Cale Jacqueline M, Bird Ian M
Department of Obstetrics and Gynecology, The University of Wisconsin-Madison, Madison, WI 53715, USA.
Biochem J. 2006 Sep 1;398(2):279-88. doi: 10.1042/BJ20060371.
eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.
内皮型一氧化氮合酶（eNOS）的活性通过酰化、蛋白质-蛋白质相互作用、细胞内运输和磷酸化等多种方式进行复杂的翻译后调控。调节eNOS活性的信号通路包括磷酸肌醇3激酶/Akt、环核苷酸依赖性激酶[蛋白激酶A（PKA）和蛋白激酶G（PKG）]、蛋白激酶C（PKC）以及细胞外信号调节激酶（ERK）。ERK在eNOS激活中的作用仍存在争议。在本研究中，我们研究了ERK1/2在转化的人脐静脉内皮细胞（HUVEC-CS）以及一种广泛用于eNOS研究的模型——瞬时转染的COS-7细胞中eNOS激活过程中的作用。用U0126预处理HUVEC-CS可增强ATP刺激的eNOS活性，且与细胞内钙离子浓度（[Ca2+]i）的变化无关。在瞬时表达绵羊eNOS的COS-7细胞中，U0126增强了A23187刺激的eNOS活性，但抑制了ATP刺激的活性。eNOS五个关键残基磷酸化的补偿性变化并不能解释A23187刺激活性的变化。然而，对于ATP而言，磷酸化的改变和[Ca2+]i的变化可能部分导致了U0126对活性的抑制。最后，构建了七个假定的ERK1/2靶点的eNOS丙氨酸突变体，并通过A23187和ATP处理来评估U0126预处理对eNOS活性的影响。在U0126预处理和ATP刺激eNOS激活后，T97A-eNOS是唯一与野生型有显著差异的构建体。在本研究中，COS-7细胞中的eNOS活性要么增强要么受到抑制，这表明MEK/ERK1/2信号通路（其中MEK是丝裂原活化蛋白激酶/ERK激酶）对eNOS的作用存在激动剂依赖性以及包括[Ca2+]i、磷酸化和可能的细胞内运输在内的复杂机制。
