Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat-kidney mitochondria

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.     

Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes.

The total activity of pyruvate dehydrogenase (PDH) complex in rat hind-limb muscle mitochondria was 76.4 units/g of mitochondrial protein. The proportion of complex in the active form was 34% (as isolated), 8-14% (incubation with respiratory substrates) and greater than 98% (incubation without respiratory substrates). Complex was also inactivated by ATP in the presence of oligomycin B and carbonyl cyanide m-chlorophenylhydrazone. Ca2+ (which activates PDH phosphatase) and pyruvate or dichloroacetate (which inhibit PDH kinase) each increased the concentration of active PDH complex in a concentration-dependent manner in mitochondria oxidizing 2-oxoglutarate/L-malate. Values giving half-maximal activation were 10 nM-Ca2+, 3 mM-pyruvate and 16 microM-dichloroacetate. Activation by Ca2+ was inhibited by Na+ and Mg2+. Mitochondria incubated with [32P]Pi/2-oxoglutarate/L-malate incorporated 32P into three phosphorylation sites in the alpha-chain of PDH; relative rates of phosphorylation were sites 1 greater than 2 greater than 3, and of dephosphorylation, sites 2 greater than 1 greater than 3. Starvation ( 48h ) or induction of alloxan-diabetes had no effect on the total activity of PDH complex in skeletal-muscle mitochondria, but each decreased the concentration of active complex in mitochondria oxidizing 2-oxoglutarate/L-malate and increased the concentrations of Ca2+, pyruvate or dichloracetate required for half-maximal reactivation. In extracts of mitochondria the activity of PDH kinase was increased 2-3-fold by 48 h starvation or alloxan-diabetes, but the activity of PDH phosphatase was unchanged.     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

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

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.     

Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria.

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates.

The flux control distribution of the net rate of state 3 respiration was determined in heart and kidney mitochondria incubated with low concentrations of pyruvate (0.5 mM) or 2-oxoglutarate (1 mM), and in conditions that led to activation of NAD-linked dehydrogenases, i.e., high substrate or Ca2+ concentrations. Control of flux was exerted by the ATP/ADP carrier (flux control coefficient, ci = 0.37) and Site 1 of the respiratory chain (ci = 0.28) when dehydrogenase activity was low. Control of the process shifted to the ATP synthase (ci = 0.32) and the Pi carrier (Ci = 0.27) when dehydrogenases were activated by high pyruvate and high Ca2+. The changes in the control exerted by the ATP/ADP carrier and the ATP synthase were not due to changes in the transmembrane potential, nor to a modification of intramitochondrial ATP/ADP ratios. Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps. The NAD-linked dehydrogenases did not exert any significant control at high Ca2+ or high pyruvate concentrations.     

Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria.

1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.     

Vasopressin and/or glucagon rapidly increases mitochondrial calcium and oxidative enzyme activities in the perfused rat liver

Mitochondria were prepared by a method including a Percoll purification step after the rapid homogenization of livers of fed rats which had been perfused either under unstimulated conditions or in the presence of vasopressin and/or glucagon. The two hormones separately or together increased the total calcium content of the mitochondria. This enhancement was accompanied by parallel increases in activities of the Ca2+-sensitive intramitochondrial enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The effects of the two hormones on total mitochondrial calcium and on the activities of the oxidative enzymes were additive. The persistent enhancements of mitochondrial calcium content and enzyme activities were partially reversed by the addition of Na+ ions to the mitochondrial incubations; these effects of Na+ were blocked by diltiazem, a selective inhibitor of Na+-induced Ca2+ release. Mitochondria from control livers were incubated in vitro with CaCl2 to achieve various calcium content, and mitochondrial enzyme activities and calcium content were measured. A good correlation was obtained between the total calcium content and the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase. The results obtained are consistent with the hypothesis that vasopressin and glucagon additively cause increases in intramitochondrial [Ca2+] and so bring about the activations of these key enzymes of mitochondrial oxidative metabolism.     

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+.

Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.     

Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria.

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.     

The calcium sensitive dehydrogenases of vertebrate mitochondria

Three important dehydrogenases in vertebrate mitochondria are activated by Ca2+ ions with half-maximal effects at about 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated within intact mitochondria when extramitochondrial Ca2+ is increased within the range of concentrations generally considered to occur in the cytoplasm of vertebrate cells. It is argued that the main role of the calcium transport system in the inner membrane of vertebrate mitochondria is to relay changes in the cytoplasmic concentration of Ca2+ into the mitochondrial matrix. In this way, hormones and other extracellular stimuli which stimulate ATP-requiring processes such as contraction and secretion through increases in the cytoplasmic concentration of Ca2+ may also increase intramitochondrial oxidative metabolism and hence the replenishment of ATP.     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.