SYNERGISTIC EFFECTS OF A CALCIUM IONOPHORE AND ACTIVATORS OF PROTEIN-KINASE-C ON EPITHELIAL PARACELLULAR PERMEABILITY

Oxidants reversibly increase the paracellular permeability of Madin Darby canine kidney (MDCK) epithelial cell monolayers, and the decrease in resistance occurs within 10 to 15 min of initiating oxidant exposure. Oxidants also initiate hydrolysis of phosphatidylinositol in MDCK cells, with resultant increases in diacylglycerol and inositol phosphates. Phorbol esters and synthetic diacylglycerols increase the paracellular permeability of MDCK monolayers with a time course similar to the oxidants. In contrast, calcium ionophores increase MDCK monolayer paracellular permeability only after 2 to 3 h of exposure. Because the products of the oxidant-initiated phospholipid hydrolysis would be likely to both activate protein kinase C and increase cell calcium, we asked if ionomycin, a calcium ionophore, and phorbol esters or diacylglycerols, activators of protein kinase C, might not act in concert to alter MDCK monolayer paracellular permeability. When ionomycin was added alone to MDCK monolayers, there was an increase in cell calcium, activation of a lumen negative current, a limited transitory decrease in transepithelial resistance, but no increase in mannitol flux across the monolayers. When phorbol dibutyrate (PDBU) or oleyl acetyl glycerol (OAG) were added to MDCK monolayers, there was no current activated, there was a progressive decrease in trans-epithelial resistance, and there was an increase in mannitol flux across the monolayers which was evident within 20 to 40 min of adding the agent. When 1-mu-M ionomycin was added to the monolayers along with PDBU or OAG, there was a synergistic increase in paracellular permeability of the monolayers when compared to addition of ionomycin, PDBU, or OAG alone. Inhibition of the lumen negative current with barium did not alter the increase in epithelial permeability. The synergistic effect of ionomycin and PDBU on paracellular permeability was paralleled by a synergistic increase in diacylglycerol formation in the cells. Agents that cause both an increase in cell calcium and activation of protein kinase C might be expected to have marked effects on epithelial paracellular permeability.