Ogazon Del Toro Alejandro, Jimenez Lidia, Hinojosa Lorena, Martínez-Rendón Jacqueline, Castillo Aida, Cereijido Marcelino, Ponce Arturo
Department of Physiology, Biophysics and Neurosciences, CINVESTAV-IPN, CDMX, C.P. 07360, Mexico.
Cardiol Res Pract. 2019 Dec 30;2019:8646787. doi: 10.1155/2019/8646787. eCollection 2019.
Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive 1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.
强心苷是一类因其在心脏组织中的作用而广为人知的化合物，其中一些已被发现是内源性产生的（内源性强心苷）。我们之前研究了内源性强心苷哇巴因对上皮细胞生理学的影响，并且我们已经表明，在纳摩尔范围内的浓度下，它会影响上皮细胞的关键特性，如紧密连接、顶端 - 基底外侧极化、间隙连接细胞间通讯（GJIC）和黏附连接。在这项工作中，我们研究了另外两种内源性表达的强心苷地高辛和海蟾蜍毒配基对GJIC以及由于紧密连接完整性（TJ）导致的跨上皮紧密程度的影响。我们通过染料转移试验评估GJIC，并通过跨上皮电阻（TER）测量以及紧密连接蛋白2和4表达的免疫组织化学和蛋白质印迹试验评估紧密连接完整性。我们发现，根据地高辛和海蟾蜍毒配基的TER测量评估，二者均改善GJIC并显著增强紧密连接的紧密程度。免疫荧光试验表明，这两种化合物均促进紧密连接蛋白 - 4在基底外侧的定位增强，但不促进紧密连接蛋白2的定位增强，而蛋白质印迹试验的光密度分析表明紧密连接蛋白4的表达显著增加。地高辛和海蟾蜍毒配基对GJIC和TER诱导的这些变化，在MDCK - R（一种经过基因改造具有不敏感的1亚基的改良MDCK细胞系）中未观察到，这表明钠钾ATP酶作为一种受体介导了两种内源性强心苷的作用。此外，两种强心苷的作用均被与PP2（一种c - Src激酶抑制剂）、PD98059（一种丝裂原细胞外激酶 - 1抑制剂）和Y - 27632（一种ROCK选择性抑制剂，一种Rho相关蛋白激酶）一起孵育所抑制，这表明所涉及的信号通路包括c - Src和ERK1/2以及Rho - ROCK。这些结果拓宽并强化了我们的总体假设，即内源性强心苷的一个非常重要的生理作用是控制上皮表型和调节细胞 - 细胞接触。
