A23187 increases permeability of MDCK monolayers independent of phospholipase activation

Abstract

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.

Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions

“…The described alterations in epithelial barrier function may result from the triggering of signal transduction pathways, e.g., growth factor activation, protein phosphorylation, phospholipase activation, and increased intracellular calcium and inositol phosphate fluxes, all of which have been described during invasion of other cell lines (e.g., HeLa and Henle-407) by S. typhimurium (10,11,15,17). Indeed, some of these components of the signalling pathways previously demonstrated or inferred to be stimulated by S. typhimurium increase paracellular permeability in MDCK cells, e.g., activation of phospholipase C and protein kinase C (3,18), increased intracellular calcium levels (16), and an increase in protein tyrosine phosphorylation (5). At present there is a paucity of information regarding the precise signalling events occurring in MDCK cells following S. typhimurium infection, while it is known that Salmonella species induce different signalling events in a variety of nonpolarized cell lines (10).…”

Possible Role of Arachidonic Acid in Stress‐Induced Cytochrome P450IA1 Activity1

“…Caffeic acid (CA) is a lipoxygenase inhibitor (Koshihara et al, 1984; Miller et al, 1989), and the antioxidant, nordihydroguaiaretic acid (NDGA), inhibits both the lipoxygenase and cytochrome P‐450 metabolic pathways (Capdevila et al, 1988; Agarwal et al, 1991). In addition, PLA 2 can be blocked by quinacrine, thereby preventing the release of AA (DeGeorge et al, 1987; Peterson and Gruenhaupt, 1990). Glucocorticosteroids such as dexamethasone or cortisol have been reported to be effective in blocking either PLC, PLA 2 , or both in different cell systems (DeGeorge et al, 1987; Peterson and Gruenhaupt, 1990; Clark et al, 1986).…”

“…In addition, PLA 2 can be blocked by quinacrine, thereby preventing the release of AA (DeGeorge et al, 1987; Peterson and Gruenhaupt, 1990). Glucocorticosteroids such as dexamethasone or cortisol have been reported to be effective in blocking either PLC, PLA 2 , or both in different cell systems (DeGeorge et al, 1987; Peterson and Gruenhaupt, 1990; Clark et al, 1986). Furthermore, the involvement of PLD can be deduced by the addition of ethanol which prevents the formation of PA and preferentially forms phosphatidylethanol (Peth) (Moehren et al, 1994; Carnero et al, 1994).…”

Physiological and Cytotoxic Effects of Ca²⁺ Ionophores on Caco-2 Paracellular Permeability: Relationship of 45Ca²⁺ Efflux to ⁵¹Cr Release

“…Specifically, this involves the application of Ca2+ ionophores (e.g. A23187 or ionomycin) to tissues followed by mea surements of the change in Rt or nonelectro lyte flux [11,14,32].…”