(55) KINETIC ANALYSIS OF REVERSIBLE IMPAIRMENT OF OXIDATIVE PHOSPHORYLATION IN HEART MITOCHONDRIA BY CALCIUM OVERLOAD

Vida Mildaziene (1,2), Ausra Marcinkeviciute (1), Rasa Baniene (1), Sarune Morkunaite (2) and Boris Kholodenko (3)
(1) Institute for Biomedical Research, Kaunas Medical Accademy, Kaunas, Lithuania
(2) Kaunas Vytautas Magnus University, Kaunas, Lithuania
(3) Thomas Jefferson University, Philadelphia, PA

Heart mitochondria sustain an exposure to much higher concentration of external Ca2+ than liver mitochondria do. At 30 mkM external Ca2+ and in the presence of the respiratory substrate, Mg2+, ATP,ADP they are still capable to phosphorylate and to maintain the membrane potential. Although the permeability transition pore remains closed, Ca2+ overload severely affects mitochondrial oxidative phosphorylation. The rate of respiration in state 3 with succinate is inhibited by 25-30%, with pyruvate+malate - by 70%. After addition of 2 mM EGTA to mitochondria already inhibited by 30 mkM Ca2+ the rate of respiration with both substrates was slowly restored and this restoration was abolished by 200 mM diltiazem indicating that 2Na+/Ca2+ efflux is responsible for the reversal. The elasticity kinetic analysis was applied to distinguish which blocks of reactions (inner membrane permeability to ions, respiratory subsystem or phosphorylation subsystem) are effected by Ca2+ in heart mitochondria oxidizing succinate and pyruvate+malate. For both substrates, Ca2+ overload resulted in inhibition of both the respiratory subsystem and the phosphorylation subsystem and in partial uncoupling of mitochondria. By measuring the kinetics of the separate subsystems of the oxidative phosphorylation machinery we showed that the strongest effect of Ca2+ on succinate oxidation is the inhibition of the phosphorylation (-86%, as a result, the membrane potential in state 3 increases from 151 to 164 mV), respiratory subsystem is inhibited moderately (-12%), and permeability to ions increases (+216%). For pyruvate + malate, inhibition of the respiratory subsystem is much more pronounced (-72%, the membrane potential in state 3 decreases from 137 mV to 125 mV), although phosphorylation is also considerably inhibited (-78%) and ion permeability increased (+50%). Metabolic control analysis was applied to quantify the shift in the control over respiration and phosphorylation fluxes induced by calcium overload. The relative contribution of the membrane potential-producing and the membrane potential-consuming blocks of reactions to the overall response of mitochondrial respiration to Ca2+ overload was determined - the values obtained are 45% and 55% for succinate oxidation, 72% and 28% for pyruvate + malate oxidation, respectively.