow us to interpret the physiology of the redox signal and lay the pavement to then address dysregulation in redox homeostasis associated with disease processes.
Nitric Oxide (NO) is a messenger involved in various physiologic processes in the brain, such as ... more Nitric Oxide (NO) is a messenger involved in various physiologic processes in the brain, such as modulation of neurotransmitter release, long term potentiation and neurovascular coupling [1]. Unlike most signaling molecules, NO diffusion is not restricted by cell membranes, which means that its concentration dynamics cannot be controlled by vesicle storage/exocytose or intracellular uptake processes [2]. Instead of being released from vesicles like classical neurotransmitters, NO is enzymatically synthesized, mainly in synaptic terminals containing NMDA receptors physically coupled to the neuronal isoform of NO synthase (nNOS). At this location NO production is triggered by activation of nNOS, via calcium influx into the post-synaptic terminal through N-Methyl-DAspartate (NMDA) receptors activated by glutamate [1].
The brain is highly rich in lipids, which accounts for roughly 50% of its dry weight. The brain l... more The brain is highly rich in lipids, which accounts for roughly 50% of its dry weight. The brain lipidome, generally characterized over half a century ago, is comprised of thousands of biochemical structures expressed differentially as a function of brain region, structure, cell type and subcellular compartment. Lipids play diverse structural and functional roles in the brain, not only due to their chemical diversity but also due to the unique hydrophobic environment that they create. This lipophilic milieu promotes interactions involving reactive oxygen and nitrogen species that may not occur, at least at a similar extent, in aqueous environments.In the present chapter, we have focused on 3 distinct types of bioactive lipids and the roles played in brain physiology and pathology: nitrated fatty acids, cholesterol and endocannabinoids. These lipids are diverse in origin and bioactivity: (1) nitrated fatty acids result from biochemical modification of dietary fatty acids by nutrients ...
The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical sc... more The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical sciences since it was promoted from atmospheric air pollutant to biological ubiquitous signaling molecule. Its unique physical chemical properties expand beyond its radical nature to include fast diffusion in aqueous and lipid environments and selective reactivity in a biological setting determined by bioavailability and reaction rate constants with biomolecules. In the brain, •NO is recognized as a key player in numerous physiological processes ranging from neurotransmission/neuromodulation to neurovascular coupling and immune response. Furthermore, changes in its bioactivity are central to the molecular pathways associated with brain aging and neurodegeneration. The understanding of •NO bioactivity in the brain, however, requires the knowledge of its concentration dynamics with high spatial and temporal resolution upon stimulation of its synthesis. Here we revise the understanding of the role of neuronal-derived •NO in brain physiology, aging and degeneration, focused on changes in the extracellular concentration dynamics of this free radical and the regulation of bioenergetic metabolism and neurovascular coupling.
The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...
Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in b... 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 Nω-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...
The brain has high energetic and metabolic demands, which requires a precise communication betwee... more The brain has high energetic and metabolic demands, which requires a precise communication between neuronal cells and the blood vessels. Neurovascular coupling (NYC) allows the allocation of resources to areas of higher energetic requirement with temporal and regional precision. One key mediator of NYC is nitric oxide ( • NO) produced in activated neurons by nNOS. Enzymatic production of • NO can be impaired in conditions like hypoxia, ageing and neurodegenerative diseases, impacting NYC. We hypothesize that low-level changes in basal • NO levels can result from modulation of circulating nitrite levels via dietary intervention with nitrate-rich foods, improving cerebrovascular function and NYC in aging and degeneration or stroke. In this work, we used gas phase chemiluminescence to quantify nitrite reduction between pH 6.0–7.4 in the presence of ascorbate, the more abundant reducing agent found in the brain extracellular space and released from neurons upon glutamatergic stimulation. Results support nitrite reduction to • NO by ascorbate as a function of pH. In vivo studies to assess the impact of nitrite-driven • NO in the functionality of NYC were implemented and preliminary data support a contribution of this mechanism to the maintenance of NYC.
Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in b... 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.
Oxidative Stress, Inflammation and Angiogenesis in the Metabolic Syndrome, 2009
ABSTRACT Free radicals-mediated oxidation of biomolecules and oxidative stress were implausible n... more ABSTRACT Free radicals-mediated oxidation of biomolecules and oxidative stress were implausible notions just a few decades ago. The discovery of superoxide dismutase in the late 1960’s triggered intense research that ultimately led to the description of the production of free radicals and oxidants by mitochondria and by several metabolic pathways in mammalian cells. In view of the threats imposed by free radicals and oxidants, life in an aerobic environment required antioxidant strategies to prevent and repair potential oxidative damage to vital cell components. As a corollary of these discoveries Helmut Sies formulated the concept of oxidative stress, emphasizing the balance in the dynamic equilibrium between oxidants and antioxidants. Later, it was recognized that free radicals and oxidants are not only noxious cellular stressors but also play an essential role in cellular signalling and redox regulation of metabolic process. In particular, it has been appreciated the involvement of free radical and oxidants in discreet redox pathways, suggesting that specific mechanisms have evolved for free radicals and oxidants signalling. This and other observations, arguing against a global imbalance between oxidants and antioxidants, led to an update of the notion of oxidative stress in order to emphasize discreet and compartmentalized cellular redox circuits. The updated concept of oxidative stress may thus help project novel therapeutic approaches selectively directed to targets and disease conditions.
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Papers by J. Laranjinha