The Ca2+-calmodulin-Ca2+/calmodulin dependent protein kinase II signaling pathway is involved in oxidative stressinduced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes

TOLEDO, FD; PEREZ, LM ; BASIGLIO, CL; OCHOA, JE; SÁNCHEZ POZZI, EJ; ROMA, MG

ARCHIVES OF TOXICOLOGY.

SPRINGER

Lugar: Berlin; Año: 2014 vol. 88 p. 1695 - 1709

0340-5761

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 inthese 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 mitochondrialROS 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 werepartially 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 subsequentexacerbation of OS from mitochondrial origin and apoptotic cell death.