Liu F F, Diep K, Hill R P
Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, Canada.
Radiother Oncol. 1996 Jul;40(1):75-83. doi: 10.1016/0167-8140(96)01733-1.
We have demonstrated previously the relationship between intracellular pH (pHi) level and heat survival in mammalian cells. To explore this in further detail, we studied thermosensitivity in CCL 39 and their variant PS120 cells, which lack Na+/H+ antiport function.
CCL39 and PS120 cells were heated with or without amiloride, or 5-(N-ethyl(-N-isopropyl) amiloride (EIPA), inhibitors of Na+/H+ antiport function. Antiport activity and pHi measurements were made using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). A clonogenic assay was used to assess survival after heating.
Enhanced cytotoxicity was observed when CCL39 cells were heated with either EIPA (15 microM) or amiloride (2.5 mM) at pHe7.3 in the presence of NaHCO3. Under the same conditions, thermal enhancement of PS120 cells was observed only with amiloride at 2.5 mM. When the cells were heated at pHe 6.5 in bicarbonate-free medium, both EIPA and amiloride enhanced thermal cytotoxicity in CCL39 cells, but only the higher dose of amiloride sensitized the variant PS120 cells. Surviving fraction was related to pHi, but the data fell into two clusters, depending on whether or not both Na+/H+ antiport and the Na(+)-dependent HCO3-/Cl- exchangers were functioning.
We confirm that Na+/H+ antiport function can mediate thermosensitivity, and corroborate a linear correlation between pHi level and log survival after heating, but suggest that this relationship is complicated by other factors such as membrane exchanger function, and extracellular pH levels during heating.
我们之前已经证明了哺乳动物细胞内pH（pHi）水平与热耐受性之间的关系。为了更详细地探究这一点，我们研究了CCL 39细胞及其缺乏Na+/H+逆向转运功能的变体PS120细胞的热敏感性。
用或不用氨氯吡脒或5-（N-乙基-N-异丙基）氨氯吡脒（EIPA，Na+/H+逆向转运功能抑制剂）对CCL39和PS120细胞进行加热处理。使用荧光染料2,7-双（羧乙基）-5（6）-羧基荧光素（BCECF）进行逆向转运活性和pHi测量。采用克隆形成试验评估加热后的细胞存活率。
在存在NaHCO3的情况下，当CCL39细胞在pH7.3条件下用EIPA（15 microM）或氨氯吡脒（2.5 mM）加热时，观察到细胞毒性增强。在相同条件下，仅在2.5 mM氨氯吡脒存在时观察到PS120细胞的热增强作用。当细胞在无碳酸氢盐培养基中于pH6.5条件下加热时，EIPA和氨氯吡脒均增强了CCL39细胞的热细胞毒性，但只有较高剂量的氨氯吡脒使变体PS120细胞敏感。存活分数与pHi相关，但数据分为两组，这取决于Na+/H+逆向转运和Na+依赖性HCO3-/Cl-交换体是否都在发挥作用。
我们证实Na+/H+逆向转运功能可介导热敏感性，并证实pHi水平与加热后的对数存活率之间存在线性相关性，但表明这种关系因其他因素如膜交换体功能和加热期间的细胞外pH水平而变得复杂。
