Fiber-optic measurement of intracellular pH in intact rat liver using pH-sensitive dyes

Abstract

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.