Confocal laserscan microscopy with a dual-excitation device was used to record intracellular pH (pHi) regulation in rat proximal convoluted tubules microperfused in vivo. Cells were loaded with the pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Single cells could be distinguished within the tubules and separate measurements were possible. Application of an NH4Cl pulse by peritubular perfusion caused an immediate increase in intracellular pH. Intraluminal injection of NH4Cl led to a slower increase in intracellular pH. In both cases, cessation of perfusion led to an immediate acidification. Peritubular perfusion with 300 microM 4,4'-diisothiocanatodihydrostilbene-2,2'-disulphonic acid (H2DIDS) caused an intracellular alkalinisation. Microperfusion, pH-sensitive dyes and confocal laserscan microscopy provide a new non-invasive method to measure intracellular pH effectively in individual cells of near-surface structures of the intact kidney.
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