Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo
J. Clin. Invest. Daniel R. Clayburgh, et al. 116:2682 doi:10.1172/JCI29218 [Go to this article.]

Figure 2
TNF and LIGHT mediate epithelial barrier dysfunction via MLCK activity. (A) Either TNF or LIGHT treatment increases MLC phosphorylation in jejunal epithelia. PIK prevented TNF- and LIGHT-mediated increases in MLC phosphorylation. (B) Either TNF or LIGHT causes increased BSA flux. PIK prevented increases in BSA flux after TNF or LIGHT treatment, indicating that epithelial barrier dysfunction after TNF or LIGHT treatment requires MLCK. (C) TNF causes net water secretion, while LIGHT increases net water absorption. PIK restored net absorption after TNF treatment, although a significant quantitative defect remained (P = 0.01). PIK reduced normal water absorption to control levels in animals treated with LIGHT. MLCK-mediated barrier dysfunction is therefore responsible for the changes in water flux due to LIGHT but does not completely account for changes due to TNF. (D) Immunofluorescent localization of occludin (red), F-actin (green), and nuclei (blue) in the jejunal epithelium shows that in control mice (top panels), occludin was localized to apical cell-cell junctions. After either TNF or LIGHT injection (middle and bottom panels), intracellular occludin inclusions were also present (white arrows). Scale bar: 5 μm. (E) Immunofluorescent localization of occludin (red), F-actin (green), and nuclei (blue) in the jejunal epithelium of mice treated with the MLCK inhibitor PIK demonstrates that occludin was localized exclusively at apical cell-cell junctions, indicating that PIK treatment prevented TNF- and LIGHT-mediated occludin internalization. Scale bar: 5 μm.