Figure 4

Overexpression of dominant-negative AMPK (dnAMPK) mutant inhibits eNOS phosphorylation, NO production and tube formation by activation of TRPV1 in ECs. (A) BAECs were transfected with vector (1 µg) or dnAMPK mutant (K45R, 1 µg) for 24 h, then with evodiamine (1 µmol/L) for an additional 15 min. Cellular lysates underwent immunoblotting to detect the levels of phosphorylated AMPK at Thr172, AMPK, phosphorylated eNOS at Ser635 and Ser1179 and eNOS. (B, C) Transfected BAECs were treated with 1 µmol/L evodiamine for 24 h. The level of nitrite in cultured medium was analyzed by Griess assay (B). Tube formation in vitro; bar graphs indicate the number of branch points in five randomly selected microscopy views (C). Data are mean ± SD from five independent experiments. *P < 0.05 versus nontreated group, ^#P < 0.05 versus evodiamine-treated group.