Mayol V, Dignat-George F, Gerbi A, Martin-Vasallo P, Lesaule G, Sampol J, Maixent J M
Laboratoire de Recherche Cardiologique, Faculté de Médecine, Marseille, France.
J Hypertens. 1998 Feb;16(2):145-50. doi: 10.1097/00004872-199816020-00003.
The catalytic alpha and smaller beta subunits of the plasma membrane Na,K-ATPase occur in various molecular forms (alpha1, alpha2, alpha3, beta1 and beta2). The alpha isoforms of the enzyme have varying affinities for ouabain and exist in different tissues with particular distribution patterns.
To document the existence of isoforms of the Na,K-ATPase in cultured human umbilical vein endothelial cells.
Microsomal fractions were prepared by differential ultracentrifugation from primary cultures of human umbilical vein endothelial cells and from such cells obtained after three passages. Na,K-ATPase activity was assayed using the coupled assay method and sensitivity to ouabain was determined in the presence of varying concentrations of ouabain. Specific antibodies for the various Na,K-ATPase isoforms were used to label these different proteins by immunocytochemistry in endothelial cells and by Western blotting in isolated membranes.
In plotting the dose-response curves for Na,K-ATPase activity in response to ouabain we assumed the existence of two independent sites exhibiting different affinities for ouabain (in the micromol/l and the nmol/l ranges). The contribution of low-affinity sites was threefold that of high-affinity sites. After three passages in culture, a specific increase in Na,K-ATPase activity of the high-affinity sites was observed compared with that of cells from primary cultures. Confocal microscopy revealed the existence of beta1, beta2, and alpha1 subunit proteins in human umbilical endothelial cells. Staining for alpha3 isoform was less pronounced and no obvious alpha2 was detected.
These findings suggest that human umbilical vein endothelial cells contain beta1, beta2, a large amount of alpha1 isoform with an apparently low affinity for ouabain, and a lesser amount of high-affinity sites, which may correspond to the alpha3 protein.
质膜钠钾ATP酶的催化α亚基和较小的β亚基存在多种分子形式（α1、α2、α3、β1和β2）。该酶的α同工型对哇巴因具有不同的亲和力，并以特定的分布模式存在于不同组织中。
证实培养的人脐静脉内皮细胞中钠钾ATP酶同工型的存在。
通过差速超速离心从人脐静脉内皮细胞原代培养物以及传代三次后的细胞中制备微粒体部分。使用偶联测定法测定钠钾ATP酶活性，并在不同浓度的哇巴因存在下测定对哇巴因的敏感性。使用针对各种钠钾ATP酶同工型的特异性抗体，通过免疫细胞化学在内皮细胞中以及通过蛋白质印迹法在分离的膜中对这些不同的蛋白质进行标记。
在绘制钠钾ATP酶活性对哇巴因的剂量反应曲线时，我们假设存在两个对哇巴因具有不同亲和力的独立位点（在微摩尔/升和纳摩尔/升范围内）。低亲和力位点的贡献是高亲和力位点的三倍。在培养传代三次后，与原代培养的细胞相比，观察到高亲和力位点的钠钾ATP酶活性有特异性增加。共聚焦显微镜显示人脐静脉内皮细胞中存在β1、β2和α1亚基蛋白。α3同工型的染色不太明显，未检测到明显的α2。
这些发现表明，人脐静脉内皮细胞含有β1、β2、大量对哇巴因亲和力明显较低的α1同工型以及少量高亲和力位点，后者可能对应于α3蛋白。
