title: Mechanism of unoprostone-induced cytosolic Ca2+ mobility in cultured porcine corneal endothelial cells.
abstract: The effect of unoprostone isopropyl on the intracellular free Ca(2+) signaling ([Ca(2+)](i)) in cultured porcine corneal endothelial cells was evaluated by using fura-2-AM as a Ca(2+) probe. In Ca(2+)-containing buffer and Ca(2+)-free buffer, unoprostone increased [Ca(2+)](i) concentration dependently from 28.2 to 0.282microM, diluted from original concentration to 1/100, 1/1000 and 1/10,000. The response was inhibited on extracellular Ca(2+) removal. In Ca(2+)-free buffer, pretreatment of the cells with unoprostone inhibited the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin-induced [Ca(2+)](i) increase and the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release by 96 and 95%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited unoprostone-1-induced [Ca(2+)](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 A(2) inhibitor aristolochic acid. After incubation of the cells with unoprostone in Ca(2+)-free buffer, the addition of 5mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores caused by unoprostone further induced capacitative Ca(2+) entry. These results suggest that unoprostone-induced [Ca(2+)](i) increase in corneal endothelial cells results from Ca(2+) influx from external buffer and release of Ca(2+) from intracellular stores from the endoplasmic reticulum and mitochondria Ca(2+) stores followed by capacitative Ca(2+) entry. Unoprostone-induced intracellular Ca(2+) release was also modulated by phospholipase A(2) and C-coupled events.
authors.affiliation: Department of Ophthalmology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Kwou-Yeung
authors.initials: KY
authors.lastname: Wu
authors.forename: Hwei-Zu
authors.initials: HZ
authors.lastname: Wang
authors.forename: Show-Jen
authors.initials: SJ
authors.lastname: Hong
mesh_terms: D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010740:Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology; D034421:Sus scrofa
chemical_list: D000959:Antihypertensive Agents; D001548:Benzalkonium Compounds; D010726:Phosphodiesterase Inhibitors; D011310:Preservatives, Pharmaceutical; C072630:isopropyl unoprostone; D015237:Dinoprost; D010740:Phospholipases; D002118:Calcium