Prostaglandins Protect Human Intestinal Cells Against Ethanol Injury by Stabilizing Microtubules (Role of Protein Kinase C and Enhanced Calcium Efflux)

Prostaglandins (PG) protect gastrointestinalcells against damage induced by ethanol (EtOH) and othernoxious agents, a process termed cytoprotection. Thepresent study investigated the relationships between microtubule (MT) stability, protein kinase C(PKC) activation, and calcium efflux as a possiblemechanism of PG's protective action using a humancolonic cell line (Caco-2) exposed to known damagingconcentrations of EtOH (7.5% and 10% ). Preincubation ofCaco-2 cells with 16,16-dimethyl-PGE2 (PG,2.6 μM) significantly increased PKC activity in thesecells. Pretreatment of Caco-2 cells with 50 μM OAG (asynthetic diacylglycerol and PKC activator) or 30 nM TPA(a direct PKC activator) prior to exposure to 7.5% or10% EtOH for 5 min significantly reduced cell injury, asdetermined by trypan blue exclusion, and increased MT stability, as confirmed by confocalmicroscopy. Pretreatment of Caco-2 cells with 4alpha-PDD (an inactive phorbol ester, 20 nM) failed toprevent cell injury and disruption of the MTcytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM)abolished the protective effects of PG in cells exposedto 7.5% and 10% EtOH. Incubation of Caco-2 cells withA23187 (a Ca2+ ionophore), similar to 10%EtOH, caused a significant reduction in cell viability andMT stability. Preincubation with A23187 in combinationwith PG or OAG prior to subsequent exposure to EtOHsignificantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatmentwith OAG, TPA, or PG resulted in significant increasesin calcium-45 efflux, which correlated with increasedstability of the MT cytoskeleton. These data suggest that PG possesses direct protective effectsagainst EtOH injury in Caco-2 cells and may act bystabilizing MT through the PKC signal transductionpathway and/or stimulation of calcium efflux from thecells.