Perturbation of Cell pH Regulation by H2O2 in Renal Epithelial Cells

Kaufman, Dorit; Goligorsky, Michael S.; Nord, Edward P.; Graber, Mark L.

doi:10.1006/abbi.1993.1206

Cited by 37 publications

(34 citation statements)

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“…In contrast, the pH-sensitive LysoTracker was released slowly, consistent with lysosomal alkalinization as reported previously in peroxide-treated cells. 35 Upon withdrawal of the stimulus, lysosomes quickly recaptured LysoTracker, further indication of the absence of membrane disruption under these conditions (Figure 6c). Lysosomal integrity in the presence of peroxide was also confirmed in ER-depleted/FCCP-treated HeLa cells (not shown).…”

Section: Acidic Compartmentsmentioning

confidence: 85%

A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

et al. 2004

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Oxidative stress releases intracellular calcium, which plays a pathogenic role in mammalian cell death. Here we report a search for the source of oxidative calcium in HeLa cells based on confocal epifluorescence microscopy. H 2 O 2 caused a rapid increase in cytosolic calcium, which was followed by mitochondrial Ca 2 þ loading. Combined mitochondrial uncoupling with full depletion of thapsigargin-sensitive stores abrogated inositol 1,4,5-trisphosphate-mediated calcium release but failed to inhibit H 2 O 2 -induced calcium release, observation that was confirmed in MDCK cells. Prevention of peroxide-induced acidification with a pH clamp was also ineffective, discarding a role for endosomal/lysosomal Ca 2 þ / H þ exchange. Lysosomal integrity was not affected by H 2 O 2 . Mature human erythrocytes also reacted to peroxide by releasing intracellular calcium, thus directly demonstrating the cytosolic source. Glutathione depletion markedly sensitized cells to H 2 O 2 , an effect opposite to that achieved by DTT. Iron chelation was ineffective. In summary, our results show the existence of a previously unrecognized sulfhydrylsensitive source of pathogenic calcium in the cytosol of mammalian cells.

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Section: Acidic Compartmentsmentioning

confidence: 85%

A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

et al. 2004

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“…Transepithelial resistance and intracellular ATP levels fall after exposure to hydrogen peroxide, whereas the permeability for small solutes across the monolayer increases (6, 7). Initially, a reduced intracellular ATP level was thought to cause the disruption of the junction (36), but later studies identified other potential regulators such as intracellular calcium activity (37), pH (38), PKC (39), and calcium-independent phospholipase A 2 (40) during oxidative stress. Until now, the mechanisms of tight junction reassembly after oxidative stress that lead to a reconstituted and functional epithelial cell barrier have not been investigated.…”

Section: Discussionmentioning

confidence: 99%

Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity

Meyer

Schwesinger

et al. 2001

Journal of Biological Chemistry

118

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Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. Many disease states of the kidney such as ischemia/reperfusion, inflammation, or toxic injury to the kidney and gut lead to loss of the epithelial barrier. Reactive oxygen species are directly involved in the pathophysiology of some of these diseases. Targets for hydrogen peroxide (H 2 O 2 ) 1 include DNA, proteins, and lipids. Reported effects of hydrogen peroxide on renal epithelial cell lines include DNA damage with induction of apoptosis or necrosis (1-3), decreased activity of membrane transporters (4), and membrane lipid peroxidation (5). Hydrogen peroxide decreases transepithelial resistance (TER) and increases transcellular permeability of MDCK cell monolayers (6, 7), which is also well documented in non-kidney epithelial cell lines (8 -11) and endothelial cell lines (12-15). However, the biochemical and subcellular effects of hydrogen peroxide on tight junction (TJ) proteins have not been studied, nor have they been distinguished from effects due to partial ATP depletion. Short-term depletion and repletion of intracellular ATP in cultured cells has been used to study the disassembly and/or reassembly of junctions as a model of organ ischemia and reperfusion (16 -20). However, little is known about the reassembly of the tight junction after exposure to reactive oxygen species due to the lack of a reproducible model system. We have now developed and analyzed a model of reversible hydrogen peroxide-induced disassembly and reassembly of the TJ in vitro and show that the reassembly pathway, as monitored by physiological, biochemical, and immunocytochemical parameters, depends upon tyrosine kinase activity. Furthermore, the cellular and biochemical consequences of H 2 O 2 exposure are distinct from those caused by ATP depletion-repletion. EXPERIMENTAL PROCEDURESCell Culture and Materials-Madin-Darby canine kidney cells (MDCK, type II) were obtained from American Type Tissue Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 50 IU/ml penicillin, and 50 g/ml streptomycin. Cells were incubated at 37°C in 5% CO 2 and were passaged weekly. DMEM was from Cellgro (Herndon, VA), streptomycin, penicillin, and fetal calf serum from Sigma. Plasticware was from ...

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“…Therefore, extensive PARP activation can consume a large amount of NAD and release a substantial amount of protons in a very short period; this may have the potential to cause cellular acidification after DNA damage. Indeed, oxidative damage that also activates PARP has earlier been shown to cause rapid acidification of certain cells that are pathophysiologically exposed to oxidants, such as cardiac myocytes or myoblasts (14), aortic endothelial cells (15,16), renal epithelial cells (17), and hippocampal neurons (18). Neuronal acidification has also been reported in other models that indirectly implicate oxidant damage, such as cerebral ischemia (19) or treatment with N-methyl-D-glucamine (20).…”

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confidence: 99%

“…Neuronal acidification has also been reported in other models that indirectly implicate oxidant damage, such as cerebral ischemia (19) or treatment with N-methyl-D-glucamine (20). In the majority of these studies, acidification was attributed to inhibition of the proton export mainly through Na ϩ ͞H ϩ exchangers (NHE) (15)(16)(17) or through the neuronal Ca 2ϩ ͞H ϩ exchanger (20). In other studies, acidification was suggested to be caused by passive influx of protons or release of protons from other subcellular compartments (17) or caused by oxidant-mediated inhibition of glycolysis and resultant hydrolysis of ATP (14).…”

mentioning

confidence: 99%

“…In the majority of these studies, acidification was attributed to inhibition of the proton export mainly through Na ϩ ͞H ϩ exchangers (NHE) (15)(16)(17) or through the neuronal Ca 2ϩ ͞H ϩ exchanger (20). In other studies, acidification was suggested to be caused by passive influx of protons or release of protons from other subcellular compartments (17) or caused by oxidant-mediated inhibition of glycolysis and resultant hydrolysis of ATP (14). Whereas these studies focused on the role of oxidative damage in acidification, we examined whether acidification is a unique response to oxidants or whether it would occur with other DNA-damaging agents that activate PARP.…”

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confidence: 99%

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Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

Affar

Shah

Dallaire

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate ␤-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of ␤-nicotinamide adenine dinucleotide.

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Perturbation of Cell pH Regulation by H2O2 in Renal Epithelial Cells

Cited by 37 publications

References 0 publications

A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity

Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

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