2018-01-302018-01-302014-03-110340-5761http://hdl.handle.net/2133/10485Oxidative stress is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both oxidative stress from mitochondrial origin and cell death. Intracellular Ca2+ elevations play 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) signalling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidizing, 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 clyclosporin A (CsA)-sesitive manner, revealing the involvement of MPTPs in tBOOH-induced 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 oxidative stress-induced induced MPTP formation, and the subsequent exacerbation of oxidative stress from mitochondrial origin and apoptotic cell death.application/pdf1695 - 1709engopenAccessOxidative Stresstert-Butyl HydroperoxideCa2+/calmodulin-dependent Protein Kinase IIMitochondrial Permeability Transition PoreApoptosisCytochrome cCaspasasarticleSpringerGerman Society for Experimental and Clinical Pharmacology and Toxicology (DGPT)Universidad Nacional de RosarioToledo, Flavia D.Pérez, Leonardo M.Basiglio, Cecilia LorenaOchoa, Justina E.Sánchez Pozzi, Enrique J.Roma, Marcelo GabrielAttribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)