Figure 1.
Capsaicin alters the TER of epithelial monolayers in a concentration-dependent and reversible manner.
(A) TER was assessed in MDCK monolayers exposed to ethanol (vehicle) control (crosses), 300 µM (triangles), 100 µM (circles) or 30 µM (squares) capsaicin. (B) After exposure to capsaicin at the concentrations and for the lengths of time indicated, monolayers in (A) were lysed and subjected to western blot analysis. (C) MDCK monolayers incubated with ethanol control (crosses), 300 µM capsaicin (squares) or 0.1 µM LatA (triangles). Values represent mean ± S.D. Asterisks indicated significant difference from control at the same time point; *, p<0.01; **, p<0.001.
Figure 2.
Capsaicin induces actin depolymerization and morphological changes.
(A) Monolayers were exposed to ethanol control, capsaicin (300 µM, 45 min), Jpk (2 µM, 60 min) or LatA (0.5 µM, 90 min) and lysed, and the G- and F-actin fractions were separated. Fractions were analyzed by SDS-PAGE and immunoblotting with an anti-actin antibody. (B) MDCK monolayers were exposed to ethanol control, capsaicin (300 µM, 45 min), LatA (0.1 µM, 30 min), Jpk (2 µM, 1 min) or CytoB (5 µg/ml, 15 min), and were fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm. (C) The fluorescence intensities of the images in (B) were analyzed. Bar: 10 µm. ***represents p<0.001 by Student’s t test.
Figure 3.
Effects of capsaicin on the distribution of TJ proteins and F-actin.
(A, B) Monolayers were exposed to vehicle (A) or 300 µM capsaicin (B) for 45 min and were labeled with each TJ antibody (green), rhodamine-phalloidin (red) and Hoechst (blue). Images were collected as a Z-series, and then deconvoluted and overlayed to display a single composite projection. Top: XY sections of merged images. Bottom: XZ sections of merged images. Scale bar: 10 µm. The corresponding TJ antibodies are listed above the images. (C) 3D projection images in a 45°-angle of claudin-1 and tricellulin staining, plus F-actin. Scale bar: 10 µm. (D) MDCK monolayers exposed to vehicle (top) or capsaicin (bottom) as above were stained with E-cadherin. The colors corresponding to each antibody are listed above the images. Scale bar: 2.5 µm. (E) Schematic explanation of the protein distributions.
Figure 4.
Capsaicin decreases occludin protein content and the protein-protein interactions involving occludin in TJ.
(A,B) MDCK monolayers were exposed to ethanol control or 300 µM capsaicin for 45 min. (A) The cellular localization of the TJ proteins occludin and claudin-1 was examined by immunofluorescence. Scale bar: 10 µm. (B) Western blot detection of occludin, tricellulin, Zo-1, claudin-1, E-cadherin and actin in the cytosol, membrane fractions and total cell extracts. The densitometic analysis of total proteins from three independent experiments performed with NIH ImageJ software. **represents p<0.01 by Student’s t test. (C) Western blot detection of occludin, claudin-1, phospho-cofilin, cofilin and E-cadherin in total extracts exposed to 300 µM capsaicin or 0.1 µM LatA for the durations indicated. (D) Distribution of TJ proteins in sucrose gradient centrifugation of lysates from monolayers (shown in the inset) after exposure to ethanol control or 300 µM capsaicin for 1 h. Fractions were collected from the top of the gradient, separated by SDS-PAGE and subjected to immunoblotting. The apparent molecular masses of BSA (66 kDa) and myosin (200 kDa) are shown above the fraction number. Representative data from three independent experiments is shown. The densitometic analyses from three independent experiments are shown. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 5.
Effects of suppression of cofilin activation or overexpresssion of TJ proteins on the capsaicin-induced cofilin dephosphorylation and decrease in occludin expression.
(A–C) Flag-HA tagged cofilin and LIMK, and EGFP-occludin and claudin-1 stable transfectants were established. Monolayers prepared from the transfectants and non-transfected MDCK cells were exposed to 300 µM capsaicin for the time indicated. After lysis, protein expression was analyzed by immunoblotting. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were pretreated with vehicle or 50 µM TFP for 30 min, and then exposed to 100 µM capsaicin for the time indicated. The levels of occludin and phospho-cofilin were analyzed by western blotting.
Figure 6.
Overexpresssion of cofilin, LIMK and occludin, but not claudin-1, significantly diminishes the capsaicin-induced decrease in TER.
(A,B) Transfectant monolayers were prepared in transwell inserts, exposed to 300 µM capsaicin, and subjected to TER measurements. Values represent mean ± S.D. Asterisks indicated significant difference from Vector control at the same time point; *, p<0.01; **, p<0.001. (C) TER was assessed in MDCK monolayers pretreated with vehicle control or 50 µM TFP for 30 min, and exposed to 100 µM capsaicin at time 0. Values represent mean ± S.D. Repeated measure ANOVA followed by Tukey’s multiple comparisons test; **p<0.01, control vs. capsaicin; ##p<0.01, TFP + capsaicin vs. capsaicin. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were treated with vehicle or 50 µM TFP for 30 min followed by 100 µM capsaicin for 15 min, then fixed and stained with rhodamine-phalloidin to detect F-actin alteration (upper panel). To see the relative distribution of F-actin (red) in 3D, images labeled with anti-claudin-1 (green) and Hoechst (blue) are also shown below. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm.
Figure 7.
Cofilin activation, decrease in occludin expression and actin alteration in the recovery phase of capsaicin treatment.
(A) Western blot detection of phospho-cofilin and occludin in total extracts exposed to 300 µM capsaicin for the times indicated. (B) MDCK monolayers were exposed to 300 µM capsaicin for the time indicated, and then fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm.
Figure 8.
Capsaicin increases TJ permeability as measured by the paracellular passage of ionic and non-ionic compounds.
(A,B,D) MDCK cells were seeded on transwells and grown for 3 days to develop monolayers. Paracellular fluxes of CF (A), FD4 (B), and insulin (D) were measured in the apical to basolateral direction. All measurements were in triplicates (n = 3) and representative data are shown. (C) Effects of 300 µM capsaicin, 1 mg/ml C10, 0.1 µM LatA on the Papp of molecules across MDCK monolayers. Papp was calculated from the above data. aEach value represents the mean ± S.D. of three independent experiments. bEnhancement ratio calculated as Papp(sample)/Papp(control).