Regarding the lipophilic probes FM 1C43 and FM 4C64, they were less useful as predictors of paracellular PE action, although EDTA did cause a slight staining in the lamina propria. of both polar probes, including 10 C and 70 kDa dextrans and lipophilic probes, visualized by distinct stripy lateral staining of enterocytes and/or accumulation in the lamina propria. In addition, EDTA caused a loss of epithelial cell polarity by opening tight junctions for diffusion of domain-specific basolateral/apical cell membrane protein markers into the opposite domains. By transmission electron microscopy, SDS caused the formation of vacuoles and vesicle-like structures at the lateral cell membranes. In contrast, EDTA led to a bulging of the whole enterocyte apex, resulting in a cobblestone appearance of the epithelium, probably caused by an extreme 17-Hydroxyprogesterone contraction of the perijunctional actomyosin ring. We conclude that the mucosal explant system is a convenient model for predicting transcellular/paracellular modes of action of novel prospective PEs. -and studies.6 In addition, a fixed culture period of 1 h was chosen for all experiments performed. This time period proved optimal for detecting immediate effects of the PEs on parameters such as apical endocytic activity, cell membrane leakage, and paracellular permeation, rather than recording accumulated long-term effects. Open in a separate window Figure 2. Extraction of microvillus membrane vesicles by SDS (top) or EDTA (bottom). Following their preparation, the microvillus membranes were extracted with the respective PEs at the indicated concentrations. After incubation and centrifugation, pellet (P) C and supernatant (S) fractions were analyzed by SDS/PAGE. Total protein was visualized by staining with Coomassie brilliant blue. Arrows indicate molecular weight-values of 17-Hydroxyprogesterone sucrose-isomaltase (250 kDa), aminopeptidase N (150 kDa) and actin (42 kDa). Figure 3 shows how exposure for 1 h to 0.05% SDS or EDTA affected the epithelial morphology of cultured mucosal explants. For both PEs, overall tissue architecture including epithelial integrity 17-Hydroxyprogesterone was generally well preserved, with the Tbp exception of the villus tips where foci of denudation were frequently observed, both with SDS and EDTA. Exfoliation of senescent enterocytes normally takes place at the villus tip, but this is also a site where barrier restitution rapidly occurs after epithelial injury.26C28 In addition to this effect EDTA, but not SDS, frequently caused the apex of the enterocytes to bulge into the lumen, creating a characteristic cobblestone appearance of the epithelial surface (Figure 3d). In the uptake experiments presented below, we mainly focused on how SDS and EDTA acted on the more well-preserved parts of the villus epithelium, that is, the enterocytes lining the sides of the villi rather than at the tip. Open in a separate window Figure 3. Hematoxylin-eosin stained sections of mucosal explants cultured for 1 h in the absence (a) or presence of 0.05% SDS (b) or 0.05% EDTA (c), as described in Methods. Both SDS and EDTA caused denudation at the tip of the villi (arrows in (b) and (c)), but the epithelium along the sides of villi and in the crypts was generally well preserved. (d) is a higher magnification image of the area circled in c and shows the characteristic cobblestone appearance of enterocytes caused by EDTA. Enterocytes (e) and lamina propria (LP) are indicated. Bars: 100 m (aCc); 20 m (d). SDS and EDTA effects on localization of basolateral C and apical cell surface markers In control explants, antibodies to the Na+/K+-ATPase exclusively labeled the basolateral sides of the enterocytes, whereas the labeling using antibodies to the two prominent brush border enzymes aminopeptidase N (ApN) and sucrose-isomaltase (SI) was confined to the apical cell surface (Figure 4). This pattern of cell membrane protein distribution demonstrates the strict maintenance of cell polarity normally upheld in intestinal epithelial cells.29,30 SDS did not affect the localization of any of the cell surface markers used, indicating that this transcellular-acting PE leaves cell polarity intact (Figure 4). In the presence of EDTA, on the other hand, patchy labeling for Na+/K+-ATPase was clearly visible at the apical surface of enterocytes and conversely, distinct traces of both ApN and SI were detectable along the lateral sides of the cells (Figure 4). This surprising result shows that the paracellular-acting PE EDTA is capable of disrupting the cell polarity of enterocytes, most likely by permitting diffusion of membrane proteins through the normally impenetrable TJs. Open in a separate window Figure 4. Effects of SDS and EDTA on surface expression of enterocyte cell membrane markers. Sections of mucosal explants, cultured for 1 h in the absence (Control) or presence of 0.05% SDS or 0.05% EDTA, were immunolabeled for the basolateral marker Na+/K+-ATPase or the apical markers ApN or.