Köttgen M, Leipziger J, Fischer K G, Nitschke R, Greger R
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
Pflugers Arch. 1994 Sep;428(2):179-85. doi: 10.1007/BF00374856.
The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)
使用pH敏感荧光指示剂2'，7'-双羧乙基-5(6)-羧基荧光素（BCECF）对HT29结肠癌细胞中的pH调节进行了研究。在对照条件下，含HCO3(-)溶液中的细胞内pH（pHi）为7.21±0.07（n = 22），不含HCO3(-)溶液中的为7.21±0.09（n = 12）。HOE-694（10 μmol/l），一种有效的Na+/H+交换抑制剂，不影响对照pHi。作为使细胞酸化的一种方法，我们使用了NH4+/NH3（20 mmol/l）预脉冲技术。平均峰值酸化是0.37±0.07 pH单位（n = 6）。在不含HCO3(-)的溶液中，HOE-694（1 μmol/l）完全阻断了酸负荷后的恢复，而在含HCO3(-)的溶液中，需要HOE-694和4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐（DIDS，0.5 mmol/l）的组合才能显示相同的效果。在含HCO3(-)和不含HCO3(-)的溶液中，酸负荷后的恢复均依赖于Na(+)。去除外部Cl-会导致快速的、可被DIDS阻断的碱化，当外部Na+与Cl-一起去除时，碱化幅度为0.33±0.03 pH单位（n = 15）和0.20±0.006 pH单位（n = 5）。这种碱化在含HCO3(-)的溶液中比在不含HCO3(-)的溶液中更快。目前的观察结果表明HT29细胞中存在三种不同的pHi调节机制：（a）Na+/H+交换器，（b）HCO3-/Cl-交换器，（c）一种依赖Na(+)的HCO3-转运体，可能是Na(+)-HCO3-/Cl-反向转运体。（摘要截短于250字）
