Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.