Modest intracellular acidification suppresses death signaling in ouabain-treated cells

Akimova, Olga A.; Pchejetski, Dimitri; Hamet, Pavel; Orlov, Sergei N.

doi:10.1007/s00424-005-1493-4

Cited by 14 publications

(16 citation statements)

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“…Recently, we discovered that the death of ouabaintreated renal epithelial and vascular endothelial cells can be abolished by modest extracellular acidification as well as by intracellular acidification caused by inhibition of the Na + /H + exchanger [33]. In the present study, we reproduced this observation (Fig.…”

Section: Effect Of Pma and Phsupporting

confidence: 90%

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

et al. 2008

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The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K+-free medium almost completely blocked Na+,K+-ATPase, increased Na (i) + content by approximately 10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K+-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na (i) + ,K (i) + -independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.

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Section: Effect Of Pma and Phsupporting

confidence: 90%

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

et al. 2008

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“…The impact of CTSs on cell viability was studied by cell attachment assay as described in detail elsewhere44. Briefly, the HUVEC were seeded into 6-well plates and treated as indicated above.…”

Section: Methodsmentioning

confidence: 99%

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

Климанова

Tverskoi

Кольцова

et al. 2017

Sci Rep

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Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies.

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“…However, it has to be noted that in epithelial Madin-Darby canine kidney (C7-MDCK) cells and vascular endothelial cells from porcine aorta (PAEC) mild intracellular acidification prevented cell death induced by ouabain and other cardiotonic steroids. The cell death-inducing action of these compounds was triggered via interaction with the Na + /K + ATPase but was independent of the inhibition of ion fluxes and the ratio of intracellular Na + to K + and is most are likely accompanied by the de novo synthesis of pH i -sensitive genes involved in the suppression of the cell death machinery [72]. …”

Section: Discussionmentioning

confidence: 99%

The Putative Role of the Non-Gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H⁺/K⁺ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

Jakab

Hofer

Ravasio

et al. 2014

Cell Physiol Biochem

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Background/Aims: The ATP12A gene codes for a non-gastric H⁺/K⁺ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H⁺/K⁺ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H⁺/K⁺ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pH_i) after acute intracellular acid load (NH₄Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H⁺/K⁺ ATPase inhibitor SCH28080 (100 µM) diminishes K⁺-dependent pH_i recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. Conclusion: In summary we show that in HL-60 cells ATP12A is a functionally active H⁺/K⁺ ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K⁺ ions and apoptotic volume decrease. Its expression and/or susceptibility to the H⁺/K⁺ ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.

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Modest intracellular acidification suppresses death signaling in ouabain-treated cells

Cited by 14 publications

References 50 publications

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

The Putative Role of the Non-Gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H⁺/K⁺ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

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Modest intracellular acidification suppresses death signaling in ouabain-treated cells

Cited by 14 publications

References 50 publications

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

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The Putative Role of the Non-Gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H⁺/K⁺ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells