H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

Abstract

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

“…It is therefore possible that the PMCA has an intimate dependence on functional mitochondria. In addition, oxidants have been shown extensively to impair mitochondrial function (30,54). We therefore examined the effects of three mechanistically distinct mitochondrial inhibitors to test if these have similar effects to H 2 O 2 .…”

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

“…CCK can evoke oscillatory Ca 2+ signals and substantial mitochondrial Ca 2+ uptake in pancreatic acinar cells [4041]. H 2 O 2 can cause mitochondrial Ca 2+ release abolished by pretreatment of FCCP or CCCP, a mitochondrial uncoupler [4041]. However, in another study, mitochondrial Ca 2+ uptake did not occur in unstimulated resting cells [18].…”

Interferences of resveratrol with fura-2-derived fluorescence in intracellular free-Ca2+ concentration determinations

“…It has been previously suggested that, when working with dual wavelength-excited fluorescent dyes, if one of the lines is affected by the experimental procedures, the wavelength not affected can be successfully employed for the determinations (Gonzalez et al 2005). Following these directions, data of fluorescence at 380 nm excitation light were normalized to the basal (resting) fluorescence values.…”