Bile acid-induced depolarization of mitochondrial membrane potential preceding cell injury in cultured gastric mucosal cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1–4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR. NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

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Mitochondrial KATP channel activation reduces anoxic injury by restoring mitochondrial membrane potential

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1295 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1295-H1303 ◽

Cited By ~ 62

Author(s):

Meifeng Xu ◽

Yigang Wang ◽

Ahmar Ayub ◽

Muhammad Ashraf

Keyword(s):

Membrane Potential ◽

Cytochrome C ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion ◽

Selective Inhibitor ◽

Atp Depletion ◽

Channel Activation

Mitochondrial membrane potential (ΔΨm) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K+ (mitoKATP) channel activation prevents the collapse of ΔΨm in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed ΔΨm, as monitored by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome cimmunostaining. Activation of mitoKATP channel with diazoxide (100 μmol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized ΔΨm. This protection was blocked by 5-hydroxydecanoate (500 μmol/l), a mitoKATPchannel-selective inhibitor, but not by HMR-1098 (30 μmol/l), a putative sarcolemmal KATP channel-selective inhibitor. Dissipation of ΔΨm also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts ΔΨm and induces apoptosis, which are prevented by the activation of the mitoKATP channel. This further emphasizes the therapeutic significance of mitoKATP channel agonists in the prevention of ischemia-reperfusion cell injury.

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Rise in cytosolic Ca2+ and collapse of mitochondrial potential in anoxic, but not hypoxic, rat proximal tubules.

Journal of the American Society of Nephrology ◽

10.1681/asn.v7112348 ◽

1996 ◽

Vol 7 (11) ◽

pp. 2348-2356

Author(s):

S M Peters ◽

M J Tijsen ◽

R J Bindels ◽

C H Van Os ◽

J F Wetzels

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Cell Damage ◽

Energy State ◽

Oxygen Deprivation ◽

Proximal Tubules ◽

Rhodamine 123 ◽

Anoxic Conditions

It has been suggested that ischemic renal proximal tubular cell injury is mediated by an increase in cytosolic calcium concentrations ((Ca2+)i). However, measurements of (Ca2+)i in rat or rabbit proximal tubules exposed to hypoxia or anoxia have yielded ambiguous results. This study explored the possibility that the severity of oxygen deprivation and the energy state of the mitochondria are important determinants of (Ca2+)i. To this end, (Ca2+)i (measured with fura-2) and the mitochondrial membrane potential (measured with rhodamine 123) were studied simultaneously in individual rat proximal tubules in hypoxic and anoxic conditions. (Ca2+)i did not change during hypoxia, but increased rapidly during anoxia. Increases in (Ca2+)i were only observed in parallel with a decrease of rhodamine 123 fluorescence, which indicates a collapse of the mitochondrial membrane potential. The increase in (Ca2+)i during anoxia was prevented by incubating the tubules in a low Ca2+ medium, which did not interfere with the collapse of the mitochondrial membrane potential. Both hypoxic and anoxic incubation led to cell death, as assessed by the fluorescent dye propidium iodide. These results clearly demonstrate that the level of oxygen deprivation is critical in determining changes in (Ca2+)i. Because cell damage occurred in both hypoxic and anoxic conditions. It was concluded that an increase in (Ca2+)i is not a necessary prerequisite for the development of ischemic cell injury.

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A digitized-fluorescence-imaging study of mitochondrial Ca2+ increase by doxorubicin in cardiac myocytes

Biochemical Journal ◽

10.1042/bj2810871 ◽

1992 ◽

Vol 281 (3) ◽

pp. 871-878 ◽

Cited By ~ 38

Author(s):

E Chacon ◽

R Ulrich ◽

D Acosta

Keyword(s):

Membrane Potential ◽

Energy Conservation ◽

Fluorescence Imaging ◽

Cardiac Myocytes ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Cultured Cells ◽

Imaging Study

The objective of the present study was to investigate the role of mitochondrial Ca2+ in doxorubicin-induced cell injury. The effect of doxorubicin on cultured cells was investigated by digitized fluorescence imaging. The Ca2+ sensitive fluorescent dye fura-2 was used to estimate cytosolic, mitochondrial and total cellular Ca2+. Rhodamine 123 was used to estimate the mitochondrial membrane potential, and cellular ATP was determined by h.p.l.c. The data showed that doxorubicin induced greater-than-2-fold increases in mitochondrial Ca2+ before changes in cytosolic Ca2+ could be detected. An increase in mitochondrial Ca2+ paralleled the observed dissipation in mitochondrial membrane potential. Cellular ATP levels appeared to decrease as a result of mitochondrial dysfunction, which in turn produced greater-than-2-fold increases in cytosolic Ca2+. The data suggest that doxorubicin-induced alterations in mitochondrial Ca2+ homoeostasis are associated with a dissipation in energy conservation, which may result in cell injury.

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Tauro-β-muricholic acid restricts bile acid-induced hepatocellular apoptosis by preserving the mitochondrial membrane potential

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.07.029 ◽

2012 ◽

Vol 424 (4) ◽

pp. 758-764 ◽

Cited By ~ 23

Author(s):

Gerald Ulrich Denk ◽

Carl Philipp Kleiss ◽

Ralf Wimmer ◽

Timo Vennegeerts ◽

Florian Paul Reiter ◽

...

Keyword(s):

Bile Acid ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Hepatocellular Apoptosis

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Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes

Nature ◽

10.1038/325078a0 ◽

1987 ◽

Vol 325 (6099) ◽

pp. 78-81 ◽

Cited By ~ 388

Author(s):

John J. Lemasters ◽

James DiGuiseppi ◽

Anna-Liisa Nieminen ◽

Brian Herman

Keyword(s):

Cell Death ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Preceding Cell

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Apaf-1, Bcl-xL, Cytochrome c, and Caspase-9 Form the Critical Elements for Cerebral Vascular Protection by Erythropoietin

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000050061.57184.ae ◽

2003 ◽

Vol 23 (3) ◽

pp. 320-330 ◽

Cited By ~ 37

Author(s):

Zhao Zhong Chong ◽

Jing-Qiong Kang ◽

Kenneth Maiese

Keyword(s):

Membrane Potential ◽

Cytochrome C ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Vascular System ◽

Dna Integrity ◽

Vascular Protection ◽

Caspase 9 ◽

Cerebral Vascular

Erythropoietin (EPO) plays a prominent role in the regulation of the hematopoietic system, but the potential function of this trophic factor as a cytoprotectant in the cerebral vascular system is not known. The authors examined the ability of EPO to modulate a series of death-related cellular pathways during free radical–induced injury in cerebral microvascular endothelial cells (ECs). Endothelial cell injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, apoptotic protease–activating factor-1 (Apaf-1), and Bcl-xL expression, mitochondrial membrane potential, cytochrome c release, and cysteine protease activity. They show that constitutive EPO is present in ECs but is insufficient to prevent cellular injury. Signaling through the EPO receptor, however, remains biologically responsive to exogenous EPO administration to offer significant protection against nitric oxide–induced injury. Exogenous EPO maintains both genomic DNA integrity and cellular membrane asymmetry through parallel pathways that prevent the induction of Apaf-1 and preserve mitochondrial membrane potential in conjunction with enhanced Bcl-xL expression. Consistent with the modulation of Apaf-1 and the release of cytochrome c, EPO also inhibits the activation of caspase-9 and caspase-3–like activities. Identification of novel cytoprotective pathways used by EPO may serve as therapeutic targets for cerebral vascular disease.

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Circ_0030235 knockdown protects H9c2 cells against OGD/R-induced injury via regulation of miR-526b

PeerJ ◽

10.7717/peerj.11482 ◽

2021 ◽

Vol 9 ◽

pp. e11482

Author(s):

Yuquan Zhang ◽

Shuzhu Liu ◽

Limin Ding ◽

Dawei Wang ◽

Qiangqiang Li ◽

...

Keyword(s):

Membrane Potential ◽

Cell Viability ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Glucose Deprivation ◽

Luciferase Reporter ◽

Circular Rnas ◽

H9c2 Cells ◽

Related Factors

Backgrounds Acute myocardial infarction (MI) is the common clinical manifestation of coronary heart disease. Circular RNAs (circRNAs) act key roles in cardiomyocytes growth and angiogenesis. However, their functions in MI are not entirely clear. This research intended to investigate the role and underlying mechanisms of circ_0030235 in H9c2 cells. Methods H9c2 cells were conducted to oxygen glucose deprivation/reperfusion (OGD/R) inducement to establish the MI model. Circ_0030235 and miR-526b expression was tested and altered by qRT-PCR and transfection. Cell viability, apoptosis and reactive oxygen species (ROS) injury were tested by CCK-8 assay, TUNEL assay kit, and ROS Detection Assay Kit, respectively. Assessment of cell injury-related factors was performed by employing ELISA, Mitochondrial Viability Staining and the JC-1-Mitochondrial Membrane Potential Assay Kit. The relationship between circ_0030235 and miR-526b was analyzed by dual luciferase reporter assay. The expression of key proteins was analyzed by western blot. Results Circ_0030235 was highly expressed in OGD/R-induced H9c2 cells. OGD/R inducement cell viability, while accelerated apoptosis. Besides, the level ROS, cell injury-related factors, mitochondrial membrane potential were notably elevated by OGD/R inducement, while mitochondrial viability was remarkably declined. Whereas, these impacts were all noticeably remitted by circ_0030235 knockdown. miR-526b was a target of circ_0030235. Circ_0030235 knockdown-induced impacts were all notably abrogated by miR-526b inhibition, including the activating impacts on PI3K/AKT and MEK/ERK pathways. Conclusions This research implied that circ_0030235 knockdown might remit OGD/R-induced impacts via activation of PI3K/AKT and MEK/ERK pathways and regulation of miR-526b.

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Intramitochondrial [Ca2+] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.2.h484 ◽

1998 ◽

Vol 275 (2) ◽

pp. H484-H494 ◽

Cited By ~ 19

Author(s):

T. J. Delcamp ◽

C. Dales ◽

L. Ralenkotter ◽

P. S. Cole ◽

R. W. Hadley

Keyword(s):

Membrane Potential ◽

Guinea Pig ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Cell Injury ◽

Ventricular Myocytes ◽

Confocal Imaging ◽

Mitochondrial Depolarization ◽

Intracellular Ca ◽

A Cell

The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+ concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.

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