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We have tested the hypothesis that the rapid stimulation of Na+/H+ exchange by epidermal growth factor (EGF) is a requirement for induction of mitogenesis. BALB/c 3T3 cells exposed for 4 hr at 37 degrees C to both EGF at 1 ng/ml and either 0.2-1 mM amiloride (an inhibitor of Na+/H+ exchange) or 10 microM MK-685 (an amiloride analog and more potent inhibitor of Na+/H+ exchange) incorporated no less [methyl-3H]thymidine during a 1-hr pulse 20 hr later than did cells exposed for 4 hr to EGF alone. Control experiments utilizing low external pH (to dissociate EGF from its receptor) and anti-EGF antibodies indicated that the failure of amiloride to inhibit mitogenesis when copresent with EGF during the first 4 hr was not due to incomplete removal of EGF and complete removal of amiloride at t4. Cells incubated with 200 microM amiloride for 24 hr showed nearly complete inhibition of stimulation by EGF. In comparison, cells incubated with 10 microM MK-685 for 24 hr showed only a slight inhibition of stimulation by EGF. Incubations with amiloride or MK-685 for shorter periods of time indicated that only amiloride inhibited mitogenesis and that this inhibition happened between 4 (t4) and 10(t10) hr after EGF addition, during which time increases in RNA and protein synthesis (required for mitogenesis) occurred. Amiloride inhibited both RNA and protein syntheses in intact cells during this prereplicative period, while MK-685 was without effect. We conclude that (i) inhibition of EGF-induced mitogenesis by amiloride is due not to inhibition of EGF-stimulated Na+/H+ exchange but rather to inhibition of necessary events occurring during the hours immediately prior to the onset of DNA synthesis, these events probably being RNA and protein synthesis and (ii) in cell culture medium buffered with CO2/HCO3-, complete inhibition of EGF-stimulated Na+/H+ exchange does not inhibit EGF-induced mitogenesis and, thus, stimulation of Na+/H+ exchange is not necessary for induction of mitogenesis by EGF.

Inhibition of epidermal growth factor-induced mitogenesis by amiloride and an analog: evidence against a requirement for Na+/H+ exchange.