Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1.

Properties and peculiarities of the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal laser scanning microscopy. It was found that in human malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between indicator calibration in cell-free solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the indicator between cytosol and lipophilic cell compartemants, e.g. plasma membrane, since the indicator can even be loaded efficiently into the cells via its active acid form (instead of the acetoxymethyl ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated indicator component results in an apparent pK shift of delta pK = log(1 + epsilon.P), with P the partition coefficient and epsilon a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.