in-1, is decreased 5 Reversible TJ Open by Cofilin-Actin and Occludin in fractions 1115, which contain TJ protein complexes following capsaicin treatment. These results suggest that the decrease in the level of occludin observed in total lysates following capsaicin treatment reflects a 
decrease in occludin complex formation within TJ. Relationship between the Signaling Pathways Involved in the Capsaicin-dependent Activation of Cofilin and the Decreased Level of Occludin The analysis described above suggests that capsaicin modulates two major molecules in TJ structures: capsaicin decreases and changes the F-actin distribution, and decreases the level of occludin in TJ. Therefore, unlike the actin-depolymerizing factors that induce TJ opening through the loss of actin network, capsaicin seems to increase TJ permeability by modulating not only actin but also TJ structure, i.e., by altering the levels and interactions of the proteins constituting the TJ. Therefore, the relationship between these two signaling pathways was studied, and the contribution of F-actin and occludin to the capsaicin2883-98-9 web induced decrease in TER was assessed. First, the effect of cofilin activation on the decrease in the level of occludin was analyzed, and vice versa. Cofilin is expected to be the upstream molecule 12411425 in the capsaicin-induced F-actin reorganization. LIMK, which phosphorylates and inactivates cofilin, and cofilin were introduced into MDCK cells, and stable transfectants were established. In the cofilin stable transfectants, cofilin dephosphorylation induced by capsaicin was reduced. The same was observed in LIMK transfectants. However, the capsaicin-induced decrease in occludin was not changed. Next, occludin and claudin-1 were stably expressed in MDCK monolayers with fluorescent protein tags. The distribution of these fusion proteins was assessed by microscopic analysis: the tagged proteins showed the ring-like structures as their endogenous counterparts. Since stable occludin expression produced one clone with similar TER as non-transfected MDCK cells and other clones with a,30% increase in TER, two clones Reversible TJ Open by Cofilin-Actin and Occludin are shown for occludin. Claudin-1 expression had no effect on the decrease in occludin or cofilin dephosphorylation induced by capsaicin. Following the overexpression of occludin, the decrease in endogenous occludin induced by capsaicin was not inhibited, but exogenous EGFP-occludin overwhelmed the decrease; however, cofilin dephosphorylation was not affected. In addition, trifluoperazine inhibited capsaicin-induced cofilin dephosphorylation by blocking calcium signaling, but the occludin decrease was not affected. These observations suggest that the capsaicin-induced 12750467 cofilin dephosphorylation and decrease in the level of occludin are independent events. Cofilin Activation and a Decreased Level of Occludin Contribute to the TER Decrease Induced by Capsaicin Next, the effect of each of these two independent phenomena on the TER decrease was investigated. Vector control, cofilin and Reversible TJ Open by Cofilin-Actin and Occludin correlating the level of cofilin phosphorylation. Taken together, these observations suggest that cofilin activation and a decreased level of occludin are independent of one another but that both of them contribute significantly to the decrease in TER induced by capsaicin. The actin reorganizatino is correlated to the level of cofilin activation, suggesting that the cofilin-actin mo
