Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1beta (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with L-arginine (10 mmol/L) or tetrahydrobiopterin (BH(4); 100 micromol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 micromol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1beta induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1beta-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-arginine or BH(4) increased NO generation by L-NAME- or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH(4). The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 micromol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.