Substance P stimulates vascular endothelial cellular reducing capacity in the presence of insulin and human plasma factors

Substance P (SP) is an important tachykinin in vascular wall biology. In previous studies [Villablanca et al. (1994): Circ Res 75:1113-1120], the authors have demonstrated that SP is a stimulus for endothelial cell growth and proliferation in serum-free culture conditions with cells quiescent in the G₀-G₁ phase of the cell cycle. As mitogenic and metabolic activity may interrelate, the purpose of this study was to determine the effects of the vasoactive perivascular neuropeptide SP on changes in the metabolic function of endothelial cells, and to characterize the response, by studying cellular reducing capacity in nortic vascular endothelial cells. In addition, interactions between SP and other growth factors (insulin and non-platelet plasma factors) were investigated and compared to the responses to SP alone. Metabolic effects were determined by evaluating cellular reducing capacity by the conversion of (3-[4,5-dimethylthiazole-2-yl]-2,5diphenyltetrazolium bromide) to formazan (the MTT assay). The findings demonstrated that SP alone (10 pg/ml-25 μg/ml) inhibited cellular reducing capacity in vascular endothelial cells. In contrast, SP in the presence of insulin (10 μg/ml) stimulated endothelial reducing capacity, as compared to SP alone, by twofold on average. The effect of SP and insulin was additive at ≤0.001 μg/ml SP, and synergistic at SP concentrations ranging within 0.01-1.0 μg/ml. SP in the presence of human platelet-poor plasma (HPPP, 5%) stimulated endothelial reducing capacity, as compared to SP alone, by threefold on average. The effect of SP and HPPP was additive at ≤0.01 μg/ml SP and synergistic at SP concentrations of 0.1-25 μg/ml. Lastly, SP in the presence of insulin and HPPP stimulated endothelial metabolic activity, as compared to SP alone, by 14-fold on average. An additive response to SP, insulin, and HPPP was observed at the lowest SP concentration studied (10 μg/ml). At all other SP concentrations studied (0.0001-2,5 μg/ml), the responses to insulin, HPPP, and SP were synergistic. Our studies indicate that the vasoactive neuropeptide substance P may synergize with insulin and HPPP in regulating endothelial cell metabolism. In addition, our findings suggest that the mechanisms by which SP stimulates cellular metabolism are different from the mechanisms by which it stimulates cell growth.