Caveolin-1 is required for TNF-induced occludin internalization, barrier dysfunction, and net water secretion. (A) Caveolin-1^+/+ and caveolin-1^−/− mice were injected with vehicle or TNF as indicated, and a segment of jejunum harvested 135 min later. Sections were labeled for occludin (green), F-actin (red), and nuclei (blue). Bar, 10 µm. (B) Electron micrographs demonstrate TNF-induced perijunctional actomyosin condensation in jejunal enterocytes of caveolin-1^+/+ and caveolin-1^−/− mice. Bar, 125 nm. (C) Jejunal epithelial cells were isolated from caveolin-1^+/+ and caveolin-1^−/− mice 135 min after injection with vehicle or TNF. Cell lysates were analyzed by immunoblotting for phosphorylated and total MLC. (D) Jejunum was harvested 135 min after caveolin-1^+/+ and caveolin-1^−/− mice were injected with vehicle or TNF. Sections were labeled for phosphorylated MLC (green) and nuclei (blue). Bar, 10 µm. (E) Mucosal TNF mRNA transcripts measured by quantitative RT-PCR were increased by TNF injection in jejunum of caveolin-1^+/+ and caveolin-1^−/− mice. (F) Cell lysates were analyzed by immunoblotting for occludin, caveolin-1, clathrin heavy chain, and E-cadherin. Protein content was not affected by acute TNF exposure. (G) In vivo perfusion assays show that TNF increases paracellular flux in caveolin-1^+/+ mice but not in caveolin-1^−/− mice. (H) TNF reverses the direction of water movement from net absorption to net water secretion in caveolin-1^+/+ but not caveolin-1^−/− mice (n = 6). Error bars indicate mean ± SEM.