Preparation of individual human diploid fibroblasts and study of ion transport

Abstract

A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.

Patulin‐induced ion flux in cultured renal cells and reversal by dithiothreitol and glutathione: A scanning electron microscopy (sem) X‐ray microanalysis study

“…The rupture of terminal blebs has been shown to result in a rapid increase in nonspecific permeability to organic cations and an- ions and is coincident with cell death (26). Ouabain has been reported to induce blebbing in intact kidney (27), and both ouabain (18,23) and patulin (28) have been reported to induce volume changes in cultured cells at high doses. We can find no report, however of either ouabain-or patulin-induced blebbing in cultured cells.…”

“…Excess water was wicked off using filter paper, taking care to avoid contact with the cells. This method was chosen because it has been shown to effectively remove extracellular ions without doing osmotic damage to the cells (18). Coverslips were air dried in a dust-free environment, mounted on aluminum stubs using a carbon-base adhesive, and carbon coated in a vacuum evaporator.…”

“…For this reason, unless indicated otherwise, the data for sodium and potassium are normalized to phosphorus. The decision was made to use phosphorus rather than sulfur because phosphorus has been used to normalize results in other studies (18,20). The chloride ion was also easily detected by the X-ray method, but the variability was so great that it was not possible to assess the effects of patulin or ouabain on chloride content.…”

Measure of carbon and nitrogen stable isotope ratios in cultured cells

“…Silicon chips with attached cells were quickly washed in stirred distilled water (4°C) and then quenched in liquid nitrogen [24]. The samples were kept in liquid nitrogen at Ϫ196°C until they could be transferred to a custom-built freeze dryer pre-cooled to Ϫ80°C to prevent remodelling of water crystals in the cells.…”

Electron microprobe analysis of ouabain-exposed ciliary epithelium: PE-NPE cell couplets form the functional units

“…Although it is not possible to estimate ion concentrations in millimoles per liter from these data, the changes in intracellular contents of (Na ϩ K) or of (Na ϩ K ϩ Cl) must reflect changes in intracellular water content (1). For this reason, the sums of the measured ratios (Na/P ϩ K/P) are illustrated on occasion.…”