Weintraub W H, Machen T E
Department of Physiology/Anatomy, University of California, Berkeley 94720.
Am J Physiol. 1989 Sep;257(3 Pt 1):G317-27. doi: 10.1152/ajpgi.1989.257.3.G317.
Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.
在Fu5（一种维持多种分化功能的大鼠肝癌细胞系）中研究了细胞内pH值（pHi）的调节。使用对pH敏感的染料2',7'-双羧乙基-5(6)-羧基荧光素（BCECF）的显微荧光测定法，测量安装在显微镜载物台上的流通室中盖玻片上生长的10 - 15个细胞的pHi。在N-2-羟乙基哌嗪-N'-2-乙磺酸（HEPES）缓冲溶液中，pHi为7.14，固有缓冲能力与pHi呈负相关。氨氯吡咪（0.1 mM）在4分钟内使pHi降低0.33个pH单位。酸负荷后的恢复（使用NH4预脉冲技术或无钠溶液）被氨氯吡咪完全阻断。在HCO3-CO2缓冲溶液中，pHi为7.15，缓冲能力在pHi为6.6至7.2之间对pHi相对不敏感。氨氯吡咪仅使pHi降低0.09个单位。酸负荷后的恢复依赖于钠，在无氯溶液中发生，并且被氨氯吡咪加0.5 mM二氢-4,4'-二异硫氰酸芪-2,2'-二磺酸（H2DIDS）的组合完全阻断；当去除氨氯吡咪或H2DIDS时恢复发生。去除外部氯导致快速的、可被H2DIDS阻断的碱化，在HCO3-CO2中比在HEPES中更快。无氯碱化松弛时Clout的表观Km为4.5 mM。无氯处理期间HCO3转运速率在碱性静息pHi时增加。结论是Fu5细胞有两种依赖钠的碱加载机制和一种酸加载的Cl-HCO3交换体。在含有HCO3-CO2的溶液中，Na-H交换体占酸负荷后恢复的约40%，其余由Na-HCO3共转运体负责。碱负荷后的恢复似乎通过Cl-HCO3交换体的活性发生。
