Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs.
Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs.
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Akt1 介导粗上肢中嘌呤能依赖性 NOS3 激活。
DOI：
10.1152/ajprenal.00270.2009
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Garvin,JeffreyL
中科院分区：
文献类型：
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作者：
Silva,GuillermoB;Garvin,JeffreyL
细胞外ATP通过释放一氧化氮（NO）调节许多生理过程。ATP刺激粗升支（TAL）中的NO，但对该肾单位段细胞以及许多其他类型细胞中涉及的信号级联反应知之甚少。我们推测ATP通过刺激PI 3激酶和Akt来增强一氧化氮合酶（NOS）的活性。1）用NO敏感的染料FITC-2 DA检测TAL中的NO; 2）用荧光共振能量转移和Akt亚型的磷酸化检测Akt活性。ATP（100 μM）刺激野生型小鼠的NO [26 ± 4个任意单位（Au）]，但在NOS 3 −/−小鼠中没有（2 ± 2个Au;P< 0.04）。在NOS 1和NOS 2选择性抑制剂7-NI和1400 W存在下，ATP分别刺激NO增加30 ± 2和33 ± 3 Au（与对照相比不显著）。在PI 3激酶抑制剂LY 294002存在下，ATP增加的NO减少85%（5 ± 2 vs. 28 ± 4 Au;P< 0.02）。ATP单独增加Akt活性，这种作用被P2受体拮抗剂苏拉明显著阻断。在Akt选择性抑制剂存在下，ATP诱导的NO被阻断90 ± 4%。ATP显著刺激Akt 1在Ser 473的磷酸化，幅度为91 ± 13%，而Akt 2磷酸化保持不变，Akt 3磷酸化降低。在体内转导的TAL与显性负Akt 1显着降低ATP诱导的NO 88 ± 6%。我们的结论是，ATP通过激活TAL中的Akt 1增加NOS 3衍生的NO。
Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured1) NO in TALs using the NO-sensitive dye DAF-2 DA and2) Akt activity by fluorescence resonance energy transfer and phosphorylation of Akt isoforms. ATP (100 μM) stimulated NO in wild-type mice [26 ± 4 arbitrary units (AU)], but not in NOS3 −/− mice (2 ± 2 AU;P< 0.04). In the presence of the NOS1- and NOS2-selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30 ± 2 and 33 ± 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 ± 2 vs. 28 ± 4 AU;P< 0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90 ± 4%. ATP significantly stimulated Akt1 phosphorylation at Ser473by 91 ± 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Akt1 significantly decreased ATP-induced NO by 88 ± 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL.