Department of Physiology, National Yang-Ming University, Taipei, Taiwan.
Acta Physiol (Oxf). 2013 Oct;209(2):124-35. doi: 10.1111/apha.12157. Epub 2013 Aug 29.
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.
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.
In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα) at Serine 657 (Ser657) and PKCβ2 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, PKCβ2 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 PKCβ2 at Ser660 in apolipoprotein E (ApoE)-deficient mouse aortas but not TRPV1-deficient or ApoE/TRPV1 double-knockout mice.
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.
我们研究了瞬时受体电位香草酸 1（TRPV1），一种钙（Ca（2+））通透性非选择性阳离子通道，对牛主动脉内皮细胞（BAEC）和小鼠内皮型一氧化氮合酶（eNOS）丝氨酸 497（Thr497）磷酸化的影响及其潜在的分子机制。
采用Western blot 和免疫沉淀法评价蛋白磷酸化；采用常规试剂盒评估蛋白磷酸酶 2B（PP2B）活性；采用 Griess 法测定 NO 产生；采用管形成和 Matrigel plugs 测定血管生成。
在 BAEC 中，TRPV1 配体吴茱萸碱处理可降低 eNOS 在 Thr497 、蛋白激酶 Cα（PKCα）在 Ser657（Ser657）和蛋白激酶 Cβ2 在 Ser660 的磷酸化。吴茱萸碱增加蛋白磷酸酶 2B（PP2B）活性，并促进 PP2B-PKC 复合物的形成。用药理学拮抗剂阻断 TRPV1 激活、去除细胞外 Ca（2+）或用药理学抑制剂阻断 PI3K/Akt/钙调蛋白依赖性蛋白激酶 II/AMP 激活蛋白激酶信号通路，均可消除吴茱萸碱诱导的 eNOS 在 Thr497 、PKCα在 Ser657 、PKCβ2 在 Ser660 磷酸化及 PP2B 活性的改变，以及 PP2B-PKC 复合物的形成。抑制 PP2B 活性可部分降低吴茱萸碱诱导的内皮细胞（ECs）中 NO 生物利用度和管形成及小鼠血管生成。此外，吴茱萸碱可降低载脂蛋白 E（ApoE）缺陷型小鼠主动脉中 eNOS 在 Thr497 、PKCα在 Ser657 和 PKCβ2 在 Ser660 的磷酸化，但不能降低 TRPV1 缺陷型或 ApoE/TRPV1 双敲除型小鼠中这些蛋白的磷酸化。
ECs 中 TRPV1 的激活可能引发 Ca（2+）依赖性的 PP2B-PKC 信号，导致 ECs 及小鼠中 eNOS 在 Thr497 的去磷酸化。
