Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.

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Role of Mitochondrial Ca2+ in the Regulation of Cellular Energetics

Ions and energy in mammalian brain.

The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism.

Status of the mitochondrial pool of glutathione in the isolated hepatocyte.

Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

https://www.jneurosci.org/content/jneuro/24/36/7771.full.pdf

Generation of reactive oxygen species in the reaction catalyzed by α-ketoglutarate dehydrogenase

1 The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation 2 In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40–50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate 3 In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25% Other metabolites remain unchanged After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70% The [NAD+]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same Glucagon shifts this ratio and also that of the [NADP+]/[NADPH] couple towards a more reduced state 4 In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50% Other metabolites are not changed by glucagon Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values The [NAD+]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former 5 Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon 6 The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.

Interconversion and kinetic properties of pyruvate dehydrogenase from brain.

Kinetic and regulatory properties of heart muscle pyruvate dehydrogenase.

1
Pyruvate-dehydrogenase phosphatase was extensively purified from pig heart muscle.

2
The molecular weight was determined to be in the range between 92000 and 95000. By dodecylsulfate-gel electrophoresis there was no indication for a subunit structure of the protein.

3
The phosphatase is dependent on Mg2+ or Mn2+. Half-maximal activity was obtained, in histidine buffer, at 2.5 mM MgCl2 and 1.8 mM MnCl2, respectively.

4
Fluoride at a concentration of 0.6 mM inhibited phosphatase activity by 50% in the presence of Mg2+, but not of Mn2+. The enzyme was not affected by alkylating thiol-reagents.

5
Studies with sequestering agents suggest that pyruvate-dehydrogenase phosphatase contains a metal which, besides Mg2+ or Mn2+, is essential for its function.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb01744.x

Otto H. Wieland

Papers

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.

Interconversion and kinetic properties of pyruvate dehydrogenase from brain.

Kinetic and regulatory properties of heart muscle pyruvate dehydrogenase.

Purification and characterization of pyruvate-dehydrogenase phosphatase from pig-heart muscle.

Metabolism of isolated kidney tubules. Oxygen consumption, gluconeogenesis and the effect of cyclic nucleotides in tubules from starved rats.