Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK.
Pflugers Arch. 1998 Jul;436(4):615-22. doi: 10.1007/s004240050679.
We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pHi) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H+ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50-7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pHi values, 7. 16+/-0.05 (n=10) and 7.17+/-0.06 (n=9) respectively in nominally CO2/HCO3--free saline. At both extremes of the pH range there were small discrepancies. At acidic pHi, 6.87+/-0.09 (n=5), the intracellular HPTS measurement differed by -0.08+/-0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pHi, 7. 32+/-0.10 (n=5), HPTS measurements produced pH values that differed by +0.07+/-0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77+/-15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 microM to 2 mM, did not block activity-dependent pHi changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00-8.00.
我们评估了基于芘的比率荧光染料 8-羟基芘-1,3,6-三磺酸（HPTS），并将其与玻璃 pH 敏感微电极一起使用，以测量电压钳蜗牛神经元中的细胞内 pH（pHi）。用丙酸进行细胞内酸化，以及激活 H+通道后碱化，使染料的校准可以与 pH 微电极在 pH 6.50-7.50 范围内进行比较。HPTS 在体外校准，玻璃 pH 敏感微电极产生相似的绝对静息 pHi 值，分别为 7.16+/-0.05（n=10）和 7.17+/-0.06（n=9），在名义上不含 CO2/HCO3-的盐水中。在 pH 范围的两个极端都存在小的差异。在酸性 pHi 时，6.87+/-0.09（n=5），细胞内 HPTS 测量值与 pH 敏感微电极测量值相差-0.08+/-0.03 pH 单位。在碱性 pHi 时，7.32+/-0.10（n=5），HPTS 测量值与 pH 敏感微电极的测量值相差+0.07+/-0.04 pH 单位。一些差异可以通过凹陷尖端 pH 敏感微电极的缓慢响应来解释（时间常数 77+/-15 s，n=3）。进一步的实验表明，当 HPTS 的细胞内浓度为 200 microM 至 2 mM 时，它不会阻断活性依赖的 pHi 变化。通过在一系列 HPTS-KCl 注射期间测量细胞内氯离子，计算细胞内 HPTS 浓度。将 HPTS 与相同浓度的 2',7'-双（2-羧乙基）-5,6-羧基荧光素（BCECF）进行比较，结果表明 HPTS 在 pH 6.00-8.00 范围内产生的比率变化更大。
