Effect of extracellular Mg2+ on ROS and Ca2+ accumulation during reoxygenation of rat cardiomyocytes

2001 ◽  
Vol 280 (1) ◽  
pp. H344-H353 ◽  
Author(s):  
Mohammad N. Sharikabad ◽  
Kirsten M. Østbye ◽  
Torstein Lyberg ◽  
Odd Brørs
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Flow Cytometry ◽  
Lactate Dehydrogenase ◽  
Superoxide Anion ◽  
Oxygen Species ◽  
No Donor ◽  
Rat Cardiomyocytes ◽  
Intracellular Ros ◽  
Ldh Release

The effects of Mg2+ on reactive oxygen species (ROS) and cell Ca2+ during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2′,7′-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H2O2 and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O2 −·), respectively. Rapidly exchangeable cell Ca2+ was determined by 45Ca2+uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100–130% during reoxygenation alone and further increased 60% by increasing extracellular Mg2+concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16–24% during reoxygenation but were not affected by Mg2+. Cell Ca2+ increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg2+, 57% by 10 μM 3,4-dichlorobenzamil (DCB) (inhibitor of Na+/Ca2+ exchange), and 75% by combining Mg2+ and DCB. H2O2 (25 and 500 μM) reduced Ca2+ accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso- N-acetyl-penicillamine (1 mM) had no effect. Mg2+ reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg2+ to 5 mM increased the fluorescence of the H2O2/NO-sensitive probe DCF without increasing that of the O2 −·-sensitive probe ETH, reduced Ca2+ accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg2+, may be linked to the effect of Mg2+ on Ca2+ accumulation and/or ROS levels.

Real-time monitoring of peroxynitrite (ONOO−) in the rat brain by developing a ratiometric electrochemical biosensor

The Analyst ◽  
2019 ◽  
Vol 144 (6) ◽  
pp. 2150-2157 ◽  
Author(s):  
Feiyue Liu ◽  
Hui Dong ◽  
Yang Tian
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Rat Brain ◽  
Real Time ◽  
Superoxide Anion ◽  
Electrochemical Biosensor ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Real Time Monitoring ◽  
The Brain

As a reactive oxygen species (ROS), peroxynitrite (ONOO−) generated by nitric oxide (NO) and superoxide anion (O2˙−) plays important roles in physiological and pathological processes in the brain.

Differential responses of hydrogen peroxide, lipid peroxidation and antioxidant enzymes in Azolla microphylla exposed to paraquat and nitric oxide

Biologia ◽  
2012 ◽  
Vol 67 (6) ◽  
Author(s):  
Anjuli Sood ◽  
Charu Kalra ◽  
Sunil Pabbi ◽  
Prem Uniyal
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Lipid Peroxidation ◽  
Antioxidative Enzymes ◽  
Guaiacol Peroxidase ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Concentration Dependent Manner ◽  
No Donor ◽  
Azolla Microphylla

AbstractThe present investigation was carried out to decipher the interplay between paraquat (PQ) and exogenously applied nitric oxide (NO) in Azolla microphylla. The addition of PQ (8 μM) increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) by 1.7, 2.7, 3.9 and 1.9 folds respectively than that control in the fronds of Azolla. The amount of H2O2 was also enhanced by 2.7 times in the PQ treated plants than that of control. The supplementation of sodium nitroprusside (SNP) from 8–100 μM along with PQ, suppressed the activities of antioxidative enzymes and the amount of H2O2 compared to PQ alone. The drop in the activity of antioxidative enzymes — SOD, GPX, CAT and APX was highest (39.9%, 48.4%, 41.6% and 41.3% respectively) on the supplementation of 100 μM SNP with PQ treated fronds compared to PQ alone. The addition of NO scavengers along with NO donor in PQ treated fronds neutralized the effect of exogenously supplied NO. This indicates that NO can effectively protect Azolla against PQ toxicity by quenching reactive oxygen species. However, 200 μM of SNP reversed the protective effect of lower concentration of NO donor against herbicide toxicity. Our study clearly suggests that (i) SNP released NO can work both as cytoprotective and cytotoxic in concentration dependent manner and (ii) involvement of NO in protecting Azolla against PQ toxicity.

Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation

1997 ◽  
Vol 272 (5) ◽  
pp. L897-L902 ◽  
Author(s):  
J. J. Zulueta ◽  
R. Sawhney ◽  
F. S. Yu ◽  
C. C. Cote ◽  
P. M. Hassoun
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Endothelial Cells ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Intracellular Ros ◽  
Ros Formation ◽  
Oxide Synthase

Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.

scholarly journals Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2741-2748 ◽  
Author(s):  
P Seth ◽  
R Kumari ◽  
M Dikshit ◽  
RC Srimal
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Methylene Blue ◽  
Polymorphonuclear Leukocytes ◽  
Reactive Oxygen Species Generation ◽  
Oxygen Species ◽  
Superoxide Generation ◽  
No Donor ◽  
Radical Generation ◽  
Leukocyte Response

The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl- methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG- mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.

Nitric oxide and reactive oxygen species in the nucleus revisitedThis review is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

2010 ◽  
Vol 88 (3) ◽  
pp. 296-304 ◽  
Author(s):  
Chantale Provost ◽  
Faten Choufani ◽  
Levon Avedanian ◽  
Ghassan Bkaily ◽  
Fernand Gobeil ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Prostaglandin E ◽  
Oxygen Species ◽  
Nuclear Level ◽  
No Donor ◽  
Envelope Membranes ◽  
Endothelial Nitric Oxide

Recent work from our group showed that the nuclear envelope membranes contain several G protein-coupled receptors, including prostaglandin E2 (EP3R) and endothelin-1 (ET-1) receptors. Activation of EP3R increased endothelial nitric oxide synthase (eNOS) RNA expression in nuclei. eNOS and inducible NOS (iNOS) are reported to also be present at the nuclear level. Furthermore, reactive oxygen species (ROS) were also localized at the nuclear level. In this review, we show that stimulation with NO donor sodium nitroprusside results in an increase of intranuclear calcium that was dependent on guanylate cyclase activation, but independent of MAPK. This increase in nuclear calcium correlated with an increase in nuclear transcription of iNOS. H2O2 and ET-1 increase both cytosolic and nuclear ROS in human endocardial endothelial cells and in human aortic vascular smooth muscle cells. This increase in ROS levels by H2O2 and ET-1 was reversed by the antioxidant glutathione. In addition, our results strongly suggest that cytosolic signalization is not only transmitted to the nucleus but is also generated by the nucleus. Furthermore, we demonstrate that oxidative stress can be sensed by the nucleus. These results highly suggest that ROS formation is also generated directly by the nucleus and that free radicals may contribute to ET-1 regulation of nuclear Ca2+ homeostasis.

scholarly journals Reactive oxygen species in disease: Rebuttal of a conventional concept

2015 ◽  
Vol 1 (1) ◽  
pp. 23-27
Author(s):  
Luis Vitetta ◽  
Samantha Coulson ◽  
Anthony W Linnane
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Nucleic Acids ◽  
Superoxide Anion ◽  
Reactive Oxygen ◽  
Second Messenger ◽  
Normal Function ◽  
Ageing Process ◽  
Oxygen Species

The production of intracellular reactive oxygen species and reactive nitrogen species has long been proposed as leading to the random deleterious modification of macromolecules (i.e., nucleic acids, proteins) with an associated progressive development of the age associated systemic diseases (e.g., diabetes, Parkinson’s disease) as well as contributing to the ageing process.   Superoxide anion (hydrogen peroxide) and nitric oxide (peroxynitrite) comprise regulated intracellular second messenger pro-oxidant systems, with specific sub-cellular locales of production and are essential for the normal function of the metabolome and cellular electro-physiology.  We have posited that the formation of superoxide anion and its metabolic product hydrogen peroxide, and nitric oxide, do not conditionally lead to random damage of macromolecular species such as nucleic acids or proteins.  Under normal physiological conditions their production is intrinsically regulated that is very much consistent with their second messenger purpose of function.   We further propose that the concept of an orally administered small molecule antioxidant as a therapy to abrogate free radical activity (to control oxidative stress) is a chimera.  As such we consider that free radicals are not a major overwhelming player in the development of the chronic diseases or the ageing process.        

scholarly journals Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia

2002 ◽  
Vol 93 (4) ◽  
pp. 1411-1418 ◽  
Author(s):  
Dawn R. S. Steiner ◽  
Norberto C. Gonzalez ◽  
John G. Wood
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Leukocyte Adherence ◽  
No Donor ◽  
Substrate Limitation ◽  
Systemic Hypoxia ◽  
Microvascular Response ◽  
Spermine Nonoate

Systemic hypoxia results in oxidative stress due to a change in the reactive oxygen species (ROS)-nitric oxide (NO) balance. These experiments explored two mechanisms for the altered ROS-NO balance: 1) decreased NO synthesis by NO synthase due to limited O2 substrate availability and 2) increased superoxide generation. ROS levels and leukocyte adherence in mesenteric venules of rats during hypoxia were studied in the absence and presence of an NO donor [spermine NONOate (SNO)] and of the NO precursorl-arginine. We hypothesized that if the lower NO levels during hypoxia were due to O2 substrate limitation,l-arginine would not prevent hypoxia-induced microvascular responses. Graded hypoxia (produced by breathing 15, 10, and 7.5% O2) increased both ROS (123 ± 6, 148 ± 11, and 167 ± 3% of control) and leukocyte adherence. ROS levels during breathing of 10 and 7.5% O2 were significantly attenuated by SNO (105 ± 6 and 108 ± 3%, respectively) andl-arginine (117 ± 5 and 115 ± 2%, respectively). Both interventions reduced leukocyte adherence by similar degrees. The fact that the effects of l-arginine were similar to those of SNO does not support the idea that NO generation is impaired in hypoxia and suggests that tissue NO levels are depleted by the increased ROS during hypoxia.

Mechanistic Study of Hemoglobin Induced Platelet Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5262-5262
Author(s):  
Christine R. Carlisle ◽  
Mary Stahle ◽  
Ryan Vest ◽  
Roy Hantgan ◽  
Janet Lee ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Platelet Activation ◽  
Reactive Oxygen ◽  
Inhibitory Effect ◽  
Mechanistic Study ◽  
Blood Collection ◽  
Oxygen Species ◽  
Free Hemoglobin ◽  
No Donor

Abstract Abstract 5262 Nitric oxide (NO) has been previously shown to inhibit adenosine diphosphate (ADP)- and thrombin- mediated platelet activation. This inhibitory effect of NO is blocked by cell-free oxyhemoglobin (Villagra, Shiva et al. 2007). Cell-free hemoglobin (Hb) also appears to directly activate platelets even in the absence of ADP. However, the precise mechanisms for cell-free Hb-induced platelet activation have not been fully delineated. We hypothesized that oxyhemoglobin can promote platelet activation through one of three potential mechanisms: 1) direct apoprotein interaction with platelet receptors, 2) reactive oxygen species (ROS) production, and/or 3) scavenging of endogenous NO. Platelets were isolated from whole blood obtained from healthy volunteers (n=15) using sodium citrate blood collection tubes. Platelet activation was measured by examining immunofluorescent Pac-1 labeling by flow cytometry using activated glycoprotein IIb/IIIa as a measure of activation. In the presence of 10 uM concentration of NO donor mahmaNONOate, ADP-induced platelet activation was inhibited by 44%. Incubation of 100 uM cell-free oxyhemoglobin eliminated the inhibitory effect of NO. In addition, cell-free oxyhemoglobin (100 uM concentration) induced platelet activation by 17% above unstimulated controls even in the absence of ADP. Methemoglobin was used to test if direct Hb protein interaction with platelets was sufficient for platelet activation, independent of heme redox state. To produce methemoglobin, the ferrous iron in oxyhemoglobin was oxidized to its ferric form, thus methemoglobin is likely to preserve all direct protein interactions. However, unlike oxyhemoglobin, incubation of 100 uM methemoglobin did not induce platelet activation. Next, we tested whether reactive oxygen species production was responsible for hemoglobin-induced platelet activation. In the absence of hemoglobin, superoxide dismutase (SOD) (100 U/ml) and catalase (100 U/ml) reduced baseline platelet activation, verifying a ROS (Pignatelli, Pulcinelli et al. 1998). However, in the presence of oxyhemoglobin, SOD and catalase did not significantly abrogate the effect of Hb on platelet activation, suggesting that the ROS superoxide and hydrogen peroxide are not involved in hemoglobin-induced platelet activation. To determine if scavenging of endogenous NO was responsible for hemoglobin-induced platelet activation, platelets were incubated in the presence or absence of NO scavengers, carboxy-PTIO (5mM) and Fe-DTCS (3 mM), or nitric oxide synthase (NOS) inhibitor, LNMMA (100 uM). Results from these investigations suggest scavenging of endogenous NO was not responsible for hemoglobin-induced platelet activation. Overall our data suggest a complex mechanism for oxyhemoglobin-mediated platelet activation that is likely to involve more than a single contributing pathway. These data aid in elucidating the mechanism of platelet activation by cell-free hemoglobin and may provide novel insights to designing potential treatments to reduce the platelet activation underlying hypercoagulable states associated with a variety of pathological conditions including hemolytic anemias, stroke, and diabetes. This work was supported by NIH grants HL058091 and HL098032 Disclosures: Gladwin: Patents filed related to treating hemolysis.: Patents & Royalties. Kim-Shapiro:Patents filed related to treating hemolysis. Honororia from Sangart Inc.: Honoraria, Patents & Royalties.

scholarly journals Anticancer Activity of New Na(I) Complex on Retinoblastoma Cells via Inhibiting PI3K/AKT/mTOR Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Jun Zhang ◽  
Zhi-Nan Liu ◽  
Guo-Hua Deng
Keyword(s):  
Reactive Oxygen Species ◽  
Flow Cytometry ◽  
Mixed Solvent ◽  
Annexin V ◽  
Mtor Pathway ◽  
Oxygen Species ◽  
Flow Cytometry Analysis ◽  
Retinoblastoma Cells ◽  
Intracellular Ros ◽  
Hoechst Staining

Here, through applying 2,6-bis(4′-carboxyl-phenyl)pyridine (H2L), a rigid ligand featuring both carboxylic acid and pyridine groups, a new coordination polymer containing Na(I) has been generated with the reaction between H2L ligand and NaNO3 in a water and DMF mixed solvent, and its chemical composition is [Na2L]n. Furthermore, the antiproliferative activity of Na(I) complex against the HXO-Rb44 retinoblastoma cells was detected with CCK-8 assay. Hoechst staining along with Annexin V-FITC/PI revealed that Na(I) complex induces the HXO-Rb44 retinoblastoma cells apoptosis. Flow cytometry analysis of reactive oxygen species (ROS) showed that Na(I) complex significantly increases the level of intracellular ROS. Importantly, western blot analysis revealed that Na(I) complex might induce apoptosis through inactivation of PI3K/AKT/mTOR pathway.

scholarly journals Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2741-2748 ◽  
Author(s):  
P Seth ◽  
R Kumari ◽  
M Dikshit ◽  
RC Srimal
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Methylene Blue ◽  
Polymorphonuclear Leukocytes ◽  
Reactive Oxygen Species Generation ◽  
Oxygen Species ◽  
Superoxide Generation ◽  
No Donor ◽  
Radical Generation ◽  
Leukocyte Response

Abstract The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl- methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG- mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.

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