A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters.

Abstract

The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (GTE), is the result of cell volume decrease. CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission [F360/510]) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ([Ca2+]i). The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in [Ca2+]i, in F360/510, and in GTE. Hypertonicity increased, and hypotonicity decreased F360/510 and GTE, without having an effect on [Ca2+]i, and the changes in F360/510 and in GTE correlated linearly. Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F360/510 reflect changes in dye concentration. Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F360/510 reflect changes in size of the attached cells. Ionomycin increased [Ca2+]i, F360/510, and GTE, but the increases in [Ca2+]i preceded those in F360/510 and GTE. The calcium chelator BAPTA blocked the ionomycin-induced increase in [Ca2+]i, F360/510, and in GTE. Preincubation with 4-acetamido-4'isothiocyanatostilbene-2,2'disulfonic acid (SITS) augmented the ionomycin-induced increase in [Ca2+]i, but blocked the increases in F360/510 and in GTE. Pretreatment of cells with hypertonic solution abrogated the increases in F360/510 and in GTE in response to ionomycin, but had little effect on the ionomycin-induced increase in [Ca2+]i. On the basis of these results we suggest that the ionomycin-induced increase in GTE is mediated by [Ca2+]i-dependent chloride secretion and osmotic water loss.