Figure 4.

Inhibitors of caveolar endocytosis prevent TNF-induced occludin internalization. (A) Wild-type mice were injected with vehicle or TNF as indicated. A segment of jejunum was perfused with 50 µM dynasore, 60 µM amiloride, 200 µM chlorpromazine (CPZ), 2 mM MβCD, 50 µM L-t-LacCer, or 50 µM D-e-LacCer and harvested 135 min later. Sections were labeled for occludin (grayscale images; green in merge), F-actin (red), and nuclei (blue). Bar, 20 µm. (B) Morphometric analysis of the number of occludin-containing vesicles per enterocyte in wild-type mice injected with vehicle (white bars) or TNF (gray bars) in jejunal segments perfused with saline or the indicated inhibitors. (C) Wild-type mice were injected with TNF, and a segment of jejunum perfused with Alexa Fluor 594–conjugated WGA (50 µg/ml). Perfused segments were harvested 135 min after TNF treatment. Sections were labeled for actin (top; green) or occludin (bottom; green) and nuclei (blue). Perfusion with amiloride prevented endocytosis of WGA (red) but not occludin. Bar, 10 µm. (D) Wild-type mice were injected with TNF, and a segment of jejunum perfused with DyLight 594–conjugated IgG (40 µg/ml). Perfused segments were harvested 135 min after TNF treatment. Sections were labeled for actin (top; green) or occludin (bottom; green) and nuclei (blue). Perfusion with chlorpromazine prevented endocytosis of IgG (red) but not occludin. Bar, 10 µm. (E) Sections of jejunum from mice injected with vehicle or TNF and perfused with saline, dynasore, MβCD, or L-t-LacCer harvested 135 min after TNF injection were labeled for phosphorylated MLC (green) and nuclei (blue). Bar, 20 µm. Error bars indicate mean ± SEM.