Prostaglandins protect human intestinal cells against ethanol injury by stabilizing microtubules: role of protein kinase C and enhanced calcium efflux.
Prostaglandins (PG) protect gastrointestinal cells against damage induced by ethanol (EtOH) and other noxious agents, a process termed cytoprotection. The present study investigated the relationships between microtubule (MT) stability, protein kinase C (PKC) activation, and calcium efflux as a possible mechanism of PG's protective action using a human colonic cell line (Caco-2) exposed to known damaging concentrations of EtOH (7.5% and 10%). Preincubation of Caco-2 cells with 16,16-dimethyl-PGE2 (PG, 2.6 microM) significantly increased PKC activity in these cells. Pretreatment of Caco-2 cells with 50 microM OAG (a synthetic diacylglycerol and PKC activator) or 30 nM TPA (a direct PKC activator) prior to exposure to 7.5% or 10% EtOH for 5 min significantly reduced cell injury, as determined by trypan blue exclusion, and increased MT stability, as confirmed by confocal microscopy. Pretreatment of Caco-2 cells with 4 alpha-PDD (an inactive phorbol ester, 20 nM) failed to prevent cell injury and disruption of the MT cytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM) abolished the protective effects of PG in cells exposed to 7.5% and 10% EtOH. Incubation of Caco-2 cells with A23187 (a Ca2+ ionophore), similar to 10% EtOH, caused a significant reduction in cell viability and MT stability. Preincubation with A23187 in combination with PG or OAG prior to subsequent exposure to EtOH significantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatment with OAG, TPA, or PG resulted in significant increases in calcium-45 efflux, which correlated with increased stability of the MT cytoskeleton. These data suggest that PG possesses direct protective effects against EtOH injury in Caco-2 cells and may act by stabilizing MT through the PKC signal transduction pathway and/or stimulation of calcium efflux from the cells.
Banan
A
A
Theodore Cooper Surgical Research Institute, Department of Surgery, Saint Louis University Health Sciences Center, Missouri 63104, USA.
Smith
G S
GS
Deshpande
Y
Y
Rieckenberg
C L
CL
Kokoska
E R
ER
Miller
T A
TA
D018938:Caco-2 Cells; D020013:Calcium Signaling; D002492:Central Nervous System Depressants / Q000633:toxicity*; D003106:Colon / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D015232:Dinoprostone / Q000494:pharmacology*; D000431:Ethanol / Q000633:toxicity*; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects* / Q000473:pathology / Q000648:ultrastructure; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction
D002492:Central Nervous System Depressants; D000431:Ethanol; D011493:Protein Kinase C; D015232:Dinoprostone