Fisslthaler B, Dimmeler S, Hermann C, Busse R, Fleming I
Institut für Kardiovaskuläre Physiologie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main, Germany.
Acta Physiol Scand. 2000 Jan;168(1):81-8. doi: 10.1046/j.1365-201x.2000.00627.x.
Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.
流体剪切力通过一种不需要细胞内游离钙离子浓度（[Ca2+]i）升高的机制激活内皮型一氧化氮合酶（eNOS），并且对几种激酶抑制剂敏感。虽然有人提出eNOS的磷酸化调节酶活性，但eNOS激活的机制仍不清楚。在这里，我们证明流体剪切力引发eNOS酪氨酸和丝氨酸残基的磷酸化。使用渥曼青霉素或其下游靶点Akt（蛋白激酶B）的显性负性突变体抑制磷脂酰肌醇3激酶（PI3K），可阻止eNOS持续的丝氨酸磷酸化和激活。通过抑制丝氨酸/苏氨酸磷酸酶增强eNOS磷酸化，可使eNOS活性增加约两倍，这通过细胞内环鸟苷酸的积累来评估，而不增加细胞内游离钙离子浓度。这些数据表明，剪切力激活了一条涉及PI3K和丝氨酸/苏氨酸激酶Akt的信号通路，该通路使eNOS磷酸化。这种磷酸化直接增加静息[Ca2+]i时的eNOS活性，从而使剪切力诱导的eNOS激活明显不依赖于Ca2+。
