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SERINE PROTEASES DECREASE INTESTINAL EPITHELIAL PERMEABILITY BY A PKCzeta MEDIATED MECHANISM

VA Swystun, WK MacNaughton
Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta

Fluid balance in the gut is maintained by the regulated movement of ions and proteins across the epithelium. Ions are actively transported across the cell membranes and can move passively at the paracellular junctions; larger molecules such as proteins are restricted from transepithelial movement unless paracellular epithelial disruption has occurred due to infection and/or inflammation. Disrupted control of ion and protein movement across the epithelium contributes significantly to the pathologies of inflammatory intestinal diseases.
We have previously demonstrated that the digestive serine proteases trypsin, elastase and chymotrypsin applied to the apical (luminal) surface of intestinal epithelial cell monolayers decrease the paracellular permeabilities of Na⁺, Cl and a 3000MW dextran molecule. Viewed as a possible barrier-protective effect, we investigated the mechanisms involved. Changes in transepithelial electrical resistance (R_TE) across intestinal epithelial cells (SCBN) were measured in Ussing chamber experiments, employing several activators and inhibitors of signaling pathways. Activators of protease activated receptors (PARs)-1, -2 and -4 were without effect. Inhibitors of phosphoinositol-specific phospholipase-C (PLC), U73122 (50 µM) and ET-18-OCH₃ (10 µM), attenuated the trypsin-induced increase in R_TE by 92 ± 33 and 78 ± 24 % respectively. Protein kinase C enzymes are downstream effectors of PLC. Activation of typical and novel PKC isoforms with PMA (1 µM) did not increase R_TE and two broad spectrum inhibitors of PKCs did not inhibit the trypsin-induced increase in R_TE. However, an inhibitor specific for the atypical isoform PKCzeta (myristoylated pseudosubstrate inhibitor) significantly decreased baseline R_TE and prevented the trypsin-induced increase in R_TE (786 ± 118 ohm x cm² vs.138 ± 36 ohm x cm²; n = 18 and 9; p < 0.01). In accordance with the Ca²⁺-independence of PKCzeta activation, pre-treatment of the epithelial cells with the Ca²⁺-modulating agents BAPTA-AM or 2-APB did not affect the trypsin-mediated increase in R_TE.
These data indicate a novel role for luminal digestive serine proteases in the regulation of intestinal epithelial barrier function. The effect Is independent of PARs and is mediated by PLC and PKCzeta.
VA Swystun is supported by a CAG-CCFC Postdoctoral Fellowship

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