Ionic events induced by epidermal growth factor: Evidence that hyperpolarization and stimulated cation influx play a role in the stimulation of cell growth

Charybdotoxin, a blocker of K ⁺ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca ²⁺ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca ²⁺](i) response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (>3 x 10 ^-8 M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na ⁺ and Ca ²⁺. Pretreatment with 10 μM SC38249 induced only minor changes of the intracellular Ca ²⁺ release by EGF (the process responsible for the initial phase of the [Ca ²⁺](i) and membrane potential responses) and blocked the persistent, second phase [Ca ²⁺](i) and the hyperpolarization responses, both dependent on Ca ²⁺ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca ²⁺](i) sustained by Ca ²⁺ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.