Mitochondrial membrane potential and hydroethidine‐monitored superoxide generation in cultured cerebellar granule cells

Budd, Samantha L.; Castilho, Roger F.; Nicholls, David G.

doi:10.1016/s0014-5793(97)01088-0

Cited by 217 publications

(185 citation statements)

References 21 publications

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“…When ethidium is produced exceeding the binding capacity of mitochondrial DNAs, it binds genomic DNAs and gives rise to bright nuclear fluorescence signal. 51 In the present study, RGCs of ocular hypertensive mice revealed primarily cytoplasmic staining of DHE, suggesting that glaucoma or elevation of IOP evokes only moderate superoxide production.…”

Section: Discussionsupporting

confidence: 46%

Neuroglobin Is an Endogenous Neuroprotectant for Retinal Ganglion Cells against Glaucomatous Damage

Wei

Cho

et al. 2011

The American Journal of Pathology

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Neuroglobin (NGB), a newly discovered member of the globin superfamily, may regulate neuronal survival under hypoxia or oxidative stress. Although NGB is greatly expressed in retinal neurons, the biological functions of NGB in retinal diseases remain largely unknown. We investigated the role of NGB in an experimental model of glaucoma, a neurodegenerative disorder that usually involves elevation of intraocular pressure (IOP). Elevated IOP is thought to induce oxidative stress in retinal ganglion cells (RGCs), thereby causing RGC death and, eventually, blindness. We found that NGB plays a critical role in increasing RGC resistance to ocular hypertension and glaucomatous damage. Neuroglobin (NGB) is a novel member of the globin superfamily that is distantly related to hemoglobin and myoglobin. 1 A highly conserved protein in evolution, human and mouse Ngb, both comprised of 151 amino acids, are 94% identical. 2,3 In mammals, expression of NGB is found in the brain, retina, and other nerve tissues, predominantly in neurons but absent from glial cells. 4 -7 Distribution of NGB protein in the normal human retina is very similar to what has been described in mice: NGB is detected primarily in the plexiform layers and photoreceptor inner segments, which are rich in mitochondria and synapses and consume high amounts of oxygen. 8,9 NGB has been shown to act as an endogenous neuroprotective molecule that enhances neuronal survival under hypoxic-ischemic insults in the brain 10 -12 ; however, its physiological functions and molecular mode of actions are not fully understood. Of note, NGB is present at a 100-fold greater concentration in the retina than in the brain, 8,[13][14][15] suggesting that the retina may be its most important site of function; however, among the many unknown roles of NGB, functional significance of NGB expression in the retina has been largely unexplored.

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Section: Discussionsupporting

confidence: 46%

Neuroglobin Is an Endogenous Neuroprotectant for Retinal Ganglion Cells against Glaucomatous Damage

Wei

Cho

et al. 2011

The American Journal of Pathology

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“…Control wells received 5 mM glucose replacement (GR) immediately after the initial medium exchange. For cell culture studies, 5 mM dihydroethidium (Budd et al, 1997) was added to the cultures 30 minutes before GD. The cultures were photographed with a fluorescence microscope 1 hour after GR, and the digitized images were analyzed and expressed as percent of neurons with an Et signal at least 50% higher than background.…”

Section: Glucose Deprivation In Cell Culturesmentioning

confidence: 99%

Prevention of Acute/Severe Hypoglycemia-Induced Neuron Death by Lactate Administration

Won

Jang

Yoo

et al. 2012

J Cereb Blood Flow Metab

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Hypoglycemia-induced cerebral neuropathy can occur in patients with diabetes who attempt tight control of blood glucose and may lead to cognitive dysfunction. Accumulating evidence from animal models suggests that hypoglycemia-induced neuronal death is not a simple result of glucose deprivation, but is instead the end result of a multifactorial process. In particular, the excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) consumes cytosolic nicotinamide adenine dinucleotide (NAD + ), resulting in energy failure. In this study, we investigate whether lactate administration in the absence of cytosolic NAD + affords neuroprotection against hypoglycemiainduced neuronal death. Intraperitoneal injection of sodium L-lactate corrected arterial blood pH and blood lactate concentration after hypoglycemia. Lactate administered without glucose was not sufficient to promote electroencephalogram recovery from an isoelectric state during hypoglycemia. However, supplementation of glucose with lactate reduced neuronal death by B80% in the hippocampus. Hypoglycemia-induced superoxide production and microglia activation was also substantially reduced by administration of lactate. Taken together, these results suggest an intriguing possibility: that increasing brain lactate following hypoglycemia offsets the decrease in NAD + due to overactivation of PARP-1 by acting as an alternative energy substrate that can effectively bypass glycolysis and be fed directly to the citric acid cycle to maintain cellular ATP levels.

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“…It is well known that lipophilic cations (E + ) are sequestered into mitochondria in a membrane potential dependent manner [21]. As the triphenylphosphonium moiety targets these compounds to mitochondria [22], it is likely that Mito-HE oxidation products should also be able to bind to mitochondria.…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Zielonka

Srinivasan

Hardy

et al. 2008

Free Radical Biology and Medicine

137

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Here we report that ferricytochrome c (cyt c 3+ ) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX ™ Red) forming highly characteristic homo-and heterodimeric products. Using a HPLC-electrochemical (EC) method, several products were detected from cyt c 3+ -catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E + -E + ; Mito-HE-Mito-HE, Mito-E + -Mito-E + ) and heterodimers (HE-E + and Mito-HE-Mito-E + ), as well as the monomeric ethidium (E + ) and mito-ethidium (Mito-E + ). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c 3+ were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E + or Mito-E + , the dimeric analogs (E + -E + and Mito-E + -Mito-E + ) were not fluorescent. Superoxide ( ) or Fremy's salt react with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E + ) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c 3+ -dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.

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Mitochondrial membrane potential and hydroethidine‐monitored superoxide generation in cultured cerebellar granule cells

Cited by 217 publications

References 21 publications

Neuroglobin Is an Endogenous Neuroprotectant for Retinal Ganglion Cells against Glaucomatous Damage

Neuroglobin Is an Endogenous Neuroprotectant for Retinal Ganglion Cells against Glaucomatous Damage

Prevention of Acute/Severe Hypoglycemia-Induced Neuron Death by Lactate Administration

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

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