Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202.
Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202.
J Biol Chem. 2010 May 14;285(20):14932-14940. doi: 10.1074/jbc.M110.109041. Epub 2010 Mar 18.
Angiotensin II (Ang II) acutely stimulates thick ascending limb (TAL) NO via an unknown mechanism. In endothelial cells, activation of Ang II type 2 receptor (AT(2)) stimulates NO. Akt1 activates NOS3 by direct phosphorylation. We hypothesized that Ang II stimulates TAL NO production via AT(2)-mediated Akt1 activation, which phosphorylates NOS3 at serine 1177. We measured NO production by fluorescence microscopy. In isolated TALs, Ang II (100 nm) increased NO production by 1.1 +/- 0.2 fluorescence units/min (p < 0.01). Ang II increased cGMP accumulation by 4.9 +/- 1.3 fmol/microg (p < 0.01). Upon adding the AT(2) antagonist PD123319 (1 microm), Ang II failed to stimulate NO (0.1 +/- 0.1 fluorescence units/min; p < 0.001 versus Ang II); adding the AT(1) antagonist losartan (1 microm) resulted in Ang II stimulating NO by 0.9 +/- 0.1 fluorescence units/min. Akt inhibitor (5 microm) blocked Ang II-stimulated NO (-0.1 +/- 0.2 fluorescence units/min versus inhibitor alone). Phospho-Akt1 increased by 72% after 5 min (p < 0.006), returning to basal after 10 min. Phospho-Akt2 did not change after 5 min but increased by 115 and 163% after 10 and 15 min (p < 0.02). Phospho-Akt3 did not change. An AT(2) agonist increased pAkt1 by 78% (p < 0.02), PI3K inhibition blocked this effect. In TALs transduced with dominant negative Akt1, Ang II failed to stimulate NO (0.1 +/- 0.2 fluorescence units/min versus 1.2 +/- 0.2 for controls; p < 0.001). Ang II increased phospho-NOS3 at serine 1177 by 130% (p < 0.01) and 150% after 5 and 10 min (p < 0.02). Ang II increased phosphoNOS3 at serine 633 by 50% after 5 min (p < 0.01). Akt inhibition prevented NOS3 phosphorylation. We concluded that Ang II enhances TAL NO production via activation of AT(2) and Akt1-dependent phosphorylation of NOS3 at serines 1177 and 633.
血管紧张素 II（Ang II）通过未知机制急性刺激升支粗段（TAL）的一氧化氮（NO）生成。在血管内皮细胞中，血管紧张素 II 型 2 型受体（AT（2））的激活可刺激 NO 的生成。Akt1 通过直接磷酸化激活 NOS3。我们假设 Ang II 通过 AT（2）介导的 Akt1 激活刺激 TAL 的 NO 生成，该激活使 NOS3 的丝氨酸 1177 磷酸化。我们通过荧光显微镜测量了 NO 的生成。在分离的 TAL 中，Ang II（100nm）使 NO 的生成增加了 1.1 +/- 0.2 荧光单位/分钟（p < 0.01）。Ang II 使 cGMP 积累增加了 4.9 +/- 1.3 fmol/microg（p < 0.01）。加入 AT（2）拮抗剂 PD123319（1μm）后，Ang II 未能刺激 NO（0.1 +/- 0.1 荧光单位/分钟；p < 0.001 与 Ang II 相比）；加入 AT（1）拮抗剂氯沙坦（1μm）后，Ang II 刺激 NO 的生成增加了 0.9 +/- 0.1 荧光单位/分钟。Akt 抑制剂（5μm）阻断了 Ang II 刺激的 NO 生成（与抑制剂单独相比为-0.1 +/- 0.2 荧光单位/分钟）。磷酸化 Akt1 在 5 分钟后增加了 72%（p < 0.006），10 分钟后恢复到基础水平。磷酸化 Akt2 在 5 分钟后没有变化，但在 10 分钟和 15 分钟后分别增加了 115%和 163%（p < 0.02）。磷酸化 Akt3 没有变化。AT（2）激动剂使 pAkt1 增加了 78%（p < 0.02），PI3K 抑制阻断了这种作用。在转导了显性失活 Akt1 的 TAL 中，Ang II 未能刺激 NO（0.1 +/- 0.2 荧光单位/分钟，而对照组为 1.2 +/- 0.2；p < 0.001）。Ang II 使丝氨酸 1177 处的磷酸化 NOS3 增加了 130%（p < 0.01）和 150%（p < 0.02），在 5 分钟和 10 分钟时。Ang II 在 5 分钟时使磷酸化 NOS3 增加了 50%（p < 0.01）。Akt 抑制阻止了 NOS3 的磷酸化。我们得出结论，Ang II 通过激活 AT（2）和 Akt1 依赖性 NOS3 的丝氨酸 1177 和 633 磷酸化增强 TAL 的 NO 生成。
