Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [3H]saxitoxin ([3H]-STX) binding in a time-dependent manner (t1/2 = 26 h). Insulin (100 nM for 4 days) increased the Bmax of [3H]STX binding by 49% without changing the KD value and also augmented the maximal influx of 22Na+ due to 560 microM veratridine by 39% without altering the EC50 value of veratridine. The stimulatory effect of insulin on 22Na+ influx was concentration-dependent with an EC50 of 3 nM, whereas insulin-like growth factor (IGF)-I had little effect at 1 nM. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine (100 microM)-induced 22Na+ influx by approximately twofold in both insulin-treated cells and untreated cells. Veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine-induced 22Na+ influx via the nicotinic receptor-ion channel complex and high-K+ (direct activation of voltage-dependent Ca2+ channels)-induced 45Ca2+ influx. Stimulatory effects of insulin on [3H]-STX binding and veratridine-induced 22Na+ influx were nullified by simultaneous treatment with either 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na+ channel alpha-subunit. These results suggest that the binding of insulin to insulin (but not IGF-I) receptors mediates the up-regulation of functional Na+ channel expression at plasma membranes; this up-regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).