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49 TRYPSIN DECREASES PARACELLULAR CONDUCTANCE OF CL- AND NA+ IN INTESTINAL EPITHELIAL CELLS VA Swystun, WK MacNaughton Epithelial cells modulate fluid dynamics in various tissues by forming a selective barrier and regulating ion transport across this barrier. Ions are actively transported transcellulary and diffuse passively through the paracellular pathway. Paracellular ion conductance may be important in the regulation of ion and fluid homeostasis. Recently, it has been shown that the serine proteases trypsin and neutrophil-derived elastase increase electrical transepithelial resistance of human lung epithelial cell monolayers. We hypothesized that this increased resistance is conferred by decreased ion conductance at the paracellular level. To test this hypothesis, we performed ion gradient experiments in Ussing chambers using a chloride-secreting intestinal epithelial cell line (SCBN).
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta
Cells were grown to confluence on 12 mm transwell membranes and mounted into Ussing chambers. Cl- gradients were established by using Cl--free (Na+-gluconate) solution in the apical chambers and normal Kreb's solution in the basolateral chambers. Na+ gradients were established by using Na+-free (NMDG-Cl) solution in the basolateral chambers and normal Ringer's solution in the apical chambers. Transcellular ion transport was inhibited with glybenclamide (200 µM) and amiloride (10 µM) in the apical chambers and bumetanide (50 µM) added to the basolateral chambers. Trypsin (2.5 µM) was then added to the apical chambers and changes in short-circuit current (ISC) were measured.
Both ion gradients resulted in increases in ISC, which were insensitive to ion channel inhibition. This ISC was significantly reduced with the addition of trypsin in the Cl- gradient from 4.7±0.4 µA/cm2 to 3.8±0.3 µA/cm2 (P<0.0001; n=10), and in the Na+ gradient trypsin decreased ISC from 10.7±1.2 µA/cm2 to 6.5±0.6 µA/cm2 (n=13; P=0.0017). The ISC in control cells not treated with trypsin did not change. These decreases in conductance were accompanied by significant increases in transepithelial resistance (RTE).
These data show that pancreatic trypsin, applied to the apical surface of intestinal epithelial cell monolayers, decreases the conductance of Na+ and Cl- at the paracellular level as indicated by decreases in ISC measured in the presence of ion gradients and a concomitant increase in RTE. This indicates a role for trypsin in modulating intestinal permeability.