Felberbauer F X, Graf J
Department of General and Experimental Pathology, University of Vienna Medical School, Austria.
Pflugers Arch. 1991 Sep;419(2):215-7. doi: 10.1007/BF00373011.
The pH-sensitive fluorescent dye 1,3-dihydroxy-pyrene-6,8-disulfonic acid (DHPDS) was used to measure intracellular pH (pHi) from the surface fluorescence of the isolated perfused rat liver. Monochromatic light from a fluorometer was focused on the liver with a fiber optic, emitted light was collected with a second fiber bundle and returned to the spectrometer. To correct for changes in intracellular dye concentration, the excitation wavelength was changed between the pH-sensitive excitation peak wavelength and the isosbestic wavelength, then a ratio was computed between fluorescence intensities at these two wavelengths. Intracellular calibration of the dye was performed by clamping the intracellular to the extracellular pH with H+/K(+)-ionophore Nigericin. This method was used to monitor transient changes in intracellular pH caused either by addition and removal of NH4Cl or by changing perfusate CO2 and HCO3- concentrations while keeping their ratio constant. The effects of these maneuvers on bileflow were studied, too. Data obtained in the perfused liver were in good agreement with those obtained in isolated liver cells except that the steady-state pHi (7.46 +/- 0.02) was slightly higher than reported values. Measurements in livers of mutant TR- rats that are defective of dye secretion revealed similar pHi values, indicating that secretion of the dye into bile canaliculi did not affect measurements. The technique appears adequate to measure pHi in the liver and will allow to study pH regulatory mechanisms in the intact organ.
使用pH敏感荧光染料1,3 - 二羟基芘 - 6,8 - 二磺酸（DHPDS），通过分离灌注的大鼠肝脏表面荧光来测量细胞内pH值（pHi）。荧光计发出的单色光通过光纤聚焦在肝脏上，发射光由第二束光纤收集并返回光谱仪。为校正细胞内染料浓度的变化，在pH敏感激发峰波长和等吸收波长之间改变激发波长，然后计算这两个波长下荧光强度的比值。通过用H⁺/K⁺离子载体尼日利亚菌素将细胞内pH钳制到细胞外pH来进行染料的细胞内校准。该方法用于监测由添加和去除NH₄Cl或改变灌注液CO₂和HCO₃⁻浓度（同时保持其比例恒定）引起的细胞内pH的瞬态变化。还研究了这些操作对胆汁流量的影响。在灌注肝脏中获得的数据与在分离的肝细胞中获得的数据高度一致，只是稳态pHi（7.46±0.02）略高于报道值。在染料分泌有缺陷的突变TR - 大鼠肝脏中的测量显示出相似的pHi值，表明染料分泌到胆小管中不影响测量。该技术似乎足以测量肝脏中的pHi，并将有助于研究完整器官中的pH调节机制。
