Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins
- 530 Downloads
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.
KeywordsRat colon Tight junctions Claudins Cholera toxin Theophylline
The study has been supported by the Deutsche Forschungsgemeinschaft (DFG FOR 721, SFB 852), the Sonnenfeld-Stiftung Berlin, the Partnership Program FU Berlin–University St. Petersburg, by Saint Petersburg University Research Grant no. 126.96.36.1991, and grant RFBR 10-04-01575.
- 10.Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, Ding X, Guandalini S, Comstock L, Goldblum SE (1995) Zonula occludens toxin modulates tight junctions through protein kinase C dependent actin reorganization, in vitro. J Clin Invest 96:710–720PubMedCrossRefPubMedCentralGoogle Scholar
- 16.Köckerling A, Fromm M (1993) Origin of cAMP-dependent Cl- secretion from both crypts and surface epithelia of rat intestine. Am J Physiol Cell Physiol 264:C1294–C1301Google Scholar
- 21.Mees ST, Mennigen R, Spieker T, Rijcken E, Senninger N, Haier J, Bruewer M (2009) Expression of tight and adherens junction proteins in ulcerative colitis associated colorectal carcinoma: upregulation of claudin-1, claudin-3, claudin-4, and beta-catenin. Int J Color Dis 24:361–368CrossRefGoogle Scholar
- 22.Mennigen R, Nolte K, Rijcken E, Utech M, Loeffler B, Senninger N, Bruewer M (2009) Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis. Am J Physiol Gastrointest Liver Physiol 296:G1140–G1149PubMedCrossRefGoogle Scholar