Mechanism of unoprostone-induced cytosolic Ca2+ mobility in cultured porcine corneal endothelial cells

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The effect of unoprostone isopropyl on the intracellular free Ca2+ signaling ([Ca2+]i) in cultured porcine corneal endothelial cells was evaluated by using fura-2-AM as a Ca2+ probe. In Ca2+-containing buffer and Ca2+-free buffer, unoprostone increased [Ca2+]i concentration dependently from 28.2 to 0.282 μM, diluted from original concentration to 1/100, 1/1000 and 1/10,000. The response was inhibited on extracellular Ca2+ removal. In Ca2+-free buffer, pretreatment of the cells with unoprostone inhibited the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-induced [Ca2+]i increase and the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca2+ release by 96 and 95%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited unoprostone-1-induced [Ca2+]i rise by 84 and 57%, respectively. The intracellular calcium release caused by unoprostone was partially inhibited by phospholipase C inhibitor (U73122) and by phospholipase A2 inhibitor aristolochic acid. After incubation of the cells with unoprostone in Ca2+-free buffer, the addition of 5 mM CaCl2 increased Ca2+ influx, implying that release of Ca2+ from internal stores caused by unoprostone further induced capacitative Ca2+ entry. These results suggest that unoprostone-induced [Ca2+]i increase in corneal endothelial cells results from Ca2+ influx from external buffer and release of Ca2+ from intracellular stores from the endoplasmic reticulum and mitochondria Ca2+ stores followed by capacitative Ca2+ entry. Unoprostone-induced intracellular Ca2+ release was also modulated by phospholipase A2 and C-coupled events.

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