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Intracellular pH (pH(i)) 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 pH(i). In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 microM) 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 pH(i) acidification during RVD. The hypotonicity-induced acidification was significantly attenuated by 100 microM 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 HCO(3)(-) efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pH(i) during RVD of human cervical cancer cells.

Type

Journal article

Journal

FEBS Lett

Publication Date

13/02/2002

Volume

512

Pages

52 - 58

Keywords

Anion Exchange Protein 1, Erythrocyte, Cell Size, Cervix Uteri, Female, Homeostasis, Humans, Hydrogen-Ion Concentration, Models, Biological, Neoplasm Proteins, Sodium-Hydrogen Antiporter, Uterine Cervical Neoplasms