Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B-dependent dephosphorylation of protein kinase C.

AIMS: We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium (Ca(2+) )-permeable non-selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. METHODS: Western blotting and immunoprecipitation were used for the evaluation of protein phosphorylation; protein phosphatase 2B (PP2B) activity was assessed by convention kit; Griess assay was for NO production; tube formation and Matrigel plug assay were used for angiogenesis. RESULTS: In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα) at Serine 657 (Ser657) and PKCß2 at Ser660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B-PKC complex. Inhibition of TRPV1 activation by the pharmacological antagonists, removal of extracellular Ca(2+) or pharmacological inhibition of PI3K/Akt/calmodulin-dependent protein kinase II/AMP-activated protein kinase signalling pathway abolished the evodiamine-induced alterations in phosphorylation of eNOS at Thr497, PKCα at Ser657, PKCß2 at Ser660 and PP2B activity, as well as the formation of a PP2B-PKC complex. Inhibition of PP2B activation partially reduced the evodiamine-induced NO bioavailability and tube formation in endothelial cells (ECs) and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα at Ser657 and PKCß2 at Ser660 in apolipoprotein E (ApoE)-deficient mouse aortas but not TRPV1-deficient or ApoE/TRPV1 double-knockout mice. CONCLUSION: TRPV1 activation in ECs may elicit a Ca(2+) -dependent effect on PP2B-PKC signalling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.

Essential role of transient receptor potential vanilloid type 1 in evodiamine-mediated protection against atherosclerosis.

AIM: We investigated whether transient receptor potential vanilloid type 1 (TRPV1) was involved in the therapeutic effect of evodiamine, a main bioactive component in the fruit of Evodiae rutaecarpa, on the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice and ApoE(-/-)TRPV1(-/-) mice. METHODS: Histopathology was examined by haematoxylin and eosin staining, levels of cytokines and mediators were evaluated by ELISA kits, and protein expression was determined by Western blotting. RESULTS: Chronic administration with evodiamine (10 mg kg(-1) body weight) reduced the size of atherosclerotic lesions and alleviated the hyperlipidaemia and systemic inflammation, as well as hepatic macrovesicular steatosis, in ApoE(-/-) mice. Treating ApoE(-/-) mice with evodiamine enhanced hepatic cholesterol clearance, as revealed by upregulation of hepatic low-density lipoprotein receptor and ATP-binding cassette (ABC) transporters ABCG5, ABCG8 and cholesterol 7α-hydrolase. Genetic deletion of TRPV1 in ApoE(-/-) mice promoted the progression of atherosclerosis; elevated the serum levels of cholesterol, cytokines and chemokines; and exacerbated hepatic macrovesicular steatosis. Moreover, genetic deletion of TRPV1 abrogated the evodiamine-evoked atheroprotection but not anti-obesity effect in ApoE(-/-) mice. CONCLUSION: Evodiamine may confer novel TRPV1-dependent atheroprotection and TRPV1-independent anti-obesity action.