Respiration-dependent calcium ion uptake by two preparations of cardiac mitochondria. Effects of palmitoyl-coenzyme A and palmitoylcarnitine on calcium ion cycling and nicotinamide nucleotide reduction state

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Abstract
Ca2+ uptake and the effect of the uptake inhibitors palmitoyl-CoA and palmitoylcarnitine were examined in 2 pareparations of dog cardiac mitochondria. Mitochondria prepared using the Nagarse technique was 2.5-fold more active in respiration-dependent Ca2+ uptake than were mitochondria isolated by using the Polytron procedure. Palmitoyl-CoA and palmitoylcarnitine inhibited Ca2+ uptake in both preparations uncompetitively, with Ki,app [apparent inhibition constant] 0.4 and 20 .mu.M. Ca2+-uptake rates were related to, or influenced by, the concentration of mitochondrial reduced nicotinamide nucleotides, with uptake slowing as this concentration decreased. When most of the nicotinamide nucleotides were oxidized, Ca2+ release and respiratory stimulation were observed. In the presence of Ruthenium Red and palmitoyl-CoA, oxidation of nicotinamide nucleotides was abolished and the time to Ca2+ release was shortened corresponding to the time of onset of nicotinamide nucleotide oxidation in the absence of Ruthenium Red. NAD(P)H oxidation in the presence of rotenone was evidently a consequence of Ca2+ reuptake; net Ca2+ release could be observed as reduced nicotinamide nucleotide concentrations declined. Although nicotinamide nucleotide oxidation occurred in the presence of rotenone, it was not linked in an apparent manner to acyl-group metabolism (palmitoylcarnitine was less effective than palmitoyl-CoA). Therefore either a bypass of the rotenone block or a direct interaction of NAD(P)H with the Ca2+-uptake process was possible. Loss of NADH occurred before respiratory stimulation, and this loss may relate to decreased coupling efficiency at sites 2 and 3 of the respiratory chain, as suggested by others.