Involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in hepatocellular mitochondrial permeability transition (MPT) and apoptosis induced by radical oxygen species (ROS).

ROMA M.G.; PÉREZ L.M.; TOLEDO F.; OCHOA E.J.; SÁNCHEZ POZZI E.J.

Barga (Lucca), Italia

Conferencia; Gordon Research Conference: Thiol-Based Redox Regulation & Signaling.; 2008

Gordon Research Conference

MPT is a key event in mitochondrial ROS generation and ROS-induced hepatocellular death via apoptosis. Although it is known that Ca2+ plays a facilitating role in these processes, its action mechanism is largely unknown. We examined the involvement of Ca2+-mediated CaMKII activation in these ROS-mediated events. The pro-oxidant agent, tert-butylhydroperoxide (tBOOH, 500 µM), elevated cytosolic Ca2+ levels by 2623%, and induced significant oxidative stress, as indicated by the increase in lipid peroxidation (+723%) and in the intracellular GSSG/GSH ratio (+131%); p values lower than 0.01. The specific MPT blocker CsA reduced tBOOH-induced lipid peroxidation to a similar extent to BAPTA (a cytosolic Ca2+ chelator), W7 or trifluoperazine (two CaM inhibitors), or KN62 (a CaMKII inhibitor); CsA showed no additivity when added together with W7 or KN-62, suggesting specific CaMKII-signaling dependency of MPT. In line with this, all these signaling modulators counteracted to a similar extent to CsA tBOOH-induced MPT, as assessed by measuring mitochondrial membrane depolarisation, a surrogate MPTP marker assessed by using tetra-methyl-rhodamine methyl ester as a fluorescent probe (ca. –30%, p values lower than 0.025). tBOOH increased apoptosis from 6±1% in controls to 50±4%, as revealed by quantifying the number of cells displaying membrane-associated FICT-anexin V fluorescence. The CaM inhibitor W7 and the CaMKII inhibitors KN62 both extensively prevented tBOOH-induced apoptosis to only 8±1% and 14±2%, respectively (p value lower than 0.01). Involvement of Ca2+-dependent (“classical”) PKC isoforms and calpains was ruled out, due to the lack of effects of their specific inhibitors, Gö6976 (2 µM) and ALLN (50 µM), respectively, on tBOOH-induced lipid peroxidation and MPT occurrence. Similarly, the involvement of calcineurin, a CsA-sensitive, CaM-dependent protein with MPT pore-opening properties, was discarded, as its specific inhibition with FK506 (1 µM) was without effect. We concluded that Ca2+ facilitates tBOOH-induced oxidative hepatocellular damage via MPT, by a mechanism involving CaM formation and further CaMKII activation.