title: Diazoxide pretreatment induces delayed preconditioning in astrocytes against oxygen glucose deprivation and hydrogen peroxide-induced toxicity.
abstract: Recent studies suggest that activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) with diazoxide can protect neurons against ischemic stress. However, it is not yet known whether astrocytes, which are more resilient against ischemia, respond similarly to diazoxide. We exposed cultured astrocytes to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) with or without pretreatment with the mK(ATP) opener diazoxide. Marked decreases in astrocyte viability were evident after 9 and 12 hr of OGD [76% +/- 3% (n = 50) and 60% +/- 1% (n = 50)] and 400 and 600 microM H2O2 [40% +/- 2% (n = 16) and 25% +/- 2% (n = 16)], respectively, compared with no treatment (100% +/- 1%). Diazoxide treatment (3 days of sequential application) dramatically reversed the negative effects of OGD and H2O2, resulting in complete blockade of astrocyte cell death. Effects of diazoxide were blocked by the mK(ATP) blocker 5-hydroxydecanoic acid (5-HD). Furthermore, incubation of astrocytes with diazoxide resulted in loss of mitochondrial membrane potential monitored by tetramethylrhodamineethylester fluorescence. Additionally, generation of reactive oxygen species was observed in response to diazoxide, assessed using the oxidation-sensitive dye hydroethidine, and this effect was abolished by antioxidants, catalase, and a superoxide dismutase mimetic, M40401. Finally, diazoxide increased the protein level of phosphorylated protein kinase C (PKC) revealed by immunoblot analysis. Our findings demonstrate that opening of mK(ATP) by diazoxide identifies a delayed preconditioning effect that is protective against two types of injury in astrocytes and that diazoxide may deliver protection via mitochondrial depolarization, free radical production, and PKC activation.
authors.affiliation: Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. crajapak@wfubmc.edu
authors.forename: Nishadi
authors.initials: N
authors.lastname: Rajapakse
authors.forename: Bela
authors.initials: B
authors.lastname: Kis
authors.forename: Takashi
authors.initials: T
authors.lastname: Horiguchi
authors.forename: James
authors.initials: J
authors.lastname: Snipes
authors.forename: David
authors.initials: D
authors.lastname: Busija
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003981:Diazoxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000172:deficiency*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D019194:Ischemic Preconditioning / Q000379:methods*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D006861:Hydrogen Peroxide; D005947:Glucose; D003981:Diazoxide