Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells

Abstract

Background/Aims: To functionally characterize intracellular pH (pH_i) regulating mechanisms, such as Na⁺-H⁺ exchanger (NHE) and Na⁺-HCO₃^- co-transporter (NBC), and further examine effects of ethanol on the pH_i regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells. Methods: OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pH_i were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique. Results: i) the steady-state pH_i value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pH_i recovery following induced-acidification was totally blocked by either removing [Na]_o⁺ or adding HOE 694 (a NHE1 specific inhibitor), which demonstrates existence of NHE1; iii) in HCO₃^-/CO₂-buffer system, the pH_i recovery following induced-acidification was entirely blocked by either removing [Na]_o⁺ or adding HOE 694 plus DIDS (a NBC specific inhibitor), which suggests existence of Na⁺- and HCO₃^–dependent acid-extruder, i.e. NBC; iv) the isoforms of the two acid extruders were NHE1, NBCn1, NBCe1 and NDCBE; v) ethanol (10-1000 mM) showed a biphasic and concentration-dependent effect on resting pH_i (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (<unterline>></unterline> 300 mM) significantly inhibited the expression of NHE1, NBCn1 and NDCBE, while up-regulated NBCe1. Conclusions: Ethanol affects pH_i in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.