Sasaki S, Ishibashi K, Nagai T, Marumo F
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Biochim Biophys Acta. 1992 Oct 6;1137(1):45-51. doi: 10.1016/0167-4889(92)90098-v.
Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.
使用荧光染料2',7'-双(2-羧乙基)-5(6)-羧基荧光素（BCECF）对非洲爪蟾卵母细胞的细胞内pH值（pHi）进行光学测量。通过与膜通透性形式（BCECF-AM）孵育使卵母细胞加载染料。在pH 7.6溶液中卵母细胞的平均pHi为7.69。环境pCO2增加会迅速降低pHi，估计缓冲能力为23.8 mM/pH单位。将环境HCO3-从5 mM改变为30 mM不会改变pHi。在无Na+溶液中孵育后，向浴中添加Na+会迅速增加pHi，并且这种反应被氨氯吡脒阻断（半数有效浓度为2 μM）。向浴中添加NH4Cl会导致pHi最初短暂升高，随后出现二次下降。二次下降被组氨酸特异性试剂焦碳酸二乙酯极大地抑制。它也被哇巴因、Ba2+和呋塞米轻微抑制，但不被氨氯吡脒抑制。这些数据表明：（1）荧光技术适用于非洲爪蟾卵母细胞的pHi测量；（2）非洲爪蟾卵母细胞具有氨氯吡脒敏感的Na+/H+交换以及对CO2、NH3和NH4+的通透性。这些观察结果可能有助于研究pHi与卵母细胞发育之间的关系以及非洲爪蟾卵母细胞中酸碱转运体的表达。
