Sodium fluxes in human fibroblasts: Effect of serum,Ca+2, and amiloride

Human fibroblasts that have been serum deprived for 4 hours have a digitoxin-insensitive Na influx of 9.5 ± 1.0 (n = 4) μmol/g prot/min which is not significantly different from the influx of 9.4 ± 0.6 (n = 3) μmol/g prot/min measured in cells arrested in the G₁/G₀ state by serum-deprivation for a period of four days. The Na influx in serum-deprived cells is rapidly stimulated (within one minute) simply by assaying the cells in medium containing 10% fetal bovine serum (FBS). The digitoxin-insensitive Na influx for cells in the presence of 10% FBS is 22.9 ± 1.1 (n = 6) μmol/g prot/min. the stimulation of Na influx in serumdeprived cells can also be achieved by the addition of the purified mitogen, epidermal growth factor (EGF). Addition of EGF to serum-deprived cells gives a maximal stimulation of Na influx of approximately 1.6-fold, with the concentration for half-maximal stimulation being 7.5 ng/ml. The stimulation of Na influx results from the activation of an amiloride-sensitive pathway, which appears to be minimally active in serum-deprived cells. Kinetic analysis of Na influx experiments in the presence of 10% FBS and varying concentrations of amiloride indicate that at infinite concentrations of amiloride the Na flux would be reduced to 8.9 μmol/g prot/min, which is comparable to the level of Na flux measured in serum-deprived cells in the presence of 5 mM amiloride. Thus, amiloride can totally inhibit the serum-stimulated component of Na influx while inhibiting less than 10% of the Na influx in serum-deprived cells. The Na influx in serum-deprived cells can also be stimulated 2.5-fold by preincubating cells in the presence of the Ca⁺ ionophore A23187 to elevate the intracellular Ca content. This stimulation of Na influx by intracellular Ca⁺² can be virtually eliminated by adding 1 mM amiloride.