Ca((2+))N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry

Our aim was to develop a method to detect extramitochondrial Ca(2+) movement and O(2) fluxes simultaneously. Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca(2+) flux. Ca(2+) movement evoked by CaCl(2) or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl(2) (50 µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca(2+) uptake) with simultaneous elevation in O(2) consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca(2+) efflux) than that of the initial CaCl(2)-induced elevation. Chelation of Ca(2+) with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca(2+) 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 Ca(2+). The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O(2) and Ca(2+) fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca(2+) fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.