Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries
Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries
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DOI：
10.1161/01.res.0000178008.46759.d0
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发表时间：
2005-08-19
影响因子：
20.1
通讯作者：
Garland, CJ
中科院分区：
文献类型：
--
作者：
Mather, S;Dora, KA;Garland, CJ
在阻力动脉中，从内皮到邻近平滑肌的超极化扩散被认为是内皮源性超极化因子（EDHF）引起的舒张的重要组成部分。为了探索内皮细胞间隙连接在EDHF介导的扩张中的作用，我们开发了一种方法，该方法最初用于将膜不渗透分子加载到培养的细胞中，以将连接蛋白（Cx）特异性抑制分子快速（约15分钟）加载到大鼠肠系膜动脉内的内皮细胞中。通过选择性地将细胞不渗透荧光染料装载到几乎所有的动脉内皮细胞中，而不影响组织形态或功能，实现了验证。内皮单层作为一个有效的屏障，防止大分子进入下面的平滑肌细胞。使用这种技术，内皮细胞负载抗体的细胞内羧基末端区域的Cx40（残基340至358）或模拟肽的细胞质环（Cx40;残基130至140），每个显着抑制EDHF介导的扩张。相反，针对Cx 37和Cx43的不同细胞内区域的多种抗体以及Cx 37的细胞内环区域的模拟肽各自没有作用。此外，加压动脉与靶向内皮细胞Cxs胞外区的抑制肽（（43）Gap 26，（40）Gap 27和（37，43）Gap 27;各300 μ mol/ L）同时腔内和腔外孵育2小时也不能改变EDHF反应。高分辨率免疫组化定位Cx40的肌内皮细胞间隙连接的内皮细胞投影结束。这些数据直接证明了Cx40在EDHF介导的大鼠肠系膜动脉扩张中的关键作用。
In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor ( EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (approximate to 15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs ((43)Gap 26, (40)Gap 27, and (37,43)Gap 27; 300 mu mol/ L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.