Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia

2002 ◽  
Vol 282 (5) ◽  
pp. C1113-C1120 ◽  
Author(s):  
Stephen W. Schaffer ◽  
Viktoriya Solodushko ◽  
David Kakhniashvili
Keyword(s):  
Cell Injury ◽  
Control Cell ◽  
Calcium Handling ◽  
Hypoxic Injury ◽  
Treated Cell ◽  
Neonatal Cardiomyocytes ◽  
Chemical Hypoxia ◽  
Calcium Loading ◽  
Taurine Deficiency ◽  
Osmotic Load

Cellular sodium excess is cytotoxic because it increases both the intracellular osmotic load and intracellular calcium concentration ([Ca2+]i). Because sodium levels rise during hypoxia, it is thought to contribute to hypoxic injury. Thus the present study tested the hypothesis that taurine-linked reductions in [Na+]i reduce hypoxia-induced cell injury. Taurine depletion was achieved by exposing isolated neonatal cardiomyocytes to medium containing the taurine analog β-Alanine. As predicted, the β-Alanine-treated cell exhibited less hypoxia-induced necrosis and apoptosis than the control, as evidenced by less swelling, shrinkage, TdT-mediated dUTP nick end labeling staining, and accumulation of trypan blue. After 1 h of chemical hypoxia, [Na+]i was 3.5-fold greater in the control than the taurine-deficient cell. Although more taurine was lost from the control cell than from the β-Alanine-treated cell during hypoxia, the combined taurine and sodium osmotic load was lower in the β-Alanine-treated cell. Taurine deficiency also reduced the degree of hypoxia-induced calcium overload. Thus the observed resistance against hypoxia-induced necrosis and apoptosis is probably related to an improvement in sodium and calcium handling.

scholarly journals TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

2021 ◽  
Vol 22 (1) ◽  
pp. 412
Author(s):  
Christopher L. Moore ◽  
Alena V. Savenka ◽  
Alexei G. Basnakian
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cell Injury ◽  
Kidney Injury ◽  
Cell Types ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hypoxic Injury ◽  
Additional Information ◽  
Tissue Compartments

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes

1989 ◽  
Vol 257 (2) ◽  
pp. C347-C354 ◽  
Author(s):  
G. J. Gores ◽  
C. E. Flarsheim ◽  
T. L. Dawson ◽  
A. L. Nieminen ◽  
B. Herman ◽  
...  
Keyword(s):  
Cell Injury ◽  
Rat Hepatocytes ◽  
Iodoacetic Acid ◽  
Cell Killing ◽  
Cell Swelling ◽  
Reductive Stress ◽  
Chemical Hypoxia ◽  
Hydroperoxide Formation ◽  
Bleb Formation ◽  
The Relationship

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that “reductive stress” caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.

Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells

1996 ◽  
Vol 271 (1) ◽  
pp. F209-F215 ◽  
Author(s):  
H. Hagar ◽  
N. Ueda ◽  
S. V. Shah
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Reactive Oxygen ◽  
Reactive Oxygen Metabolites ◽  
Strand Breaks ◽  
Hypoxic Injury ◽  
Chemical Hypoxia ◽  
Dna Strand ◽  
Oxygen Metabolites

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.

scholarly journals miR-181c-5p Exacerbates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Targeting PTPN4

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Liang Ge ◽  
Yin Cai ◽  
Fan Ying ◽  
Hao Liu ◽  
Dengwen Zhang ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Control Cell ◽  
Luciferase Reporter ◽  
Apoptosis Pathway ◽  
H9c2 Cardiomyocytes ◽  
Hypoxia Reoxygenation

Background. Activation of cell apoptosis is a major form of cell death during myocardial ischemia/reperfusion injury (I/RI). Therefore, examining ways to control cell apoptosis has important clinical significance for improving postischemic recovery. Clinical evidence demonstrated that miR-181c-5p was significantly upregulated in the early phase of myocardial infarction. However, whether or not miR-181c-5p mediates cardiac I/RI through cell apoptosis pathway is unknown. Thus, the present study is aimed at investigating the role and the possible mechanism of miR-181c-5p in apoptosis during I/R injury by using H9C2 cardiomyocytes. Methods and Results. The rat origin H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 6 hours reoxygenation) to induce cell injury. The results showed that H/R significantly increased the expression of miR-181c-5p but not miR-181c-3p in H9C2 cells. In line with this, in an in vivo rat cardiac I/RI model, miR-181c-5p expression was also significantly increased. The overexpression of miR-181c-5p by its agomir transfection significantly aggravated H/R-induced cell injury (increased lactate dehydrogenase level and reduced cell viability) and exacerbated H/R-induced cell apoptosis (greater cleaved caspases 3 expression, Bax/Bcl-2 and more TUNEL-positive cells). In contrast, inhibition of miR-181c-5p in vitro had the opposite effect. By using computational prediction algorithms, protein tyrosine phosphatase nonreceptor type 4 (PTPN4) was predicted as a potential target gene of miR-181c-5p and was verified by the luciferase reporter assay. The overexpression of miR-181c-5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes. Moreover, knockdown of PTPN4 significantly aggravated H/R-induced enhancement of LDH level, cleaved caspase 3 expression, and apoptotic cell death, which mimicked the proapoptotic effects of miR-181c-5p in H9C2 cardiomyocytes. Conclusions. These findings suggested that miR-181c-5p exacerbates H/R-induced cardiomyocyte injury and apoptosis via targeting PTPN4 and that miR-181c-5p/PTPN4 signaling may yield novel strategies to combat myocardial I/R injury.

scholarly journals Long Non-Coding RNA H19 Protects H9c2 Cells against Hypoxia-Induced Injury by Targeting MicroRNA-139

2017 ◽  
Vol 44 (3) ◽  
pp. 857-869 ◽  
Author(s):  
Li-Cheng Gong ◽  
Hai-Ming Xu ◽  
Gong-Liang Guo ◽  
Tao Zhang ◽  
Jing-Wei Shi ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Injury ◽  
Myocardial Cell ◽  
Mtor Pathway ◽  
Migration And Invasion ◽  
H9c2 Cells ◽  
Hypoxic Injury ◽  
Downstream Target ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Background/Aims: Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time; ischemia is primarily caused due to hypoxia. The present study evaluated the effects of long non-coding RNA H19 on hypoxic rat H9c2 cells and mouse HL-1 cells. Methods: Hypoxic injury was confirmed by measuring cell viability, migration and invasion, and apoptosis using MTT, Transwell and flow cytometry assays, respectively. H19 expression after hypoxia was estimated by qRT-PCR. We then measured the effects of non-physiologically expressed H19, knockdown of miR-139 with or without H19 silence, and abnormally expressed Sox8 on hypoxia-induced H9c2 cells. Moreover, the interacted miRNA for H19 and downstream target gene were virtually screened and verified. The involved signaling pathways and the effects of abnormally expressed H19 on contractility of HL-1 cells were explored via Western blot analysis. Results: Hypoxia induced decreases of cell viability, migration and invasion, increase of cell apoptosis and up-regulation of H19. Knockdown of H19 increased hypoxia-induced injury in H9c2 cells. H19 acted as a sponge for miR-139 and H19 knockdown aggravated hypoxia-induced injury by up-regulating miR-139. Sox8 was identified as a target of miR-139, and its expression was negatively regulated by miR-139. The mechanistic studies revealed that overexpression of Sox8 might decrease hypoxia-induced cell injury by activating the PI3K/AKT/mTOR pathway and MAPK. Besides, H19 promoted contractility of HL-1 cells. Conclusion: These findings suggest that H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated up-regulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK.

Nox as a target for diabetic complications

2013 ◽  
Vol 125 (8) ◽  
pp. 361-382 ◽  
Author(s):  
Yves Gorin ◽  
Karen Block
Keyword(s):  
Diabetic Complications ◽  
Cell Injury ◽  
Transforming Growth Factor ◽  
Current Knowledge ◽  
Critical Role ◽  
Control Cell ◽  
Renin Angiotensin System ◽  
Present Review ◽  
Nadph Oxidases ◽  
Complications Of Diabetes

Oxidative stress has been linked to the pathogenesis of the major complications of diabetes in the kidney, the heart, the eye or the vasculature. NADPH oxidases of the Nox family are a major source of ROS (reactive oxygen species) and are critical mediators of redox signalling in cells from different organs afflicted by the diabetic milieu. In the present review, we provide an overview of the current knowledge related to the understanding of the role of Nox in the processes that control cell injury induced by hyperglycaemia and other predominant factors enhanced in diabetes, including the renin–angiotensin system, TGF-β (transforming growth factor-β) and AGEs (advanced glycation end-products). These observations support a critical role for Nox homologues in diabetic complications and indicate that NADPH oxidases are an important therapeutic target. Therefore the design and development of small-molecule inhibitors that selectively block Nox oxidases appears to be a reasonable approach to prevent or retard the complications of diabetes in target organs. The bioefficacy of these agents in experimental animal models is also discussed in the present review.

scholarly journals A model of propagating calcium-induced calcium release mediated by calcium diffusion.

1989 ◽  
Vol 93 (5) ◽  
pp. 963-977 ◽  
Author(s):  
P H Backx ◽  
P P de Tombe ◽  
J H Van Deen ◽  
B J Mulder ◽  
H E ter Keurs
Keyword(s):  
Calcium Release ◽  
Cardiac Cells ◽  
Calcium Handling ◽  
Coupled Equations ◽  
Local Fluctuations ◽  
Calcium Loading ◽  
Method Of Solution ◽  
Spatial Coordinates ◽  
Predictor Corrector ◽  
Calcium Induced Calcium Release

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading.

scholarly journals Hydrogen Sulfide Protects against Chemical Hypoxia-Induced Injury via Attenuation of ROS-Mediated Ca2+Overload and Mitochondrial Dysfunction in Human Bronchial Epithelial Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Cai-Xia Liu ◽  
Yu-Rong Tan ◽  
Yang Xiang ◽  
Chi Liu ◽  
Xiao-Ai Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Sulfide ◽  
Cell Injury ◽  
Fluorescence Probe ◽  
Environmental Pollutants ◽  
Cell Counting ◽  
Dependent Manner ◽  
Mitochondria Membrane Potential ◽  
Chemical Hypoxia ◽  
The Impact

Oxidative stress induced by hypoxia/ischemia resulted in the excessive reactive oxygen species (ROS) and the relative inadequate antioxidants. As the initial barrier to environmental pollutants and allergic stimuli, airway epithelial cell is vulnerable to oxidative stress. In recent years, the antioxidant effect of hydrogen sulfide (H2S) has attracted much attention. Therefore, in this study, we explored the impact of H2S on CoCl2-induced cell injury in 16HBE14o- cells. The effect of CoCl2on the cell viability was detected by Cell Counting Kit (CCK-8) and the level of ROS in 16HBE14o- cells in response to varying doses (100–1000μmol/L) of CoCl2(a common chemical mimic of hypoxia) was measured by using fluorescent probe DCFH-DA. It was shown that, in 16HBE14o- cells, CoCl2acutely increased the ROS content in a dose-dependent manner, and the increased ROS was inhibited by the NaHS (as a donor of H2S). Moreover, the calcium ion fluorescence probe Fura-2/AM and fluorescence dye Rh123 were used to investigate the intracellular calcium concentration ([Ca2+]i) and mitochondria membrane potential (MMP) in 16HBE14o- cells, respectively. In addition, we examined apoptosis of 16HBE14o- cells with Hoechst 33342. The results showed that the CoCl2effectively elevated the Ca2+influx, declined the MMP, and aggravated apoptosis, which were abrogated by NaHS. These results demonstrate that H2S could attenuate CoCl2-induced hypoxia injury via reducing ROS to perform an agonistic role for the Ca2+influx and MMP dissipation.

Ability of prostaglandin to reduce ethanol injury to dispersed chief cells from guinea pig stomach

1989 ◽  
Vol 256 (4) ◽  
pp. G704-G714 ◽  
Author(s):  
J. A. Cherner ◽  
L. Naik ◽  
A. Tarnawski ◽  
T. Brzozowski ◽  
J. Stachura ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Guinea Pig ◽  
Cell Injury ◽  
Protective Action ◽  
Control Cell ◽  
Ethanol Exposure ◽  
Fast Green ◽  
Chief Cells ◽  
Pepsinogen Secretion ◽  
Guinea Pig Stomach

To determine whether prostaglandin exerts a direct action on individual gastric epithelial cells that protects them from ethanol-induced injury, dispersed chief cells from guinea pig stomach were pretreated with 16,16-dimethyl-prostaglandin E2 (dmPGE2) or placebo before incubation with ethanol or control. Cell injury was assessed in terms of exclusion of Fast Green dye, release of lactate dehydrogenase, alterations of ultrastructure, and pepsinogen secretion stimulated by a variety of secretagogues. Of chief cells 60 +/- 2% were stained by Fast Green if incubated with 10% ethanol for 1 h after pretreatment with placebo, whereas only 38 +/- 1% of cells showed Fast Green staining when pretreated with 2.6 microM dmPGE2 before ethanol exposure. Similarly, 63 +/- 2% of cellular lactate dehydrogenase was released from chief cells pretreated with placebo compared with 36 +/- 4% of lactate dehydrogenase released from cells pretreated with 2.6 microM dmPGE2 (P less than 0.01). The prostaglandin's protective effect persisted throughout a 6-h incubation with ethanol. Scanning and transmission electron micrographs demonstrated disintegration of chief cells pretreated with placebo before ethanol exposure, whereas ultrastructural architecture was relatively preserved among chief cells pretreated with dmPGE2. Preincubation with 8 or 10% ethanol inhibited the subsequent stimulation of pepsinogen secretion caused by carbachol, cholecystokinin, A23187, 12-O-tetradecanoylphorbol 13-acetate, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. Pretreatment with dmPGE2 did not reduce the ethanol-induced inhibition of secretion stimulated by any of these secretagogues. These data indicate that dmPGE2 significantly reduces ethanol-induced damage to dispersed chief cells in terms of alterations of membrane permeability and ultrastructure but does not prevent the ethanol-induced impairment of pepsinogen secretion. These findings provide evidence that dmPGE2 exerts a direct but limited protective action on the gastric chief cell, independent of vascular, paracrine, or neural actions.

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