scholarly journals Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Marta Tajes ◽  
Gerard ILL-Raga ◽  
Ernest Palomer ◽  
Eva Ramos-Fernández ◽  
Francesc X. Guix ◽  
...  
Keyword(s):  
Cell Death ◽  
Time Course ◽  
Primary Cultures ◽  
Blood Perfusion ◽  
No Production ◽  
Irreversible Damage ◽  
No Donor ◽  
Human Neuroblastoma ◽  
Ischemic Tissue

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemiain vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells withH2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

Caspase Activation Is Required for Nitric Oxide–Mediated, CD95(APO-1/Fas)–Dependent and Independent Apoptosis in Human Neoplastic Lymphoid Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4311-4320 ◽  
Author(s):  
Katerina Chlichlia ◽  
Marcus E. Peter ◽  
Marian Rocha ◽  
Carsten Scaffidi ◽  
Mariana Bucur ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Present Report ◽  
Lymphoid Cells ◽  
Jurkat Cells ◽  
Target Cells ◽  
No Production ◽  
No Donor ◽  
Synthase Inhibitor ◽  
Inhibitor Of Protein Synthesis

Abstract Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1β converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.

scholarly journals Neuroprotective Effect of Bergamot Juice in 6-OHDA-Induced SH-SY5Y Cell Death, an In Vitro Model of Parkinson’s Disease

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 326 ◽  
Author(s):  
Nadia Ferlazzo ◽  
Santa Cirmi ◽  
Alessandro Maugeri ◽  
Caterina Russo ◽  
Giovanni Enrico Lombardo ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Death ◽  
Nuclear Translocation ◽  
Neuroprotective Effect ◽  
Neuroblastoma Cells ◽  
Specific Cell ◽  
Protective Effects ◽  
Human Neuroblastoma

Much evidence suggests that both oxidative stress and apoptosis play a key role in the pathogenesis of Parkinson’s disease (PD). The present study aims to evaluate the protective effect of bergamot juice (BJ) against 6-hydroxydopamine (6-OHDA)- or H2O2-induced cell death. Treatment of differentiated SH-SY5Y human neuroblastoma cells with 6-OHDA or H2O2 resulted in cell death that was significantly reduced by the pre-treatment with BJ. The protective effects of BJ seem to correlate with the reduction of intracellular reactive oxygen species and nitric oxide generation caused by 6-OHDA or H2O2. BJ also attenuated mitochondrial dysfunction, caspase-3 activation, imbalance of pro- and anti-apoptotic proteins, MAPKs activation and reduced NF-ĸB nuclear translocation evoked by neurotoxic agents. Additionally, BJ exhibited excellent antioxidant capability in cell-free assays. Collectively, our results suggest that BJ exerts neuroprotective effect through the interplay with specific cell targets and its antioxidant activity, making it worthy of consideration for the management of neurodegenerative diseases.

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

2005 ◽  
Vol 289 (3) ◽  
pp. C717-C726 ◽  
Author(s):  
My N. Helms ◽  
Ling Yu ◽  
Bela Malik ◽  
Dean J. Kleinhenz ◽  
C. Michael Hart ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Single Channel ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
No Production ◽  
Open Probability ◽  
No Donor

Several studies have shown that nitric oxide (NO) inhibits Na+ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na+ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 μM dexamethasone and 10% bovine serum. After 1.5 μM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound ( P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability ( Po) from 0.186 ± 0.043 to 0.045 ± 0.009 ( n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na+ transport is by altering iNOS production of NO.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

2005 ◽  
Vol 24 (11) ◽  
pp. 573-580 ◽  
Author(s):  
N Mohorko ◽  
N Kregar-Velikonja ◽  
G Repovs ◽  
M Gorensek ◽  
M Bresjanac
Keyword(s):  
Cell Death ◽  
Cell Transplantation ◽  
Fluorescence Intensity ◽  
Time Course ◽  
In Vitro Study ◽  
Host Cells ◽  
Hoechst 33342 ◽  
Overnight Incubation ◽  
Donor Cells

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 mg/mL of H342. H342 labeled / 50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week.Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 mg/mL of H342. Although a 12-hour incubation of rBMSC in 1 mg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.

scholarly journals The PSD95–nNOS interface

2005 ◽  
Vol 168 (1) ◽  
pp. 117-126 ◽  
Author(s):  
Jiong Cao ◽  
Jenni I. Viholainen ◽  
Caroline Dart ◽  
Helen K. Warwick ◽  
Mark L. Leyland ◽  
...  
Keyword(s):  
Cell Death ◽  
No Production ◽  
No Donor ◽  
Calcium Dependent ◽  
Upstream Regulator ◽  
Neuroprotective Drugs ◽  
Downstream Effectors ◽  
Nos Inhibitors ◽  
Excitotoxic Cell Death ◽  
The Relationship

The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress–generated NO and activation of p38, and the significance of the PSD95–nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95–nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95–nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.

scholarly journals An increase in nitric oxide produced by rat peritoneal neutrophils is not involved in cell apoptosis

1995 ◽  
Vol 4 (3) ◽  
pp. 222-228 ◽  
Author(s):  
I. M. Fierro ◽  
C. Barja-Fidalgo ◽  
R. M. Canedo ◽  
F. Q. Cunha ◽  
S. H. Ferreira
Keyword(s):  
Nitric Oxide ◽  
Cell Apoptosis ◽  
No Synthase ◽  
The Other ◽  
Polymorphonuclear Neutrophils ◽  
No Production ◽  
Spontaneous Release ◽  
No Donor ◽  
Carrageenin Injection

Polymorphonuclear neutrophils (PMN) obtained from carrageenin-stimulated peritoneal cavities of rats, but not blood PMN, spontaneously produced nitric oxide (NO) when incubatedin vitro. Incubation of the cells with the NO synthase inhibitors, L-imino-ethyl-L-ornithine (L-NIO) or NG-monomethyl-L-arginine (L-NMMA), inhibited NO production. This inhibition could be reversed by L-arginine. Incubation of PMN with lipopolysaccharide (LPS) failed to enhance NO production. Pretreatment of the rats with dexamethasone (DEXA) prior to carrageenin injection or incubation of PMN with the glucocorticoidin vitropartially inhibited the spontaneous release of NO. On the other hand, when PMN obtained from DEXA pretreated rats were incubatedin vitrowith DEXA, NO synthase activity and hence NO generation were almost abolished. A similar inhibition was also observed following the addition of L-NIO or cycloheximide to cultures of carrageenin-elicited PMN. The NO production by PMN did not appear to be related to cell viability or apoptosis. Indeed, neither the blockade of NO generation by L-NIO nor the incubation of the neutrophils with a NO donor, S-nitroso-acetylpenicillamine (SNAP) modified the pattern of LDH release or DNA fragmentation. In summary, it appears that PMN migration triggers a continuous NO synthesis, and that NO produced by these cells is not related to their apoptosis.

scholarly journals The The Effects of Centella asiatica Extract (CAE) on Methamphetamine-Induced Neurotoxicity via Human Neuroblastoma Cell Line

2021 ◽  
Vol 16 ◽  
pp. 1-9
Author(s):  
Mazatulikhma Mat Zain Mat Zain ◽  
Nursyamila Shamsuddin ◽  
Mohd Shihabuddin Ahmad Noorden
Keyword(s):  
Cell Death ◽  
Neuroblastoma Cell ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Centella Asiatica ◽  
Human Neuroblastoma Cell ◽  
Asiatic Acid ◽  
Dependent Manner ◽  
Human Neuroblastoma

Methamphetamine (METH) was reported to caused neurotoxicity and cell death, in vitro. Centella asiatica or ‘pegaga’ is a native tropical herb with antioxidant and neuroprotective activities. Although the effects of Centella asiatica against oxidative stress and neuronal cell death have been reported in previous studies, however, the potential effects of Centella asiatica against psychostimulant methamphetamine (METH) are limited. Therefore, this study was aimed to evaluate the effects of Centella asiatica extract (CAE) against METH on all-trans retinoic acid, RA-differentiated human neuroblastoma, SH-SY5Y cells. The RA-differentiated SH-SY5Y cells were used to resemble dopaminergic neuronal-like cells. Cell viability was quantitatively assessed by 3-(4,5-dimethylthiazol-2-yl)-2 tetrazolium bromide, MTS assay.  CAE at varying concentrations from 1pg/mL to 1mg/mL significantly decreased the viability of the undifferentiated SH-SY5Y cells in a concentration-dependent manner. At 1mg/mL of CAE, significantly increased the viability of differentiated SH-SY5Y cells. Meanwhile, CAE at 100µg/mL and 1mg/mL significantly reversed the METH-induced neuronal cell death. The results revealed that promising treatment of CAE on METH-induced neurotoxicity is mediated by its high content of asiaticoside, asiatic acid, madecassoside and madecassic acid. Taken together, this study may suggest CAE as a potential therapeutic treatment for METH-induced neurotoxicity, in vitro.

scholarly journals Down Regulation of Macrophage Activation in Brugia pahangi-Infected Jirds (Meriones unguiculatus)

1998 ◽  
Vol 66 (3) ◽  
pp. 1063-1069 ◽  
Author(s):  
C. Nasarre ◽  
J. L. Krahenbuhl ◽  
T. R. Klei
Keyword(s):  
Inflammatory Response ◽  
Macrophage Activation ◽  
Time Course ◽  
Meriones Unguiculatus ◽  
No Production ◽  
Mongolian Gerbils ◽  
Down Regulation ◽  
Brugia Pahangi ◽  
Mouse System

ABSTRACT The macrophage is a major component of the inflammatory response induced by lymphatic tissue-dwelling filariae. Intraperitoneal (i.p.) infections with Brugia pahangi in Mongolian gerbils, or jirds (Meriones unguiculatus), induce a peritoneal inflammatory response characterized by accumulation of numerous macrophages and fewer eosinophils. This inflammatory response is associated with the release of microfilariae by female worms. The aim of this study was to investigate the activation state of the peritoneal macrophages during the course of i.p. infections with either male or female worms. Activation was determined by a toxoplasmacidal assay and assays which measured the production of tumor necrosis factor (TNF)-like activity and nitric oxide (NO) production. The development of these assays with jirds was initially conducted in parallel with the mouse system, which served as a positive control. Jird macrophages became activated to kill Toxoplasma gondii by in vivo immunization with Mycobacterium bovis BCG in a pattern similar to that of mouse macrophages. However, unlike the mouse system, supernatants from purified protein derivative- or concanavalin A-stimulated jird splenocytes plus lipopolysaccharide failed to activate jird macrophages in vitro or induce NO production. These results indicate that factors involved in jird macrophage activation may differ from those demonstrated in the mouse system and other systems. i.p. infections of 15 days in duration with either male or female worms induced macrophage activation as measured byToxoplasma killing and TNF production. These responses decreased as the infection progressed to the chronic period on a time course that parallels the down regulation of experimentalB. pahangi granulomas. There was no evidence of NO production by activated jird macrophages. These data indicate that macrophage function is down modulated during filarial infection and suggest that mechanisms involved in macrophage deactivation are related to those that induce down modulation of the systemic granulomatous inflammatory response in the jird. This response is not dependent on the microfilarial stage of the parasite and is also independent of mechanisms which induce peritoneal accumulations of macrophages.

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