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

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Abstract
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.