Modest intracellular acidification suppresses death signaling in ouabain-treated cells
Olga A Akimova1, Dimitri Pchejetski, Pavel Hamet
1Centre de recherche, Centre hospitalier de l'Université de Montréal, CHUM - Hôtel-Dieu, Montreal, Canada.
Abstract:
The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na(+), K(+) -ATPase, but independently of inhibition of Na(+), K(+) -ATPase-mediated ion fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.4 to 7.0, i.e. under conditions when pH(i) was decreased from approximately 7.2 to 6.9. The rescue of ouabain-treated C7-MDCK cells was also detected under selective intracellular acidification caused by inhibition of Na(+)/H(+) exchanger. In these cells, neither Na(+), K(+) pump activity nor [(3)H]-ouabain binding was significantly affected by modest acidification. The death of ouabain-treated cells was independent of inhibition of RNA and protein synthesis with actinomycin D and cycloheximide. In contrast, both compounds sharply attenuated the protective action of acidified medium. Thus, our results show that very modest intracellular acidification is sufficient to inhibit the Na(+) (i)/K(+) (i)-independent death signal triggered in epithelial and endothelial cells by CTS. They also suggest that the protective action of acidification is mediated by de novo expression of genes involved in inhibition of the cell death machinery.
Insights
Cardiotonic steroids (CTS) like ouabain trigger cell death independently of ion fluxes. Modest intracellular acidification protects epithelial and endothelial cells from CTS-induced death, suggesting a role for gene expression in this protective mechanism.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- The precise mechanisms underlying cell death induced by cardiotonic steroids (CTS), such as ouabain, are not fully elucidated.
- Previous research indicated ouabain induces cell death in epithelial and endothelial cells through interaction with Na(+), K(+)-ATPase, independent of ion flux inhibition.
Purpose of the Study:
- To investigate the role of intracellular pH in ouabain-induced cell death.
- To explore the protective effects of modest intracellular acidification on CTS-treated cells.
Main Methods:
- Utilized Madin-Darby canine kidney (C7-MDCK) and porcine aortic endothelial cells.
- Manipulated extracellular and intracellular pH using medium acidification and Na(+)/H(+) exchanger inhibition.
- Assessed cell viability, Na(+), K(+)-ATPase activity, and [(3)H]-ouabain binding.
- Investigated the role of RNA and protein synthesis using actinomycin D and cycloheximide.
Main Results:
- Extracellular medium acidification from pH 7.4 to 7.0, leading to intracellular pH decrease, suppressed ouabain-induced death in C7-MDCK and endothelial cells.
- Intracellular acidification via Na(+)/H(+) exchanger inhibition also protected cells from ouabain toxicity.
- These protective effects were observed without significant alterations in Na(+), K(+)-ATPase activity or ouabain binding.
- The protective action of acidification was dependent on de novo RNA and protein synthesis.
Conclusions:
- Modest intracellular acidification effectively inhibits the Na(+)(i)/K(+)(i)-independent cell death pathway triggered by cardiotonic steroids in epithelial and endothelial cells.
- The protective mechanism appears to involve the de novo expression of genes that regulate the cell death machinery.
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