Lee Shiao-Pieng, Chao Shih-Chi, Huang Shu-Fu, Chen Yen-Lin, Tsai Yi-Ting, Loh Shih-Hurng
Division of Oral and Maxillofacial Surgery, Department of Dentistry, School of Dentistry, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan.
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Cell Physiol Biochem. 2018;47(5):2056-2068. doi: 10.1159/000491473. Epub 2018 Jul 5.
BACKGROUND/AIMS: To functionally characterize intracellular pH (pHi) regulating mechanisms, such as Na+-H+ exchanger (NHE) and Na+-HCO3- co-transporter (NBC), and further examine effects of ethanol on the pHi regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells.
OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pHi were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique.
i) the steady-state pHi value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pHi 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 pHi 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 pHi (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (
Ethanol affects pHi in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.
背景/目的：对细胞内pH（pHi）调节机制（如钠氢交换体（NHE）和钠-碳酸氢根协同转运体（NBC））进行功能特性分析，并进一步研究乙醇对人口腔表皮样癌（OEC-M1）细胞中pHi调节机制的影响。
OEC-M1细胞由三军总医院提供。使用pH敏感荧光染料BCECF通过显微分光荧光测定法检测pHi的变化。通过蛋白质印迹技术检测转运体的亚型。
i）在酸碱冲击后，稳态pHi值从碱性（7.35∼7.49）转变为酸性（7.0∼7.03）；ii）在HEPES缓冲系统中，去除细胞外[Na]⁺或添加HOE 694（一种NHE1特异性抑制剂）可完全阻断诱导酸化后pHi的恢复，这表明存在NHE1；iii）在HCO₃⁻/CO₂缓冲系统中，去除细胞外[Na]⁺或添加HOE 694加DIDS（一种NBC特异性抑制剂）可完全阻断诱导酸化后pHi的恢复，这表明存在依赖钠和碳酸氢根的酸排出体，即NBC；iv）两种酸排出体的亚型为NHE1、NBCn1、NBCe1和NDCBE；v）乙醇（10 - 1000 mM）通过相应改变NHE1和NBC的活性，对静息pHi呈现双相且浓度依赖性的影响（即先升高后降低）；vi）用乙醇处理24小时（<300 mM）可显著抑制NHE1、NBCn1和NDCBE的表达，同时上调NBCe1。
乙醇通过改变OEC-M1细胞中NHE1和NBC亚型的功能及表达，以浓度依赖性方式影响pHi。
