The Ca2+-calmodulin-Ca2+/calmodulin-dependent protein kinase II pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in rat hepatocytes
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Oxidative 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.
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