Markov Alexander G, Falchuk Evgeny L, Kruglova Natalia M, Rybalchenko Oksana V, Fromm Michael, Amasheh Salah
Institute of General Physiology, St. Petersburg State University, St. Petersburg, Russia.
Pflugers Arch. 2014 Nov;466(11):2059-65. doi: 10.1007/s00424-014-1460-z. Epub 2014 Feb 11.
Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.
已确定紧密连接蛋白Claudin主要决定肠道上皮屏障特性。虽然单个Claudin的功能作用已得到详细表征，但关于其与天然肠上皮分泌机制相互作用的信息却很少。因此，分析了霍乱毒素和茶碱对大鼠结肠的影响，包括检测封闭Claudin。将组织标本剥离黏膜下层并安装在尤斯灌流小室中，记录短路电流（ISC）和跨上皮电阻（TER）。同时，分析Claudin的表达和定位，并采用苏木精-伊红染色以及光镜和电镜进行组织学研究。茶碱可使结肠组织标本中的ISC显著增加。同时，观察到TER降低。相比之下，霍乱毒素未引起ISC显著增加，而在120分钟后检测到TER增加。蛋白质免疫印迹法显示，与霍乱毒素孵育后，Claudin-3和-4增加，茶碱可使Claudin-4增加。同样，共聚焦激光扫描显微镜显示，与霍乱毒素孵育后，紧密连接复合物内Claudin-3和-4的信号增加，与茶碱孵育后，Claudin-4的信号增加。形态学分析显示紧密连接复合物无总体变化，但与霍乱毒素和茶碱孵育后，细胞间隙明显增宽。我们得出结论，霍乱毒素和茶碱对天然结肠制剂中封闭紧密连接蛋白有不同影响，这可能在体外协同促进转运功能。
