IBIMOL   23987
INSTITUTO DE BIOQUIMICA Y MEDICINA MOLECULAR PROFESOR ALBERTO BOVERIS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Nitric oxide regulation of heart mitochondrial bioenergetics and calcium handling
Autor/es:
ZAOBORNYJ T; SIVAKUMARAN V; O´ROURKE B
Reunión:
Conferencia; 17th European Bioenergetics Conference - EBEC 2012; 2012
Resumen:
Nitric oxide (NO) is a free radical produced within the heart by NO synthases (NOS) located in different compartments. The localization of mitochondrial NOS within the organelles in charge of energy metabolism allows for a tight control of respiration, ATP and superoxide anion production, apoptosis, mitochondrial biogenesis, mitoKATP activation and MPT formation. In turn, Ca2+ plays a central role in energy supply and demand matching by transmitting changes in excitation-contraction coupling to mitochondria. The uptake of Ca2+ is accomplished by the mitochondrial Ca2+ uniporter (MCU) and the extrusion by the mitochondrial Na+/Ca2+ exchanger. Ca2+ activates NADH production in the Krebs cycle and triggers MPT in cell death. Heart mtNOS activity was measured in permeabilized myocytes and in energized mitochondria and was shown to depend on mitochondrial Ca2+ concentration. In this study, the effects of NO on Ca2+ influx were characterized by exposing isolated guinea pig heart mitochondria to physiological and pathological concentrations of NO released from two NO donors: GSNO and SNAP. Effective NO concentrations released by the donors were assessed by EPR. Mitochondrial NO production, electrochemical potential and Ca2+ uptake were followed simultaneously in a fluorometer (Quantamaster, Photon Technologies International) using DAF-FM, TMRE, Calcium Green and fura-FF as probes. Isolated mitochondria O2 consumption was assessed using a XF96 analyzer (Seahorse Bioscience). State 4 O2 uptake was not modified by 500 uM GSNO or SNAP. The addition of Ca2+ to the respiration medium produced a 20% enhancement in state 4 O2 consumption and this effect was abolished by GSNO or SNAP. Control energized mitochondria were submitted to 10 M Ca2+ pulses up to a final concentration of 80-100 uM (200-450 nmol/mg protein), showing no significant alterations in matrix volume and electrochemical potential. In the presence of GSNO or SNAP (25 to 100 uM), Ca2+ uptake was slower and extramitochondrial Ca2+ concentration increased. When single 50 uM Ca2+ pulses were added, mitochondria treated with NO donors for 2 min showed a decreased (40-50%) accumulation rate of Ca2+ with an IC50 of around 400 uM (180 nM NO). The addition of L-arginine or NOS inhibitors (L-NAME and nNOS inhibitor I) to control mitochondria produced slight changes in Ca2+ uptake and in DAF-FM signal. These results suggest that Ca2+ and NO may act as signals that coordinate changes in cytosolic workload and mitochondrial energy metabolism in the heart.