RESUMO
Our aim was to develop a method to detect extramitochondrial Ca2+ movement and O2 fluxes simultaneously. Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca2+ flux. Ca2+ movement evoked by CaCl2 or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl2 (50 µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca2+ uptake) with simultaneous elevation in O2 consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca2+ efflux) than that of the initial CaCl2-induced elevation. Chelation of Ca2+ with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca2+ movement was observed, not only in isolated liver mitochondria, but also in tissue homogenates. In all samples, the transition to anoxia resulted in immediate increase in the level of extramitochondrial Ca2+. The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O2 and Ca2+ fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca2+ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.