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.

Increased Alcohol Consumption in Mice Lacking Sodium Bicarbonate Transporter NBCn1

“…7 and 8). This downregulation is in line with alcohol-induced decrease in NBCn1 expression in aorta smooth muscles 42 and oral epidermoid carcinoma cells 43 . Acute alcohol exposure to these peripheral cells results in overall intracellular alkalosis in the presence of CO 2 /HCO 3 − , thus reducing acid extrusion.…”

“…Because NBCn1 is an acid extruder, its decrease after alcohol exposure is conceivable. However, this decrease will likely have variable impact on pH i regulation as these cells express other acid extruders such as V-ATPase, Na/H exchangers, and NBCe1, in addition to NBCn1 [42][43][44] . In contrast, in neurons where NBCn1 is the major acid extruder, NBCn1 downregulation will significantly cause intracellular acidification and subsequently depress neuronal excitability, as shown in KO neurons.…”

“…The above data from KO mice support the importance of NBCn1 for alcohol-related www.nature.com/scientificreports www.nature.com/scientificreports/ behaviors, but there is a limitation to understanding how NBCn1 dysfunction and subsequent intracellular acidosis in neurons are associated with alcohol consumption. Studies show that alcohol changes pH i in several peripheral cells [42][43][44] and this change is mediated by downregulation of acid-extruding proteins including NBCn1. These reports led us to the hypothesis that alcohol consumption downregulates NBCn1 in neurons.…”

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

“…The solution was replaced with a pipette instead of a perfusion system (including a peristaltic pump and suction). The experimental procedure is similar to microspectrofluorimetry, and the details are described in our previous study[23].…”

“…However, a reversed pH gradient of pH i ≥ 7.2 and pH e ≤ 7.1 has been demonstrated in cancer cells. This reversed pH gradient is caused the overexpression and increased set-point of the acid extrusion mechanism[12,20-23]. This dysregulated pH i feature further promotes tumor progression, invasion and metastasis[21,24-26].…”