Characteristics of Ca2+ transport by corn mitochondria

Mitochondria isolated from corn (Zea mays L.) coleoptiles by an improved procedure which yields functionally intact preparations are much more active in respiration-coupled Ca²⁺ accumulation than those employed in most earlier studies. Ca²⁺ uptake by these mitochondria is phosphate-dependent and is accompanied by decrease in Δψ, H⁺ extrusion and increase in the rate of respiration. A sigmoidal plot with a Hill coefficient of 2.22 was obtained when the rates of Ca²⁺ uptake were plotted as a function of free Ca²⁺ concentration. The K_0.5 for Ca²⁺ influx was about 31 μM and a V_max of 140 nmol Ca²⁺ per min per mg was attained at a free-Ca²⁺ concentration of about 120 μM. Ca²⁺ uptake is sensitive to inhibition by ruthenium red and Mg²⁺. The external free-Ca²⁺ concentration maintained at steady state was about 2 μM and was independent of the respiratory substrate and of external Na⁺, but was increased by exogenous Mg²⁺. In addition, this preparation of corn mitochondria has shown a much higher ability for Ca²⁺ retention in the presence of phosphate and NAD(P)H oxidants than liver mitochondria.     

Control of glutamate oxidation in brain and liver mitochondrial systems

1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-¹⁴C]- and [5-¹⁴C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD⁺, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD⁺, resulting in preferential oxidation of α-oxoglutarate.     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

Distinct characteristics of Ca-induced depolarization of isolated brain and liver mitochondria

Ca²⁺-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca²⁺ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca²⁺; 20 μM Ca²⁺ was required to depolarize liver mitochondria. Ca²⁺ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca²⁺-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

Inhibition of mitochondrial swelling by 19-nor-ethynyltestosterone acetate and other steroids

The swelling of rat liver mitochondria observed after addition of Ca²⁺, phosphate or valinomycin under suitable experimental conditions is inhibited by 19-nor-ethynyl-testosterone acetate (NEA) in the concentration range from 3 to 60 μm. The inhibition is proportional to NEA concentration and occurs when swelling is supported by oxidation of NAD-linked substrates (malate-glutamate), or endogenous substrate. Little or no inhibition occurs when swelling is supported by succinate oxidation. These observations suggest a site-specific effect near the NADH-flavoprotein portion of the respiratory chain. NEA also inhibits slightly the ATP-dependent contraction of Ca²⁺ swollen mitochondria, indicating a secondary effect on the energy-transfer mechanism. In contrast to these effects, NEA does not significantly affect: (a) H⁺ ejection after Ca²⁺ uptake supported by succinate oxidation; (b) valinomycin-induced swelling supported by ATP addition; (c) Na-acetate-induced swelling, in which the permeability of membranes to Na⁺ is rate limiting; and (d) loss of endogenous mitochondrial pyridine nucleotide. Other steroids such as androsterone, 17β-estradiol, and testosterone derivatives affect mitochondrial swelling like NEA, though to a lesser extent. Effects (a) and (d) are at variance with a previously postulated increase of mitochondrial permeability by steroids, accompanied by swelling. The studies which led to this postulate were carried out at steroid concentrations above 200 μm, where nonspecific effects on membrane permeability may well occur.     

The effect of calcium on the oxidation of acetaldehyde by rat liver mitochondria.

Isolated liver mitochondria oxidized acetaldehyde in the following order: State 4< state 3< valinomycin. Ca²⁺, in concentrations greater than 0.10 mM, inhibited the oxidation of acetaldehyde by isolated liver mitochondria under all conditions. Valinomycin-stimulated oxidation of acetaldehyde was more sensitive to inhibition by Ca²⁺ than were the state 3 or 4 rates of acetaldehyde oxidation. Acetaldehyde could support an energy-dependent uptake of Ca²⁺ at rates about 20 percent that found with succinate. Ruthenium red, an inhibitor of Ca²⁺ translocation, almost completely prevented the inhibition by Ca²⁺, under all conditions. The addition of externally added NAD⁺ or NADH provided complete relief against the inhibitions by Ca²⁺ of the state 4 and 3 rates of acetaldehyde oxidation. Although some relief was also observed with the valinomycin-stimulated system, significant inhibition persisted. Cations such as Zn²⁺, Cu²⁺, or Hg²⁺ also inhibited acetaldehyde oxidation, whereas Mg²⁺ and Mn²⁺ were without effect. These cations also blocked glutamate oxidation and presumably inhibit acetaldehyde oxidation by preventing reoxidation of NADH. The greater sensitivity of the ionophore-stimulated oxidation of acetaldehyde to inhibition by Ca²⁺ may reflect release of intramitochondria K⁺, which is known to occur in the presence of Ca²⁺, suggesting that acetaldehyde oxidation is influenced by the cation environment within the mitochondria.     

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B₁ (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca²⁺ efflux and swelling in mitochondria in the absence of phosphate, Mg²⁺ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca²⁺ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg²⁺ added together, and partially inhibited with phosphate plus either ATP or Mg²⁺. Between 0 and 7 μM added Ca²⁺ and in the presence of phosphate, ATP and Mg²⁺, tri-Calciphor stimulated the uptake of Ca²⁺ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca²⁺. Thus depending on the assay conditions, two different effects involving Ca²⁺ movements and mitochondria are observed with tri-Calciphor.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca²⁺ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate.2. Plots relating the initial rate of calcium ion (Ca²⁺) uptake and the decrease in membrane potential (Δψ) to the Ca²⁺ concentration show a half-maximal change at less than 10 μM Ca²⁺ and a saturation above 20 μM Ca²⁺.3. Plots relating the initial rate of Ca²⁺ uptake to Δψ are linear.4. Addition of Ca²⁺ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca²⁺ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca²⁺ efflux.5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca²⁺ ion is transferred across the membrane with one net positive charge in present conditions.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Effects of prostaglandins on the interaction of Ca2+ with mitochondria

Ca²⁺ stimulates the binding of a variety of prostaglandins (PG) to liver mitochondria. Optimal binding is observed at slightly acidic pH and at concentrations of Ca²⁺ between 200 and 500 μm. The stimulation of the binding requires the active transport of Ca²⁺ in mitochondria and is only observed in the absence of permeant anions. The maximal amount of PG bound is about 3 nmol/mg of mitochondrial protein. All PG tested induce efflux of the Ca²⁺ taken up by mitochondria without impairing the ability of mitochondria to actively accumulate it. Optimal stimulation of the efflux of Ca²⁺ requires concentrations of PG higher than those used in the PG-binding experiments and is associated with an evident uncoupling of the respiration that follows a Ca²⁺-induced O₂ uptake jump. The “uncoupling” by PG is explained by postulating the entrance of protonated PG into mitochondria, followed by their exit from the organelle as 2:1 complexes with Ca²⁺.     

The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other micro-organisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD.Mitochondria actively take up low concentrations of Ca²⁺ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca²⁺ concentrations in excess of 200 μM in the presence or absence of phosphate.Exogenous NADH is unique in supporting the uptake of large amounts of Ca²⁺ in the presence of phosphate and in showing an unusual ‘uncoupled’ response in the absence of phosphate.A sigmoidal relationship occurs between initial velocity of Ca²⁺ uptake and Ca²⁺ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 μM Ca²⁺.     

Effect of calmodulin antagonists on Ca2+ uptake by boar spermatozoa

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both ⁴⁵Ca²⁺ uptake and net Ca²⁺ uptake are increased by these drugs. Drug stimulated Ca²⁺ uptake is blocked by verapamil (1 mM), by ruthenium red (25 μM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca²⁺ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca²⁺-ATPase. It is proposed that calmodulin is involved in the control of Ca²⁺ entry in boar spermatozoa. Most entering Ca²⁺ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca²⁺ extrusion does not appear to be regulated by calmodulin.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

The physical and chemical properties of a chicken egg white glycoprotein purified by nondenaturing methodology

The oxidation of ethanol by the liver produces acetaldehyde, which is a highly reactive compound. Low concentrations of acetaldehyde inhibited mitochondrial respiration with glutamate, β-hydroxybutyrate, or α-ketoglutarate as substrates, but not with succinate or ascorbate. High concentrations led to respiratory inhibition with all substrates. Inhibition of succinate- and ascorbate-linked oxidation by acetaldehyde correlates with the inhibition of the activities of succinic dehydrogenase and cytochrome oxidase. A site more sensitive to acetaldehyde appears to be localized prior to the NADH-ubiquinone oxidoreductase segment of the respiratory chain. Acetaldehyde inhibits energy production by the mitochondria, as evidenced by its inhibition of respiratory control, oxidative phosphorylation, the rate of phosphorylation, and the ATP-³²P exchange reaction. Energy utilization is also inhibited, in view of the decrease in both substrate- and ATP-supported Ca²⁺ uptake, and the reduction in Ca²⁺-stimulated oxygen uptake and ATPase activity. The malate-aspartate, α-glycerophosphate, and fatty acid shuttles for the transfer of reducing equivalents, and oxidation by mitochondria, were highly sensitive to acetaldehyde. Acetaldehyde also inhibited the uptake of anions which participate in the shuttles. The inhibition of the shuttles is apparently caused by interference with NAD⁺-dependent state 3 respiration and anion entry and efflux. Ethanol (6–80 mm) had no significant effect on oxygen consumption, anion uptake, or mitochondrial energy production and utilization. The data suggest that acetaldehyde may be implicated in some of the toxic effects caused by chronic ethanol consumption.     

Interactions between prostaglandin E 1 and calcium at the level of the mitochondrial membrane

Low concentrations of PGE₁ facilitate the exit of actively accumulated Ca²⁺ from rat liver mitochondria. The effect is evident at pH 6,4 and disappears at neutral pH. Ca²⁺ bound to the mitochondrial membrane in the absence of energy is not discharged by PGE₁.Under conditions that lead to its active accumulation, Ca²⁺ stimulates the binding of PGE₁ to mitochondria. The effect is concentration dependent (maximal at 500 μM Ca²⁺), is evident only at slightly acid pH, and is transitory. The binding of PGE₁ reaches a maximum between 30 sec and 2 min and then declines very rapidly, returning to the baseline 2–5 min after the addition of Ca²⁺. The maximal amount of PGE₁ bound is 1.3 nmoles per mg of mitochondrial protein, i.e., about 1% of the Ca²⁺ taken up by mitochondria. No PGE₁ is bound when permeant anions are tranported into mitochondria together with Ca²⁺. Sr²⁺ and Mn²⁺ also stimulate the binding of PGE₁.Aspirin and indomethacin are powerful inhibitors of the binding of PGE₁ to mitochondria. This effect appears to be secondary to the inhibition of mitochondrial Ca²⁺ transport by the antiinflammatory drugs.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria

Phosphoenolpyruvate partially inhibits the accumulation of Ca²⁺ in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca²⁺ uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca²⁺ uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca²⁺ uptake completely while partially inhibiting NADH-supported Ca²⁺ uptake. The oxaloacetate inhibition of NADH-supported Ca²⁺ uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca²⁺ uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca²⁺ uptake.     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.