Insights on the Multifaceted Roles of Wild-Type and Mutated Superoxide Dismutase 1 in Amyotrophic Lateral Sclerosis Pathogenesis.

Amyotrophic Lateral Sclerosis (ALS) is a progressive motor neurodegenerative disease. Cell damage in ALS is the result of many different, largely unknown, pathogenetic mechanisms. Astrocytes and microglial cells play a critical role also for their ability to enhance a deranged inflammatory response. Excitotoxicity, due to excessive glutamate levels and increased intracellular Ca2+ concentration, has also been proposed to play a key role in ALS pathogenesis/progression. Reactive Oxygen Species (ROS) behave as key second messengers for multiple receptor/ligand interactions. ROS-dependent regulatory networks are usually mediated by peroxides. Superoxide Dismutase 1 (SOD1) physiologically mediates intracellular peroxide generation. About 10% of ALS subjects show a familial disease associated with different gain-of-function SOD1 mutations. The occurrence of sporadic ALS, not clearly associated with SOD1 defects, has been also described. SOD1-dependent pathways have been involved in neuron functional network as well as in immune-response regulation. Both, neuron depolarization and antigen-dependent T-cell activation mediate SOD1 exocytosis, inducing increased interaction of the enzyme with a complex molecular network involved in the regulation of neuron functional activity and immune response. Here, alteration of SOD1-dependent pathways mediating increased intracellular Ca2+ levels, altered mitochondria functions and defective inflammatory process regulation have been proposed to be relevant for ALS pathogenesis/progression.

Superoxide Dismutase-1 Intracellular Content in T Lymphocytes Associates with Increased Regulatory T Cell Level in Multiple Sclerosis Subjects Undergoing Immune-Modulating Treatment.

Reactive oxygen species (ROS) participate in the T-cell activation processes. ROS-dependent regulatory networks are usually mediated by peroxides, which are more stable and able to freely migrate inside cells. Superoxide dismutase (SOD)-1 represents the major physiological intracellular source of peroxides. We found that antigen-dependent activation represents a triggering element for SOD-1 production and secretion by human T lymphocytes. A deranged T-cell proinflammatory response characterizes the pathogenesis of multiple sclerosis (MS). We previously observed a decreased SOD-1 intracellular content in leukocytes of MS individuals at diagnosis, with increasing amounts of such enzyme after interferon (IFN)-b 1b treatment. Here, we analyzed in depth SOD-1 intracellular content in T cells in a cohort of MS individuals undergoing immune-modulating treatment. Higher amounts of the enzyme were associated with increased availability of regulatory T cells (Treg) preferentially expressing Foxp3-exon 2 (Foxp3-E2), as described for effective Treg. In vitro administration of recombinant human SOD-1 to activated T cells, significantly increased their IL-17 production, while SOD-1 molecules lacking dismutase activity were unable to interfere with cytokine production by activated T cells in vitro. Furthermore, hydrogen peroxide addition was observed to mimic, in vitro, the SOD-1 effect on IL-17 production. These data add SOD-1 to the molecules involved in the molecular pathways contributing to re-shaping the T-cell cytokine profile and Treg differentiation.

The Effects of Angiotensin II or Angiotensin 1-7 on Rat Pial Microcirculation during Hypoperfusion and Reperfusion Injury: Role of Redox Stress.

Renin-angiotensin systems produce angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7), which are able to induce opposite effects on circulation. This study in vivo assessed the effects induced by Ang II or Ang 1-7 on rat pial microcirculation during hypoperfusion-reperfusion, clarifying the mechanisms causing the imbalance between Ang II and Ang 1-7. The fluorescence microscopy was used to quantify the microvascular parameters. Hypoperfusion and reperfusion caused vasoconstriction, disruption of blood-brain barrier, reduction of capillary perfusion and an increase in reactive oxygen species production. Rats treated with Ang II showed exacerbated microvascular damage with stronger vasoconstriction compared to hypoperfused rats, a further increase in leakage, higher decrease in capillary perfusion and marker oxidative stress. Candesartan cilexetil (specific Ang II type 1 receptor (AT1R) antagonist) administration prior to Ang II prevented the effects induced by Ang II, blunting the hypoperfusion-reperfusion injury. Ang 1-7 or ACE2 activator administration, preserved the pial microcirculation from hypoperfusion-reperfusion damage. These effects of Ang 1-7 were blunted by a Mas (Mas oncogene-encoded protein) receptor antagonist, while Ang II type 2 receptor antagonists did not affect Ang 1-7-induced changes. In conclusion, Ang II and Ang 1-7 triggered different mechanisms through AT1R or MAS receptors able to affect cerebral microvascular injury.

Metabolism Regulation and Redox State: Insight into the Role of Superoxide Dismutase 1.

Energy metabolism and redox state are strictly linked; energy metabolism is a source of reactive oxygen species (ROS) that, in turn, regulate the flux of metabolic pathways. Moreover, to assure redox homeostasis, metabolic pathways and antioxidant systems are often coordinately regulated. Several findings show that superoxide dismutase 1 (SOD1) enzyme has effects that go beyond its superoxide dismutase activity and that its functions are not limited to the intracellular compartment. Indeed, SOD1 is secreted through unconventional secretory pathways, carries out paracrine functions and circulates in the blood bound to lipoproteins. Striking experimental evidence links SOD1 to the redox regulation of metabolism. Important clues are provided by the systemic effects on energy metabolism observed in mutant SOD1-mediated amyotrophic lateral sclerosis (ALS). The purpose of this review is to analyze in detail the involvement of SOD1 in redox regulation of metabolism, nutrient sensing, cholesterol metabolism and regulation of mitochondrial respiration. The scientific literature on the relationship between ALS, mutated SOD1 and metabolism will also be explored, in order to highlight the metabolic functions of SOD1 whose biological role still presents numerous unexplored aspects that deserve further investigation.

Dual Role of Reactive Oxygen Species in Muscle Function: Can Antioxidant Dietary Supplements Counteract Age-Related Sarcopenia?

Sarcopenia is characterized by the progressive loss of skeletal muscle mass and strength. In older people, malnutrition and physical inactivity are often associated with sarcopenia, and, therefore, dietary interventions and exercise must be considered to prevent, delay, or treat it. Among the pathophysiological mechanisms leading to sarcopenia, a key role is played by an increase in reactive oxygen and nitrogen species (ROS/RNS) levels and a decrease in enzymatic antioxidant protection leading to oxidative stress. Many studies have evaluated, in addition to the effects of exercise, the effects of antioxidant dietary supplements in limiting age-related muscle mass and performance, but the data which have been reported are conflicting. In skeletal muscle, ROS/RNS have a dual function: at low levels they increase muscle force and adaptation to exercise, while at high levels they lead to a decline of muscle performance. Controversial results obtained with antioxidant supplementation in older persons could in part reflect the lack of univocal effects of ROS on muscle mass and function. The purpose of this review is to examine the molecular mechanisms underlying the dual effects of ROS in skeletal muscle function and the analysis of literature data on dietary antioxidant supplementation associated with exercise in normal and sarcopenic subjects.

Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1G93A) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells.

The constitutive secretion of antioxidant Cu-Zn Superoxide dismutase (SOD1) has been widely demonstrated in many cellular lines. In addition, we showed that as well as the basal SOD1 secretion, this enzyme is also exported through depolarization of excitable cells by high extracellular K concentration. Recent data showed that SOD1 was able to activate muscarinic M1 receptor producing the activation, via phospholipase C, of ERK1-2 and AKT pathways. It is also known that about 20% of familial amyotrophic lateral sclerosis (fALS) is due to mutations in the gene coding for SOD1. The aim of the present research is to evaluate whether, analogously to wild type SOD1 (SOD1wt), the mutated form of SOD1G93A is able to activate ERK1-2 and AKT through muscarinic M1 receptor in SK-N-BE as well as in motoneuron like NSC-34. Our results demonstrated that in NSC-34 and SK-N-BE cells mutated SOD1G93A carried out a more evident activation of ERK1-2 and AKT and a stronger increase of intracellular calcium levels compared to SOD1WT; we also demonstrated that these effects are mediated by the M1 receptor as shown using pirenzepine, a specific M1 inhibitor and the calcium chelator BAPTA. Of note, M1 receptor pathway activation by SOD1G93A, but not by SOD1WT, is associated with both an increase of reactive oxygen species and a cytotoxic effect.

Quercetin Increases MUC2 and MUC5AC Gene Expression and Secretion in Intestinal Goblet Cell-Like LS174T via PLC/PKCα/ERK1-2 Pathway.

The main dietary flavonoid quercetin, is known to preserve the integrity of gastrointestinal barrier and to have anti-inflammatory, anti-cancer, anti-fibrotic, and other beneficial properties. Many of the biological effects of quercetin appear to be associated to the modulation of cell signaling pathways, rather than to its antioxidant activity. In spite of the large number of data available on the molecular and cellular mechanisms by which quercetin exerts its biological effects, including protection of intestinal barrier function, there is a lack of data about the role of this substance on the expression and/or the secretion of mucins released by intestinal goblet cells. Here we investigated the effects of quercetin on the secretion and the gene expression of the main intestinal gel-forming mucins, MUC2 and MUC5AC, and the signaling mechanisms underlined, in human intestinal goblet cell-like LS174T. We found that quercetin increases intracellular Ca2+ levels and induces MUC2 and MUC5AC secretion in a Ca2+-dependent manner. Quercetin also induces mRNA levels of both secretory mucins. Quercetin stimulation of LS174T cells increases phosphorylation levels of extracellular signal regulated kinase (ERK)1-2 and protein kinase C (PKC) α and the induction of MUC2 and MUC5AC secretion and mRNA relies on phospholipase C (PLC), PKC, and ERK1-2 signaling pathways since the PLC inhibitor U73122, the PKC inhibitor bisindolylmaleimide (BIM) and the ERK1-2 pathway inhibitor PD98059, all revert the stimulatory effects of quercetin. We also demonstrated that the induction of mucin gene expression by quercetin is not limited to goblet cells. Indeed, quercetin induces mRNA levels of MUC2 and MUC5AC via PKCα/ERK1-2 pathway also in the human intestinal epithelial Caco-2 cells. These data highlight a novel mechanism thereby quercetin, regulating the secretory function of intestinal goblet cells and mucin levels in enterocytes may exert its protective effects on intestinal mucosal barrier.

Human miR-26a-5p regulates the glutamate transporter SLC1A1 (EAAT3) expression. Relevance in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. MicroRNA plays pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. Increasing evidence suggests the involvement of microRNAs in the pathogenesis of neurodegenerative diseases, including MS. We have already found that the expression of a specific miRNA, hsa-mir-26a-5p (miR-26a), changed during INF-ß treatment in responder Relapsing-Remitting MS patients. Functional annotations of mir-26a targets revealed that a number of genes were implicated in Glutamate Receptor Signaling pathway, which is notoriously altered in neurodegenerative diseases as MS. In this study, the different potential targets were subjected to a validation test based on luciferase reporter constructs transfected in an oligodendroglial cell line. In this functional screening, miR-26a was able to interact with SLC1A1 3' UTR suppressing the reporter activity. Transfection of a miR-26a mimic was then shown to decrease the endogenous SLC1A1 mRNA. Afterward, we have evaluated in blood platelets from interferon-ß treated Multiple Sclerosis patients the expression of miR-26a and SLC1A1, finding not only their converse expression, but also a responsiveness to interferon-ß therapy. Overall, these data suggest that mir-26a and SLC1A1 may play a role in the MS pathogenesis, and may be potential targets for the development of new biomarkers and/or therapeutic tools.

The Cu, Zn Superoxide Dismutase: Not Only a Dismutase Enzyme.

The Cu,Zn superoxide dismutase (SOD1) is an ubiquitary cytosolic dimeric carbohydrate free molecule, belonging to a family of isoenzymes involved in the scavenger of superoxide anions. This effect certainly represents the main and well known function ascribed to this enzyme. Here we highlight new aspects of SOD1 physiology that point out some inedited effects of this enzyme in addition to the canonic role of oxygen radical enzymatic dismutation. In the last two decades our research group produced many data obtained in in vitro studies performed in many cellular lines, mainly neuroblastoma SK-N-BE cells, indicating that this enzyme is secreted either constitutively or after depolarization induced by high extracellular K+ concentration. In addition, we gave many experimental evidences showing that SOD1 is able to stimulate, through muscarinic M1 receptor, pathways involving ERK1/2, and AKT activation. These effects are accompanied with an intracellular calcium increase. In the last part of this review we describe researches that link deficient extracellular secretion of mutant SOD1G93A to its intracellular accumulation and toxicity in NSC-34 cells. Alternatively, SOD1G93A toxicity has been attributed to a decrease of Km for H2O2 with consequent OH radical formation. Interestingly, this last inedited effect of SOD1G93A could represent a gain of function that could be involved in the pathogenesis of familial Amyotrophic Lateral Sclerosis (fALS).

Reactive Oxygen Species Derived from NOX3 and NOX5 Drive Differentiation of Human Oligodendrocytes.

Reactive oxygen species (ROS) are signaling molecules that mediate stress response, apoptosis, DNA damage, gene expression and differentiation. We report here that differentiation of oligodendrocytes (OLs), the myelin forming cells in the CNS, is driven by ROS. To dissect the OL differentiation pathway, we used the cell line MO3-13, which display the molecular and cellular features of OL precursors. These cells exposed 1-4 days to low levels of H2O2 or to the protein kinase C (PKC) activator, phorbol-12-Myristate-13-Acetate (PMA) increased the expression of specific OL differentiation markers: the specific nuclear factor Olig-2, and Myelin Basic Protein (MBP), which was processed and accumulated selectively in membranes. The induction of differentiation genes was associated with the activation of ERK1-2 and phosphorylation of the nuclear cAMP responsive element binding protein 1 (CREB). PKC mediates ROS-induced differentiation because PKC depletion or bis-indolyl-maleimide (BIM), a PKC inhibitor, reversed the induction of differentiation markers by H2O2. H2O2 and PMA increased the expression of membrane-bound NADPH oxidases, NOX3 and NOX5. Selective depletion of these proteins inhibited differentiation induced by PMA. Furthermore, NOX5 silencing down regulated NOX3 mRNA levels, suggesting that ROS produced by NOX5 up-regulate NOX3 expression. These data unravel an elaborate network of ROS-generating enzymes (NOX5 to NOX3) activated by PKC and necessary for differentiation of OLs. Furthermore, NOX3 and NOX5, as inducers of OL differentiation, represent novel targets for therapies of demyelinating diseases, including multiple sclerosis, associated with impairment of OL differentiation.

The IFN-ß 1b effect on Cu Zn superoxide dismutase (SOD1) in peripheral mononuclear blood cells of relapsing-remitting multiple sclerosis patients and in neuroblastoma SK-N-BE cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease leading to axonal injury. Even if the etiology of MS is still unknown the disease begins with inflammation involving autoreactive T lymphocytes activation in genetically susceptible subjects. Interferon beta-1b (IFN ß 1b) is one of the most used drug in the MS therapy. The results obtained in this study show that the concentration of SOD1 in CSF of relapsing-remitting MS (RR-MS) patients, evaluated by enzyme-linked immunosorbent assay (ELISA), is decreased compared to pathological controls. Moreover, the Western blotting analysis demonstrated that SOD1 in human peripheral blood mononuclear cells (PBMC) in healthy controls was significantly higher compared to MS subjects before starting DMT therapy. In addition IFN ß 1b therapy causes an increase of intracellular SOD1 protein as well as mRNA levels in PBMC. Moreover, the treatment of neuroblastoma SK-N-BE cells with IFN ß 1b increased SOD1 protein and mRNA levels; these data also suggest that neuroprotective effect of this physiological molecule is, at least in part, carried out through its effect on SOD1. This study demonstrate that DMT therapy is able to increase SOD1 expression in PBMC of RR-MS patients. Therefore, the effectiveness of DMT therapy can be ascribed, at least in part, to an increased levels of this antioxidant enzyme as further confirmed by in vitro studies in SK-N-BE cells.

NOX signaling in molecular cardiovascular mechanisms involved in the blood pressure homeostasis.

Blood pressure homeostasis is maintained by several mechanisms regulating cardiac output, vascular resistances, and blood volume. At cellular levels, reactive oxygen species (ROS) signaling is involved in multiple molecular mechanisms controlling blood pressure. Among ROS producing systems, NADPH oxidases (NOXs), expressed in different cells of the cardiovascular system, are the most important enzymes clearly linked to the development of hypertension. NOXs exert a central role in cardiac mechanosensing, endothelium-dependent relaxation, and Angiotensin-II (Ang-II) redox signaling regulating vascular tone. The central role of NOXs in redox-dependent cardiovascular cell functions renders these enzymes a promising pharmacological target for the treatment of cardiovascular diseases, including hypertension. The aim of the present review is to focus on the physiological role of the cardiovascular NOX-generating ROS in the molecular and cellular mechanisms affecting blood pressure.

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes.

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2-protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2-protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.

Effects of berberine and red yeast on proinflammatory cytokines IL-6 and TNF-α in peripheral blood mononuclear cells (PBMCs) of human subjects.

BACKGROUND AND AIMS: Obesity is a condition associated with chronic or acute inflammatory response characterized by an increase of proinflammatory cytokine levels. Peripheral blood mononuclear cells (PBMCs) migrate in adipose tissue inducing synthesis and secretion of adipocytokines as IL-6 and TNF-α. The aim of this study was to investigate the effect of berberine (a natural alkaloid) and red yeast (a natural antioxidant) on IL-6 and TNF-α cytokines release and gene expression, in circulating lipopolisaccarides (LPS) stimulated PBMCs. METHODS AND RESULTS: PBMCs isolated from whole blood of healthy donors were stimulated with LPS to induce cytokines production; simultaneously cells were treated with increasing doses of berberine and red yeast. The substances were administered alone or in association. IL-6 and TNF-α protein levels in the culture medium and their mRNA levels were assessed by ELISA and real time PCR, respectively. Berberine and red yeast treatment prevented the LPS induction of IL-6 release in the culture medium of PBMCs. In addition, berberine plus red yeast treatment showed a synergic inhibitory effect on IL-6 release at low concentration. Berberine and red yeast showed an inhibitory effect also on LPS induction of TNF-α release exerting a synergic effect mainly at high concentrations. On the contrary, berberine and red yeast did not significantly affect IL-6 and TNF-α mRNA levels induced by LPS. In this case, only concomitant treatment of PBMCs with high doses of berberine and red yeast inhibits LPS induced IL-6 or TNF-α mRNA levels. CONCLUSIONS: The results of our study show that both berberine and red yeast were able to carry out anti-inflammatory action through an inhibition of proinflammatory IL-6 and TNF-α protein release. Moreover, when given in combination these substances were able to inhibit IL-6 and TNF-α gene expression in PBMCs activated by LPS. Therefore, these substances could represent a useful pharmacological treatment to reduce the proinflammatory status accompanied with obesity.

Small non-coding RNA signature in multiple sclerosis patients after treatment with interferon-ß.

BACKGROUND: Non-coding small RNA molecules play pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. In human diseases, the roles of the non-coding small RNAs in specific degradation or translational suppression of the targeted mRNAs suggest a potential therapeutic approach of post-transcriptional gene silencing that targets the underlying disease etiology. The involvement of non-coding small RNAs in the pathogenesis of neurodegenerative diseases such as Alzheimer's , Parkinson's disease and Multiple Sclerosis has been demonstrated. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. The current standard treatment for SM is interferon ß (IFNß) that is less than ideal due to side effects. In this study we administered the standard IFN-ß treatment to Relapsing-Remitting MS patients, all responder to the therapy; then examined their sncRNA expression profiles in order to identify the ncRNAs that were associated with MS patients' response to IFNß. METHODS: 40 IFNß treated Relapsing-Remitting MS patients were enrolled. We analyzed the composition of the entire small transcriptome by a small RNA cloning method, using peripheral blood from Relapsing-Remitting MS patients at baseline and 3 and 6 months after the start of IFNß therapy. Real-time qPCR from the same patients group and from 20 additional patients was performed to profile miRNAs expression. RESULTS: Beside the altered expression of several miRNAs, our analyses revealed the differential expression of small nucleolar RNAs and misc-RNAs.For the first time, we found that the expression level of miR-26a-5p changed related to INF-ß response. MiR-26a-5p expression was significantly higher in IFN-ß treated RRMS patients at 3 months treatment, keeping quite stable at 6 months treatments. CONCLUSIONS: Our results might provide insights into the mechanisms of action of IFN-ß treatment in MS and provide fundamentals for the development of new biomarkers and/or therapeutic tools.

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization, production and secretion.

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. Copper-zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the "non-T" cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.

Cu-Zn superoxide dismutase activates muscarinic acetylcholine M1 receptor pathway in neuroblastoma cells.

Muscarinic receptors (mAChRs) control several neuronal functions and are widely expressed in the central nervous system (CNS): M1 subtype represents the predominant mAChR in the CNS. Previously, we showed that antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) is secreted by many cellular lines and specifically interacts with cell surface membrane of human neuroblastoma SK-N-BE cells thus activating phospholipase C (PLC) transduction pathway and increasing intracellular calcium concentration ([Ca(2+)](i)). In addition, we demonstrated that a small amount of SOD1 is contained in large core dense vesicles and that it is secreted in response to depolarization induced by elevated extracellular K(+) concentration. In the present study, we investigated the involvement of muscarinic M1 receptors in SOD1-induced activation of PLC transduction pathway. We showed that, in SK-N-BE cells, SOD1 was able to activate muscarinic M1 receptor producing a phosphorylation of ERK 1/2 and Akt in dose- and time-dependent manner. Interestingly, in the presence of the M1 antagonist pirenzepine, ERK 1/2 and Akt phosphorylation induced by SOD1 was remarkably prevented. This effect was mimicked by knocking-down M1 receptor using two sequences of RNA silencing (siRNA). At functional level, siRNAs against M1 receptor were able to prevent the increase in [Ca(2+)](i) induced by SOD1. The same inhibitory effect on [Ca(2+)](i) changes was produced by the M1 antagonist pirenzepine. Collectively, the results of this study demonstrated that SOD1 could activate a transductional pathway through the involvement of M1 muscarinic receptor.

Reactive oxygen species regulate the levels of dual oxidase (Duox1-2) in human neuroblastoma cells.

Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox) (NOX2), or of the other membrane subunit of NADPH oxidase, p22(phox), blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.

Cigarette smoke condensate causes a decrease of the gene expression of Cu-Zn superoxide dismutase, Mn superoxide dismutase, glutathione peroxidase, catalase, and free radical-induced cell injury in SH-SY5Y human neuroblastoma cells.

Cigarette smoking condensate (CSC) contains oxidant compounds able to generate superoxide. The aim of the present study was to investigate the effect of the exposure to CSC on: (1) free radical production, (2) the gene expression of the antioxidant enzymes Cu-Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), Glutathione Peroxidase (GPx), and catalase (CAT), and (3) cell survival in human neuroblastoma SH-SY5Y cells. The results showed that exposure (24 h) to different concentrations (10-150 µg/ml) of CSC caused a dose dependent cell injury that was coupled to the maximal increase of free radical production. These events were prevented by the addition to the incubation medium of the scavenger Vitamin E (50 µM). Furthermore, CSC exposure caused a reduction of the gene expression of the antioxidant enzymes SOD1, SOD2, GPx, and CAT that was counteracted by Vitamin E (50 µM). These results suggest that CSC exposure can induce a free radical overcharge that may be responsible for the inhibition of antioxidant enzymes expression and cell injury in SH-SY5Y human neuroblastoma cells. In fact the scavenger vitamin E can block both cell injury and inhibition of SOD1, SOD2, GPx, and CAT induced by CSC exposure.

Effects of propionyl-L-carnitine on ischemia-reperfusion injury in hamster cheek pouch microcirculation.

BACKGROUND AND PURPOSE: Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia-reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. METHODS: The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (V(RBC)) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2'-7'-dichlorofluorescein (DCF), respectively. RESULTS: In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and V(RBC) decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. CONCLUSIONS: pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase.