Effect of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate

Abstract

1. The reduction of mitochondrial NAD(P) by 2-oxoglutarate was monitored as a measure of 2-oxoglutarate dehydrogenase activity in its intramitochondrial locale. In the absence of ADP, steady-state reduction of NAD(P) by 0.5 mM-2-oxoglutarate in the presence of 0.5 mM-L-malate was markedly increased by extramitochondrial Ca2+, with 50% activation at pCa 6.58, when the Na+ concentration was 10 mM, the Pi concentration ws 5 mM and the added Mg2+ concentration was 1 mM. Omission of Pi resulted in 50% activation at pCa 6.77; omission of Mg2+ resulted in 50% activation at pCA greater than or equal to 7.3. 2. The activation of 2-oxoglutarate dehydrogenase could be reversed on addition of an excess of EGTA. The rate of inactivation was dependent on the concentration of Na+, with K0.5 2.5 mM, which is consistent with the rate of withdrawal of Ca2+ from the mitochondria being the limiting factor. 3. The steady-state reduction of cytochrome c by 2-oxoglutarate (0.5 mM) also showed a marked dependence on pCa in the absence of ADP; in the presence of an excess of ADP, no such effect of Ca2+ was detectable. 4. Mitochondria from the hearts of senescent rats showed an undiminished rate of dehydrogenase activation by Ca2+ but a rate of inactivation by excess EGTA that was diminished by 40%. Direct studies of Ca2+ egress with Arsenazo III confirmed a decrement in rate with old age. 5. Studies of 2-oxoglutarate dehydrogenase activity as a function of the mitochondrial context of Ca2+, as measured by atomic-absorption spectrophotometry, showed half-maximal activation at a mitochondrial content of 1.0 nmol of Ca2+/mg of protein, and saturation at 3 nmol/mg. 6. These findings support the model advanced by Denton, Richards & Chin [(1978) Biochem. J. 176, 899-906], of a control of the tricarboxylate cycle by intramitochondrial Ca2+, and demonstrate the range of mitochondrial Ca2+ content over which this may occur. In addition, they raise the possibility of a disturbance of this control mechanism in old age.

Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration

“…matrix adenine nucleotide pool, but also allowed the presence of free Mg2" to modulate the activity of the Ca2+-transport cycle. It is known that Mg2" ions serve profoundly to inhibit the electrophoretic Ca21 uniport of heart mitochondria (Sordahl, 1974;Crompton et al, 1976a); Mg2+ also inhibits the Na+/Ca2+ antiport (Hansford & Castro, 1981;Crompton, 1985;Rizzuto et al, 1987), although this inhibition may be relieved by ATP at constant free Mg2+ concentration (Hayat & Crompton, 1987 (Chance & Williams, 1957). Although the PDHA content is substantially higher at any given value of [Ca2+]m in the presence of ADP (Fig.…”

Mitochondrial Ca²⁺ Transport and the Role of Intramitochondrial Ca²⁺ in the Regulation of Energy Metabolism

“…At present there is very little substantiated molecular detail about either of these Ca2+ transport proteins [14], Clearly, the absence or presence of these various regu latory factors will influence the rates at which Ca2+ cy cles across the inner m embrane, and also the distribution of the Ca2+ gradient across the mem brane [2]. This was first adequately dem onstrated in studies with intact iso lated m itochondria, which effectively used the activity of the dehydrogenases themselves as probes for matrix [Ca2+] [22,23], although as already noted NAD-ICDH has proved less useful than the other two enzymes in this regard [24], This work firstly served to dem onstrate that the Ca2+-regulatory phenomena observed with extracted enzymes was fully evident when the enzymes were located within their natural environm ent, i.e. the m itochondrial matrix.…”

Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress

“…Activation of these dehydrogenases could increase the supply of NADH to the respiratory chain and, indeed, an increased reduction of NAD C by a-ketoglutarate was monitored in rat heart mitochondria (Hansford & Castro 1981). The first evidence that an increase in the intracellular calcium concentration is able to activate mitochondrial dehydrogenases resulting in an increase in the NADH level in intact cells was provided by Duchen (1992) and Pralong et al (1992), demonstrating that NADH level is increased in cells in a calciumdependent manner in response to depolarization.…”