Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)
We have previously reported that exposing cultured Madin Darby canine kidney (MDCK) cells to the polycation protamine (PRO) results in increased short-circuit current and decreased barrier integrity as measured by mannitol permeability and transepithelial electrical resistance. To further investigate the interaction of PRO with the surface of epithelial cells, we labeled PRO with [14C] with use of reductive alkylation. [14C]PRO bound to the cells in a biphasic pattern. Approximately 10% of the [14C]PRO was bound to the cells in the first 5 min, followed by an additional 10% that was bound over the next 25 min. No additional [14C]PRO bound to the cells after the initial 30 min. Binding of [14C]PRO was inhibited by "cold" PRO, which suggested specificity. Binding was also inhibited by polyanions, serum, and albumin, agents previously found to protect MDCK cells from PRO-induced injury. The binding of PRO to MDCK cells was not inhibited by incubation of the MDCK cells with neuraminidase, to remove surface sialic acid residues, or with heparinase, to remove surface heparan sulfate, even though metabolic labeling experiments demonstrated that neuraminidase decreased cell sialic acid and heparinase decreased cell heparan sulfate. Neuraminidase and heparinase offered no protection from PRO injury and had no effect themselves on mannitol permeability. Incubation of the cells with trypsin, however, blunted both the binding of PRO to the cells and the increase in mannitol permeability after exposure of the cells to PRO.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in intracellular calcium influence epithelial barrier integrity, but the mechanism of action is unknown. One possibility is that calcium may work by increasing phospholipase A2 (PLA2) and/or phospholipase C (PLG) activity. Measuring the mannitol permeability (Pmann) of cultured monolayers of Madin-Darby canine kidney (MDCK) epithelium cells as a measure of barrier integrity, we found that exposure of the monolayers to 5 and 10 microM A23187 produced an increase in Pmann whereas 1 microM A23187 did not. Exposure of MDCK cells labeled with [3H]arachidonate to A23187 resulted in an increase in both PLA2 activity, as measured by an increase in free fatty acids, and in PLC activity, as measured by an increase in diacylglycerol (DAG). The increase in DAG was due to an increase in phosphatidylcholine-specific PLC activity. The relationship of phospholipolysis to Pmann was evaluated further by the use of mepacrine and dexamethasone. Mepacrine (10 microM) decreased PLA2 activity by 60% but had no effect on increased Pmann after exposure to A23187. Preexposure of the monolayers to dexamethasone (10 microM) blocked both PLA2 activity and PLC activity and also prevented the increase in Pmann after exposure to A23187. To evaluate whether this protective effect of dexamethasone was due to PLC blockade, we incubated the cells with the protein kinase C blocker H-7. Incubation with H-7 offered no protection from increased Pmann after A23187. These results demonstrate that increased intracellular calcium decreases the barrier integrity of epithelium and increases both PLA2 and phosphatidylcholine-specific PLC activity. The increase in Pmann, however, appears to occur through mechanisms other than phospholipase activation.
Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with 45Ca2+ and changes in Ca2+ flux were monitored by the release of 45Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of 45Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of 45Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.
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