AJP - Cell Physiology, Vol 259, Issue 1 C69-C76, Copyright © 1990 by American Physiological Society
A23187 increases permeability of MDCK monolayers independent of phospholipase activation
M. W. Peterson and D. Gruenhaupt
Department of Medicine, College of Medicine, University of Iowa, Iowa City 52240.
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.
