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Anion exchanger isoform 2 operates in parallel with Na+/H+ exchanger isoform 1 during regulatory volume decrease of human cervical cancer cells

M. Shen,R. Wilkins,C. Chou,J. Ellory

Published 2002 in FEBS Letters

ABSTRACT

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.

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