AMPK is involved in the formation of TRPV1 and eNOS by TRPV1 ligand in ECs. (A, B) BAECs were treated with evodiamine (1 µmol/L) for 15 min. (C) BAECs were pretreated with compound C (10 µmol/L) for 1 h or transfected with dnAMPK mutant (K45R) for 24 h, then incubated with 1 µmol/L evodiamine for 15 min. Cell lysates were immunoprecipitated (IP) with anti-IgG, anti-AMPK or anti-TRPV1 antibodies, and precipitates were probed for TRPV1, eNOS and AMPK as indicated by immunoblotting (IB). (D) BAECs were co-transfected with pM, pVP pM-TRPV1, pVP-eNOS, pM3-VP16, pG5SEAP (secreted human alkaline phosphatase) or K45R for 48 h. Transfected cells were pretreated with or without compound C (10 µmol/L) for 1 h, then treated with or without evodiamine (1 [mol/L) for an additional 24 h. The pM3-VP16/pG5SEAP-transfected group was a positive control. The SEAP activity in culture media was determined by GreatEscAPe SEAP chemiluminescence detection kits. Data are mean ± SD from five independent experiments. *P < 0.05 versus non-treated group (A–C) or pM-TRPV1/pVP-eNOS/pG5SEAP-transfected group (D), #P < 0.05 versus evodiamine-treated group (A–C) or evodiamine-treated pM-TRPV1/pVP-eNOS/pG5SEAP group (D).