Angiotensin II Stimulates Thick Ascending Limb NO Production via AT2 Receptors and Akt1-dependent Nitric-oxide Synthase 3 (NOS3) Activation
Angiotensin II Stimulates Thick Ascending Limb NO Production via AT2 Receptors and Akt1-dependent Nitric-oxide Synthase 3 (NOS3) Activation
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DOI：
10.1074/jbc.m110.109041
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发表时间：
2010-05-14
影响因子：
4.8
通讯作者：
Garvin, Jeffrey L.
中科院分区：
文献类型：
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作者：
Herrera, Marcela;Garvin, Jeffrey L.
血管紧张素 II (Ang II) 通过未知机制急性刺激粗升肢 (TAL) NO。在内皮细胞中，Ang II 2 型受体 (AT(2)) 的激活会刺激 NO。 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 NO 的产生，该激活使 NOS3 在丝氨酸 1177 处磷酸化。我们通过荧光显微镜测量了 NO 的产生。在分离的 TAL 中，Ang II (100 nM) 使 NO 产量增加 1.1 +/- 0.2 荧光单位/分钟 (p < 0.01)。 Ang II 使 cGMP 积累增加 4.9 +/- 1.3 fmol/mu g (p < 0.01)。添加 AT(2) 拮抗剂 PD123319 (1 μM) 后，Ang II 未能刺激 NO（0.1 +/- 0.1 荧光单位/分钟；与 Ang II 相比，p < 0.001）；添加AT(1)拮抗剂氯沙坦(1μM)导致Ang II以0.9+/-0.1荧光单位/分钟刺激NO。 Akt 抑制剂 (5 μM) 阻断 Ang II 刺激的 NO（与单独抑制剂相比，-0.1 +/- 0.2 荧光单位/分钟）。 5 分钟后，Phospho-Akt1 增加 72% (p < 0.006)，10 分钟后恢复至基础值。 Phospho-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)。 Phospho-Akt3 没有改变。 AT(2) 激动剂使 pAkt1 增加 78% (p < 0.02)，PI3K 抑制可阻断这种作用。在用显性失活 Akt1 转导的 TAL 中，Ang II 未能刺激 NO（0.1 +/- 0.2 荧光单位/分钟，而对照为 1.2 +/- 0.2；p < 0.001）。 Ang II 使丝氨酸 1177 处的磷酸-NOS3 增加 130% (p < 0.01)，5 分钟和 10 分钟后增加 150% (p < 0.02)。 5 分钟后，Ang II 使丝氨酸 633 处的磷酸一氧化氮合酶 (NOS3) 增加 50% (p < 0.01)。 Akt 抑制可阻止 NOS3 磷酸化。我们得出结论，Ang II 通过激活 AT(2) 和 Akt1 依赖性 NOS3 丝氨酸 1177 和 633 磷酸化来增强 TAL NO 的产生。
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/mu g (p < 0.01). Upon adding the AT(2) antagonist PD123319 (1 mu M), 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 mu M) resulted in Ang II stimulating NO by 0.9 +/- 0.1 fluorescence units/min. Akt inhibitor (5 mu M) 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 phospho-NOS3 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.