title: The Ca²⁺-calmodulin-Ca²⁺/calmodulin-dependent protein kinase II signaling pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes.
abstract: Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²⁺ increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 μM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²⁺ sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²⁺/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²⁺/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²⁺/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.
authors.affiliation: Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET-Universidad Nacional de Rosario, Suipacha 570, 2000, Rosario, Argentina.
authors.forename: Flavia D
authors.initials: FD
authors.lastname: Toledo
authors.forename: Leonardo M
authors.initials: LM
authors.lastname: Pérez
authors.forename: Cecilia L
authors.initials: CL
authors.lastname: Basiglio
authors.forename: Justina E
authors.initials: JE
authors.lastname: Ochoa
authors.forename: Enrique J
authors.initials: EJ
authors.lastname: Sanchez Pozzi
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Roma
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D008933:Mitochondrial Swelling / Q000187:drug effects; D016877:Oxidants / Q000037:antagonists & inhibitors / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000037:antagonists & inhibitors / Q000633:toxicity
chemical_list: D002147:Calmodulin; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D016877:Oxidants; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2