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

Abstract

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.

The Cytoplasmic pH Influences Hyphal Tip Growth and Cytoskeleton-Related Organization

“…Upon refilling the hypha, tip swelling and subapical branching could be seen (R-U), followed by growth recovery. Bar,5 µm. redistribute between the cytosol and other cellular substances, such as proteins and lipids, resulting in a shift of its pKa (Opitz et al, 1994). However, the facts that dyeloaded cells with no visible sequestration were obtained and that the dye ratios changed predictably with acetic acid treatment indicate that we were estimating intracellular pH.…”

EVALUATION PROCEDURE FOR INTRACELLULAR pH DETERMINATIONS BASED ON ION SENSITIVE FLUOROPROBES AND CONFOCAL LASER MICROSCOPY

“…As reported previously (1), intracellular pH measurements (pHi) using the fluorescent pH-indicator carboxy-SNARF-1 and confocal laser scanning microscopy (CLSM) are afFected by pH-dependent redistribution processes of the protonated indicator component between cytosol and lipophilic cell compartments (e.g. plasma membrane) causing considerable changes of the intracellular calibration characteristic (äs compared to an in vitro calibration) äs well äs an apparent pK-shift of up to about l pH-unit (in human malignant glioma cells grown äs multicellular spheroids (U 118 MG)) associated with a corresponding alkaline shift of the intracellular pH measuring ränge.…”

Mitochondrial involvement in aspirin-induced apoptosis in yeast

“…The basis of this equation is discussed in Opitz et al (1994). Both test ratios and calibration curves showed appreciable experimental variation in each set of incubation conditions.…”