title: Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter.
abstract: Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20 min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca(2+) uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca(2+) uptake and suppressed the Ca(2+)-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca(2+) uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.
authors.affiliation: Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Schwartz
authors.forename: Ekhson
authors.initials: E
authors.lastname: Holmuhamedov
authors.forename: Xun
authors.initials: X
authors.lastname: Zhang
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Lovelace
authors.forename: Charles D
authors.initials: CD
authors.lastname: Smith
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D000860:Hypoxia / Q000517:prevention & control; D007501:Iron / Q000493:pharmacokinetics; D008297:Male; D008911:Minocycline / Q000494:pharmacology*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000517:prevention & control*; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D015220:Calcium Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; C034703:mitochondrial calcium uniporter; D007501:Iron; D013752:Tetracycline; D008911:Minocycline; D004318:Doxycycline; D002118:Calcium
keywords: Calcium; CsA; Doxycycline; I/R; IAA; KRH; Krebs–Ringer–HEPES; MCU; MMP; MPT; Minocycline; Mitochondria; OA-Hy; PI; ROS; Rh123; Uniporter; cis-9-octadeconyl-N-hydroxylamide; cyclosporin A; iodoacetic acid; ischemia/reperfusion; matrix metalloprotease; mitochondrial calcium uniporter; mitochondrial permeability transition; propidium iodide; reactive oxygen species; rhodamine 123