ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo
ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo
复制标题
DOI：
10.1113/jphysiol.2002.021972
复制
发表时间：
2002-11-01
影响因子：
5.5
通讯作者：
Boric, MP
中科院分区：
文献类型：
--
作者：
Figueroa, XF;González, DR;Boric, MP
在培养细胞中的研究表明，内皮型一氧化氮合酶（eNOS）的激活需要这种酶从其与小窝蛋白-1（Cav-1）的抑制性结合中解离，并且可能需要其从质膜小窝易位到其他细胞隔室。我们研究了体内NO依赖性血管舒张与eNOS从细胞膜转位相关的假设。为此，我们应用乙酰胆碱局部（10-100妈妈，10分钟）仓鼠颊囊微循环和测量NO的生产，血流量和血管直径，并通过Western印迹法评估亚细胞eNOS分布。基线NO生成为54.4 +/- 5.2 pmol min（-1）（n = 16）。ACh增加NO释放，引起微动脉和微静脉扩张和微血管流量增加。这些反应被N-G-硝基-（L）-精氨酸（30 μ m）抑制。10 μ m和100 μ m ACh诱导的NO产生最大增加分别为45 +/-20%和111 +/-33%;相应的血流量增加分别为5010%和13024%（n = 4-6）。这两种反应遵循类似的时间过程，虽然增加NO流量变化之前。在非刺激组织中，eNOS主要分布在微粒体组分中。乙酰胆碱诱导的血管舒张与eNOS易位到胞质和高尔基体富集的部分。10 μ mACh作用1.5、3.0或6.0 min后，膜结合eNOS水平分别下降-13 +/-4%、-60 +/-4%和-19 +/-17%;在相同的时间点，100 μ mACh分别使微粒体eNOS含量降低了-38 +/-9%-61 +/-16%和-40 +/-18%（n = 4-5）。在所有情况下，微粒体Cav-1含量没有变化。密切的ACh浓度依赖性和eNOS亚细胞再分布和NO释放之间的相伴支持的概念，eNOS易位从质膜的激活机制，诱导NO依赖性血管舒张在体内的一部分。
Studies in cultured cells show that activation of endothelial nitric oxide (NO) synthase (eNOS) requires the dissociation of this enzyme from its inhibitory association with caveolin-1 (Cav-1), and perhaps its translocation from plasma membrane caveolae to other cellular compartments. We investigated the hypothesis that in vivo NO-dependent vasodilatation is associated with the translocation of eNOS from the cell membrane. To this end, we applied ACh topically (10-100 mum for 10 min) to the hamster cheek pouch microcirculation and measured NO production, blood flow and vessel diameter, and assessed subcellular eNOS distribution by Western blotting. Baseline NO production was 54.4 +/- 5.2 pmol min(-1) (n = 16). ACh increased NO release, caused arteriolar and venular dilatation and elevated microvascular flow. These responses were inhibited by N-G-nitro-(L)-arginine (30 mum). The maximal increase in NO production induced by 10 mum and 100 mum ACh was 45 +/- 20 % and 111 +/- 33 %, respectively; the corresponding blood flow increases were 50 10 % and 130 24 %, respectively (n = 4-6). Both responses followed a similar time course, although increases in NO preceded flow changes. In non-stimulated tissues, eNOS was distributed mainly in the microsomal fraction. ACh-induced vasodilatation was associated with eNOS translocation to the cytosolic and Golgi-enriched fractions. After 1.5, 3.0 or 6.0 min of application, 10 mum ACh decreased the level of membrane-bound eNOS by -13 +/- 4%, -60 +/- 4% and -19 +/- 17% respectively; at the same time points, 100 mum ACh reduced microsomal eNOS content by -38 +/- 9% -61 +/- 16% and -40 +/- 18 %, respectively (n = 4-5). In all cases, microsomal Cav-1 content did not change. The close ACh concentration dependence and the concomitance between eNOS subcellular redistribution and NO release support the concept that eNOS translocation from the plasma membrane is part of an activation mechanism that induces NO-dependent vasodilatation in vivo.