Insulin is not only a growth factor for vascular cells, but also an inducer of other vasoactive substances such as endothelin-1 (ET-1) in vascular cells. The aim of the present study was to assess the role of endothelial cells (EC) in insulin mediated vascular smooth muscle cell (VSMC) proliferation. Cultured human aortic EC and VSMC were separately incubated. EC were stimulated with insulin (0 to 1000 microU/mL) for 24 h, in the presence or absence of anti-insulin-growth factor-1 (anti-IGF-1) receptor antibody (alphaIR(3)) or a nonselective ET-1 receptor antagonist (TAK044). Cell proliferation was measured by determining (3)H-thymidine uptake. Although 10 microU/mL insulin did not affect ET-1 production in the EC culture medium, a higher concentration of insulin stimulated it. Production of ET-1 in EC was activated by insulin via the IGF-1 receptor, inasmuch as alphaIR(3) blocked insulin mediated upregulation of ET-1. There was no significant difference in (3)H-thymidine incorporation in the presence of insulin (up to 1000 microU/mL) or TAK044. Culture medium from EC stimulated with insulin enhanced VSMC proliferation, which was almost totally suppressed by TAK044. Insulin induced VSMC growth dose dependently when VSMC were cultured alone. In contrast, insulin at concentrations of 100 microU/mL or lower failed to stimulate growth of co-cultured VSMC, but only at 330 microU/mL or higher concentrations stimulated VSMC growth in this system. Of interest, VSMC proliferation was greatest when L-NAME was added and co-cultured with EC. In summary, a severely hyperinsulinemic state may regulate VSMC and EC proliferation via activation of vasoactive substances such as ET-1 and nitric oxide induced by insulin.