These conditions appear to result in a reorganization of the MDCK cell junctions with minimal loss of junctional proteins. In the present study we have demonstrated that pharma cological inhibition of MEK1 and p38 signaling in proin flammatory cytokine stimulated MDCK cells functionally protects the barrier function. Several studies indicate that they MEK1 signaling increases paracellular permeability, there exists some disparity in observed cellular responses. Recently, a report demonstrated that inhibition of MEK1 renal epithelial cells are exposed to agents that produce necrosis and apoptosis investigators report a decrease in TER along with a subsequent increase in paracellular flux, we confirmed this finding in the MDCK system by using a combination of energy starvation and ATP depletion.

We find that exposure of MDCK cells to TNF IFN results in a decrease in ionic permeability which is reported as increased TER values, in fact when MDCK cells signaling did not influence expression of occludin or clau din 1 or affect tight junction function in several breast cancer cell lines. Also, a study using enteropathogenic Escherichia coli, showed that ERK1/2 was activated in T84 cells, but did induce tight junction barrier disruption as measured by TER. However, activation of MEK1 sig naling by H2O2 exposure in endothelial cells increased permeability and resulted in occludin disorganization. Similar effects were also observed in Caco 1 and MDCK cell lines. In this present study, activation of the ERK1/2 pathway by TNF IFN treatment produced altered ionic permeability and dynamic changes in junc tional protein expression and localization.

Additionally, we found that TNF alone potently decreased MDCK cell ionic permeability while having only minimal impact on paracellular flux. This suggests that the observed junc tional responses occur independent of apoptotic or necrotic mechanisms that likely elevate paracellular flux. Decreased ionic permeability in response to TNF or TNF IFN exposure coupled to the increased paracellular flux of non charged solutes when cytokines were pre sented in combination is intriguing. We find that inhibi tion of ERK1/2 signaling increased ionic permeability toward control levels as expected but inhibition of p38 signaling further decreased ionic permeability levels above cytokine treatment alone.

This suggests that activa Brefeldin_A tion of the p38 pathway is antagonizing ERK1/2 mediated effects on elevated TER in TNF IFN treated MDCK cells. While the MAP kinase inhibitors produced divergent effects on cellular ionic permeability measurements both inhibitors protected against increase paracellular flux of non charged solutes. Several recent reports reveal that ERK1/2 activation in MDCK II cells results in increased TER. For instance, a recent study of cyclosporine A treated MDCK cells produced elevated TER through a MAPK path way.