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.

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2)

“…The results showed indeed an increased GSSG extrusion in BZL-treated cells that was prevented by the MRP2 inhibitor MK571. GSSG efflux was not increased when cells were only incubated with tBOOH, a situation resembling ROS and GSSG increases by BZL (Toledo et al 2014) but not associated to MRP2 induction (unpublished results). Thus, enhanced MRP2 expression and not higher substrate availability seems to play the most important role in GSSG clearance in conditions of BZL-triggered oxidative stress.…”

“…Only DMSO (0.1% v/v) was added to control cells (C). Tert-butyl hydroperoxide (tBOOH, 500 μM, 15 min) was used as a positive control of reactive oxygen species (ROS) generation (Toledo et al 2014). The concentration-dependence of BZL effects on intracellular redox status was evaluated incubating the cells with BZL 50, 100 and 200 μM for 24 h. Then, glutathione levels were quantified as described below.…”

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells

“…Accumulating evidence has implicated that transient receptor potential canonical 3 is responsible for the increase in Ca 2+ /calmodulin-dependent kinase II (CaMKII) activity and ROS production (Kitajima et al 2011). Further study showed that mitochondrial ROS generation was mediated by Ca 2+ /CaM/CaMKII signaling pathway (Toledo et al 2014). We speculate that the increased ROS formation might be via Ca 2+ /CaM/CaMKII signaling pathway which is elicited by TRPA1 activation induced by cold stimulation.…”

Highlight report: toxicology of copper

“…However, it remains to be studied, whether these changes represent primary events caused by copper or whether they are consequences of copper-induced oxidative stress (Gaetke et al 2014). Currently neurotoxicity (Balmer et al 2014;Zimmer et al 2014;Krug et al 2013;Leist et al 2013;Krause et al 2013), hepatotoxicity (Campos et al 2014Liu et al 2014;Godoy et al 2009Godoy et al , 2013Godoy et al , 2015Schyschka et al 2013;Schliess et al 2014;Grinberg et al 2014;Ghallab 2013Ghallab , 2014a and control mechanisms of oxidative stress (Møller et al 2014;Lim et al 2014;Finazzi and Arosio 2014;Wu et al 2014;Toledo et al 2014;Li Volti et al 2006) represent cutting-edge topics in toxicology. The present review of Gaetke and colleagues is particularly interesting for everyone involved in these fields of research.…”