Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells

• Published in: Redox biology Vol. 15; no. C; pp. 394 - 404
• Main Authors: Teixeira, José, Basit, Farhan, Swarts, Herman G., Forkink, Marleen, Oliveira, Paulo J., Willems, Peter H.G.M., Koopman, Werner J.H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2018; Elsevier
• Subjects: Acidosis; Acids - metabolism; Acids - pharmacology; Animals; Antimycin A - pharmacology; Cell Survival - drug effects; Cellular Microenvironment - drug effects; Cyclohexylamines - metabolism; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Membrane potential; Membrane Potential, Mitochondrial - drug effects; Mitochondria; Mitochondria, Heart - drug effects; Mitochondria, Heart - metabolism; Permeability transition pore; Phenylenediamines - metabolism; Protein Carbonylation - drug effects; Reactive Oxygen Species - metabolism; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Research Paper; Rotenone - pharmacology; AA; CI; OXPHOS; TOC; Nec-1; pHc; pHe; mPTP; CsA; Mitochondria; TCA; TMRM; Δψ; ETC; HEt; ROT; ROS; CIII; Membrane potential; Permeability transition pore; Acidosis; Fer-1
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2017.12.018

The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Δψ), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant α-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism. [Display omitted] •Extracellular acidification induces mitochondrial dysfunction.•Extracellular acidification increases intracellular ROS levels.•Extracellular acidification stimulates protein carbonylation.•Extracellular acidification induces mPTP opening- and ROS-dependent cell death.•Acidosis-induced oxidative stress likely contributes to various pathologies.