Intracellular pH (pHi) homeostasis was investigated in human cervical cancer SiHa cells undergoing regulatory volume decrease (RVD) to determine which transport systems were involved. Using isoform-specific primers, mRNA transcripts of Na+/H+ exchanger isoform 1 (NHE1) and isoform 3 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) and the results confirmed by Western immunoblotting. From anion exchanger isoforms 1-3 (AE1-3), only the mRNA transcript of AE2 was identified by RT-PCR and the identity was confirmed by digestion with a specific restriction endonuclease. SiHa cells loaded with the fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein and resuspended in isotonic media showed a stable pHi. In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 μM) and amiloride (1 mM), showed a similar potency in enhancing the rate and extent of the hypotonicity-induced internal acidification. The absence of extracellular Na+ also substantially enhanced the acidification during RVD. These results suggest that internal acidification during RVD is mainly compensated by the operation of NHE1. Extracellular Cl- was critically necessary for the pHi acidification during RVD. The hypotonicity-induced acidification was significantly attenuated by 100 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, a concentration inhibiting more than 90% AE activity. This indicates that AE2 mediates a net Cl- influx with compensating HCO3- efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pHi during RVD of human cervical cancer cells.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology
- Cell Biology