Figueroa Xavier F, González Daniel R, Martínez Agustín D, Durán Walter N, Boric Mauricio P
Unidad de Regulación Neurohumoral, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
J Physiol. 2002 Nov 1;544(3):883-96. doi: 10.1113/jphysiol.2002.021972.
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 microM 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 microM). The maximal increase in NO production induced by 10 microM and 100 microM 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 microM ACh decreased the level of membrane-bound eNOS by -13 +/- 4 %, -60 +/- 4 % and -19 +/- 17 %, respectively; at the same time points, 100 microM 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.
在培养细胞中的研究表明，内皮型一氧化氮（NO）合酶（eNOS）的激活需要该酶与其抑制性伴侣小窝蛋白-1（Cav-1）解离，并且可能需要从质膜小窝转运至其他细胞区室。我们研究了以下假说：在体内，NO依赖性血管舒张与eNOS从细胞膜的转位有关。为此，我们将乙酰胆碱（ACh）局部应用于仓鼠颊囊微循环（10 - 100 μM，持续10分钟），测量NO生成、血流量和血管直径，并通过蛋白质印迹法评估亚细胞eNOS分布。基线NO生成量为54.4±5.2 pmol·min⁻¹（n = 16）。ACh增加了NO释放，引起小动脉和小静脉舒张，并提高了微血管血流量。这些反应被N⁺-硝基-L-精氨酸（30 μM）抑制。10 μM和100 μM ACh诱导的NO生成最大增加量分别为45±20%和111±33%；相应的血流量增加分别为50±10%和130±24%（n = 4 - 6）。两种反应具有相似的时间进程，尽管NO的增加先于血流变化。在未受刺激的组织中，eNOS主要分布在微粒体部分。ACh诱导的血管舒张与eNOS转位至富含胞质和高尔基体的部分有关。在应用1.5、3.0或6.0分钟后，10 μM ACh分别使膜结合eNOS水平降低了-13±4%、-60±4%和-19±17%；在相同时间点，100 μM ACh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含量均未改变。ACh浓度依赖性密切以及eNOS亚细胞再分布与NO释放的同时发生支持了以下概念：eNOS从质膜的转位是体内诱导NO依赖性血管舒张的激活机制的一部分。
