Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition

Recent studies have highlighted the fact that cancer cells have an altered metabolic phenotype, and this metabolic reprogramming is required to drive the biosynthesis pathways necessary for rapid replication and proliferation. Specifically, the importance of citric acid cycle-generated intermediates in the regulation of cancer cell proliferation has been recently appreciated. One function of MCTs (monocarboxylate transporters) is to transport the citric acid cycle substrate pyruvate across the plasma membrane and into mitochondria, and inhibition of MCTs has been proposed as a therapeutic strategy to target metabolic pathways in cancer. In the present paper, we examined the effect of different metabolic substrates (glucose and pyruvate) on mitochondrial function and proliferation in breast cancer cells. We demonstrated that cancer cells proliferate more rapidly in the presence of exogenous pyruvate when compared with lactate. Pyruvate supplementation fuelled mitochondrial oxygen consumption and the reserve respiratory capacity, and this increase in mitochondrial function correlated with proliferative potential. In addition, inhibition of cellular pyruvate uptake using the MCT inhibitor α-cyano-4-hydroxycinnamic acid impaired mitochondrial respiration and decreased cell growth. These data demonstrate the importance of mitochondrial metabolism in proliferative responses and highlight a novel mechanism of action for MCT inhibitors through suppression of pyruvate-fuelled mitochondrial respiration.     

Neuroprotective mechanisms of cerium oxide nanoparticles in a mouse hippocampal brain slice model of ischemia

Cerium oxide nanoparticles (nanoceria) are widely used as catalysts in industrial applications because of their potent free radical-scavenging properties. Given that free radicals play a prominent role in the pathology of many neurological diseases, we explored the use of nanoceria as a potential therapeutic agent for stroke. Using a mouse hippocampal brain slice model of cerebral ischemia, we show here that ceria nanoparticles reduce ischemic cell death by approximately 50%. The neuroprotective effects of nanoceria were due to a modest reduction in reactive oxygen species, in general, and ~ 15% reductions in the concentrations of superoxide (O₂^•−) and nitric oxide, specifically. Moreover, treatment with nanoceria markedly decreased (~ 70% reduction) the levels of ischemia-induced 3-nitrotyrosine, a modification to tyrosine residues in proteins induced by the peroxynitrite radical. These findings suggest that scavenging of peroxynitrite may be an important mechanism by which cerium oxide nanoparticles mitigate ischemic brain injury. Peroxynitrite plays a pivotal role in the dissemination of oxidative injury in biological tissues. Therefore, nanoceria may be useful as a therapeutic intervention to reduce oxidative and nitrosative damage after a stroke.     

Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O₂⁻) by a diffusion-controlled reaction to form peroxynitrite (ONOO⁻), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO⁻ induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O₂⁻ scavenging with light irradiation to block ONOO⁻ production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O₂⁻, and ONOO⁻ levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O₂⁻, total ROS, and ONOO⁻ levels were increased after SNP treatment, but O₂⁻, total ROS, and ONOO⁻ levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O₂⁻, protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO⁻ synthesis.     

Inhibition of protein tyrosine phosphatases by amino acid, peptide, and protein hydroperoxides: potential modulation of cell signaling by protein oxidation products

Reaction of radicals in the presence of O2, or singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. They can be detected in cells and are poorly removed by enzymatic defenses. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, with this resulting in inactivation of some thiol-dependent enzymes. In light of these data, we hypothesized that inactivation of protein tyrosine phosphatases (PTPs), by hydroperoxides present on oxidized proteins, may contribute to cellular and tissue dysfunction by modulation of phosphorylation-dependent cell signaling. We show here that PTPs in cell lysates, and purified PTP-1B, are inactivated by amino acid, peptide, and protein hydroperoxides in a concentration- and structure-dependent manner. Protein hydroperoxides are particularly effective, with inhibition occurring with greater efficacy than with H2O2. Inactivation involves reaction of the hydroperoxide with the conserved active-site Cys residue of the PTPs, as evidenced by hydroperoxide consumption measurements and a diminution of this effect on blocking the Cys residue. This inhibition of PTPs, by oxidized proteins containing hydroperoxide groups, may contribute to cellular dysfunction and altered redox signaling in systems subject to oxidative stress.     

Extracellular superoxide dismutase in vessels and airways of humans and baboons

Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO^·) species, is a primary mediator of vascular relaxation. During NO^·′ diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO^·. High levels of extracellualar superoxide dismutase in vessels could, therefore, be essential to enable NO' to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.     

Inhibition of human surfactant protein A function by oxidation intermediates of nitrite

Nitration of protein tyrosine residues by peroxynitrite (ONOO⁻) has been implicated in a variety of inflammatory diseases such as acute respiratory distress syndrome (ARDS). Pulmonary surfactant protein A (SP-A) has multiple functions including host defense. We report here that a mixture of hypochlorous acid (HOCl) and nitrite (NO₂⁻) induces nitration, oxidation, and chlorination of tyrosine residues in human SP-A and inhibits SP-A’s ability to aggregate lipids and bind mannose. Nitration and oxidation of SP-A was not altered by the presence of lipids, suggesting that proteins are preferred targets in lipid-rich mixtures such as pulmonary surfactant. Moreover, both horseradish peroxidase and myeloperoxidase (MPO) can utilize NO₂⁻ and hydrogen peroxide (H₂O₂) as substrates to catalyze tyrosine nitration in SP-A and inhibit its lipid aggregation function. SP-A nitration and oxidation by MPO is markedly enhanced in the presence of physiological concentrations of Cl⁻ and the lipid aggregation function of SP-A is completely abolished. Collectively, our results suggest that MPO released by activated neutrophils during inflammation utilizes physiological or pathological levels of NO₂⁻ to nitrate proteins, and may provide an additional mechanism in addition to ONOO⁻ formation, for tissue injury in ARDS and other inflammatory diseases associated with upregulated ^•NO and oxidant production.     

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

The chemical biology of nitric oxide: implications in cellular signaling

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.     

Signaling and stress: The redox landscape in NOS2 biology

Nitric oxide (NO) has a highly diverse range of biological functions from physiological signaling and maintenance of homeostasis to serving as an effector molecule in the immune system. However, deleterious as well as beneficial roles of NO have been reported. Many of the dichotomous effects of NO and derivative reactive nitrogen species (RNS) can be explained by invoking precise interactions with different targets as a result of concentration and temporal constraints. Endogenous concentrations of NO span five orders of magnitude, with levels near the high picomolar range typically occurring in short bursts as compared to sustained production of low micromolar levels of NO during immune response. This article provides an overview of the redox landscape as it relates to increasing NO concentrations, which incrementally govern physiological signaling, nitrosative signaling and nitrosative stress-related signaling. Physiological signaling by NO primarily occurs upon interaction with the heme protein soluble guanylyl cyclase. As NO concentrations rise, interactions with nonheme iron complexes as well as indirect modification of thiols can stimulate additional signaling processes. At the highest levels of NO, production of a broader range of RNS, which subsequently interact with more diverse targets, can lead to chemical stress. However, even under such conditions, there is evidence that stress-related signaling mechanisms are triggered to protect cells or even resolve the stress. This review therefore also addresses the fundamental reactions and kinetics that initiate signaling through NO-dependent pathways, including processes that lead to interconversion of RNS and interactions with molecular targets.     

Diazeniumdiolation of protamine sulfate reverses mitogenic effects on smooth muscle cells and fibroblasts

After vascular interventions, endothelial cells are typically injured or lacking, resulting in decreased NO synthesis to maintain vascular health. Moreover, inflammation as a result of the tissue injury and/or the presence of an implanted foreign polymer such as a vascular graft causes excessive generation of reactive oxygen species (ROS) (e.g., superoxide), which can react with NO. The combination of the above creates a general decline in NO bioavailability, as well as oxidative stress due to less available NO to scavenge ROS. Localized NO delivery is an attractive solution to alleviate these issues; however, NO donors typically exhibit unpredictable NO payload release when using nitrosothiols or the risk of nitrosamine formation for synthetic diazeniumdiolates. The objective of this study was therefore to synthesize an NO donor from a biological peptide that could revert to its native form upon NO release. To this effect, protamine sulfate (PS), an FDA-approved peptide with reported vasodilator and anticoagulant properties, was diazeniumdiolated to form PS/NO. PS/NO showed diazeniumdiolate-characteristic UV peaks and NO release in physiological solutions and was capable of scavenging radicals to decrease oxidative stress. Furthermore, PS/NO selectively inhibits the proliferation of smooth muscle cells and adventitial fibroblasts, thereby reversing reported mitogenic properties of PS. Endothelial cell growth, on the other hand, was promoted by PS/NO. Finally, PS retained its anticoagulant properties upon diazeniumdiolation at clinically relevant concentrations. In conclusion, we have synthesized an NO prodrug from a biological peptide, PS/NO, that selectively inhibits proliferation of smooth muscle cells and fibroblasts, retains anticoagulant properties, and reverts back to its native PS form upon NO payload release.     

Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis

Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd²⁺ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd²⁺-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd²⁺. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd²⁺-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd²⁺-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.     

Hemodynamic effects of metalloporphyrin catalytic antioxidants: structure-activity relationships and species specificity

Superoxide plays a role in blood pressure regulation in certain vascular diseases, however, its involvement in regulating basal blood pressure is uncertain. Vascular superoxide concentrations are limited by extracellular superoxide dismutase (EC-SOD), which is highly expressed in the vasculature of most animal species. Metalloporphyrins are low molecular weight, synthetic, redox-active, catalytic antioxidants that act as SOD mimetics. We evaluated the effects of metalloporphyrins on blood pressure in different animal species. The metalloporphyrin AEOL10113 (5–10 μg/kg iv), but not native or polyethylene glycol-CuZnSOD, caused a dose-dependent reduction in blood pressure in anesthetized rats. AEOL10113 had no effect on blood pressure in mice (wild-type or EC-SOD knockouts), guinea pigs, dogs, or baboons at doses up to 5 mg/kg iv Structure-activity studies indicated that metalloporphyrins with high SOD activity were more effective in lowering rat blood pressure than low-activity analogs. The blood pressure effect of AEOL10113 was not attributable to the release of manganese, nor was it affected by inhibitors of nitric oxide synthase (L-NAME) and guanylate cyclase (ODQ, 8-bromo-cGMP, and methylene blue) or nitric oxide scavengers (HbA_o). Chlorpheniramine attenuated the effect, suggesting that the blood pressure response in rats is related to histamine release rather than the protection of nitric oxide.     

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.     

Mechanism and regulation of peroxidase-catalyzed nitric oxide consumption in physiological fluids: Critical protective actions of ascorbate and thiocyanate

Catalytic consumption of nitric oxide (NO) by myeloperoxidase and related peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how peroxidases consume NO in physiological fluids, in which multiple reactive enzyme substrates and antioxidants are present. Notably, ascorbate has been proposed to enhance myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by myeloperoxidase and related peroxidases (lactoperoxidase, horseradish peroxidase) by acting as a competitive substrate for protein-bound redox intermediates and by efficiently scavenging peroxidase-derived radicals (e.g., urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during inflammation. We show that NO consumption by myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However, thiocyanate effectively inhibits this process and yields hypothiocyanite at the expense of NO consumption. Hypothiocyanite can in turn form NO-consuming radicals, but thiols (albumin, glutathione) readily prevent this. Conversely, where ascorbate is absent, glutathione enhances NO consumption by urate radicals via pathways that yield S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating peroxidase-catalyzed NO consumption.     

Mechanisms and kinetic profiles of superoxide-stimulated nitrosative processes in cells using a diaminofluorescein probe

In this study, we examined the mechanisms and kinetic profiles of intracellular nitrosative processes using diaminofluorescein (DAF-2) as a target in RAW 264.7 cells. The intracellular formation of the fluorescent, nitrosated product diaminofluorescein triazol (DAFT) from both endogenous and exogenous nitric oxide (NO) was prevented by deoxygenation and by cell membrane-permeable superoxide (O₂⁻) scavengers but not by extracellular bovine Cu,Zn-SOD. In addition, the DAFT formation rate decreased in the presence of cell membrane-permeable Mn porphyrins that are known to scavenge peroxynitrite (ONOO⁻) but was enhanced by HCO₃⁻/CO₂. Together, these results indicate that nitrosative processes in RAW 264.7 cells depend on endogenous intracellular O₂⁻ and are stimulated by ONOO⁻/CO₂-derived radical oxidants. The N₂O₃ scavenger sodium azide (NaN₃) only partially attenuated the DAFT formation rate and only with high NO (>120 nM), suggesting that DAFT formation occurs by nitrosation (azide-susceptible DAFT formation) and predominantly by oxidative nitrosylation (azide-resistant DAFT formation). Interestingly, the DAFT formation rate increased linearly with NO concentrations of up to 120–140 nM but thereafter underwent a sharp transition and became insensitive to NO. This behavior indicates the sudden exhaustion of an endogenous cell substrate that reacts rapidly with NO and induces nitrosative processes, consistent with the involvement of intracellular O₂⁻. On the other hand, intracellular DAFT formation stimulated by a fixed flux of xanthine oxidase-derived extracellular O₂⁻ that also occurs by nitrosation and oxidative nitrosylation increased, peaked, and then decreased with increasing NO, as previously observed. Thus, our findings complementarily show that intra- and extracellular O₂⁻-dependent nitrosative processes occurring by the same chemical mechanisms do not necessarily depend on NO concentration and exhibit different unusual kinetic profiles with NO dynamics, depending on the biological compartment in which NO and O₂⁻ interact.     

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.     

Inhibition of Neuronal Nitric Oxide Synthase by 7-Nitroindazole Protects Against MPTP-Induced Neurotoxicity in Mice

Abstract: Several studies suggest that nitric oxide (NO^•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7-nitroindazole (7-NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7-NI dose-dependently protected against MPTP-induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovanillic acid and 3,4-dihydroxyphenylacetic acid. 7-NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO^• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO⁻), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. Our results suggest that NO^• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.     

Biosynthesis of silver nanoparticles using aqueous extract of Phyllanthus acidus L. fruits and characterization of its anti-inflammatory effect against H2O2 exposed rat peritoneal macrophages

The green synthesis and characterization of silver nanoparticles (AgNPs) derived from plants impart ecological and economic benefits to AgNPs. In addition, AgNPs have potential therapeutic roles in cytoprotectivity and anti-inflammation. The present work utilizes the aqueous extract of Phyllanthus acidus fruits for the production of AgNPs from aqueous silver nitrate solution. The synthesized AgNPs were characterized spectrophotometrically Fourier transform infrared spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, scanning electron microscopy and transmission electron microscopy analysis. The characterized AgNPs showed potent anti-inflammatory activity by scavenging nitric oxide and superoxide anions. In addition, blunting of the expression of pro-inflammatory cytokine interleukin 1 beta (IL-1β) assayed both by ELISA and Western blot, using H₂O₂ – induced inflammation in rat peritoneal macrophages. Furthermore, short-term exposure to P. acidus-mediated green-synthesized AgNPs did not affect the viability of peritoneal macrophages, as assessed by MTT assay. Our findings indicate that P. acidus-mediated green-synthesized AgNPs could be a potential therapeutics to treat inflammatory diseases.