Haynes M P, Sinha D, Russell K S, Collinge M, Fulton D, Morales-Ruiz M, Sessa W C, Bender J R
Division of Cardiovascular Medicine and Molecular Cardiobiology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812, USA.
Circ Res. 2000 Oct 13;87(8):677-82. doi: 10.1161/01.res.87.8.677.
17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.
17β-雌二醇（E₂）是内皮型一氧化氮合酶（eNOS）的快速激活剂。这一激活事件的产物NO是心血管稳态的一个基本决定因素。我们之前证明，E₂刺激的内皮细胞释放NO可在胞质Ca²⁺不增加的情况下发生。据我们所知，在此我们首次证明，E₂通过磷脂酰肌醇3（PI3）-激酶-Akt途径快速诱导eNOS的磷酸化和激活。用人内皮细胞系EA.hy926进行E₂处理（10 ng/mL），导致NO生成增加，PI3-激酶抑制剂LY294002和雌激素受体拮抗剂ICI 182,780可消除这种增加。E₂刺激丝氨酸473上的Akt快速磷酸化。正如血管内皮生长因子所示，eNOS是E₂激活的Akt底物，丝氨酸1177上的eNOS快速磷酸化证明了这一点，丝氨酸1177是eNOS激活和对静息细胞Ca²⁺水平敏感性增强的关键残基。腺病毒介导的EA.hy926转导证实了Akt的功能参与，因为激酶缺陷型、显性负性Akt消除了E₂刺激的NO释放。膜不透性的E₂-BSA缀合物显示可结合内皮细胞膜位点，也诱导Akt快速磷酸化并随之导致eNOS磷酸化。因此，膜雌激素受体的结合通过PI3-激酶-Akt依赖性途径导致内皮细胞快速释放NO。这部分解释了对胞质Ca²⁺通量需求的降低，并描述了一条与心血管病理生理学相关的重要途径。
