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

Abstract

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.

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

“…This approach, as originally proposed by Grynkiewicz et al (1985), would minimize problems due (c) (Hansford & Cohen, 197-;Hansford, 1981), but also exploit the indo-1 technique to measure the important parameter [Ca2+1m. Addition of Ruthenium Red, and inhibition of the Ca2+ uniporter (Moore, 1971), results in a rapid decrease in fluorescence, corresponding to loss of Ca2+ from the mitochondrial matrix.…”

“…Each of these dehydrogenase systems is present within the permeability barrier of the mitochondrial inner membrane, leading (Sheu & Fozzard, 1982;McCormack & England, 1983), would elicit enhanced dehydrogenase activity, and enhanced energy transduction by oxidative phosphorylation. Evidence in favour of this proposal has been recently reviewed (Denton & McCormack, 1985;Hansford, 1985).Although there is good agreement on the relationship between the fraction of pyruvate dehydrogenase existing in the active dephosphorylated form (PDHA; see reviews by Reed, 1981;Wieland, 1983) in isolated cardiac mitochondria and the concentration of free ionized Ca2" ([Ca2+]O) in the suspending medium (Hansford & Cohen, 1978;Hansford, 1981) al., 1982;Hansford & Castro, 1982), and the insertion of the metallochromic indicator Arsenazo III into the mitochondria by a permeabilization/resealing protocol (Hayat & Crompton, 1987). The precision of the 'nullpoint' technique is limited by the small absorbance changes seen with the extramitochondrial metallochromic indicator when mitochondrial Ca2" loads low enough to be of relevance to dehydrogenase regulation are investigated, whereas the permeability/resealing protocol may, theoretically at least, perturb the matrix composition, especially with respect to metal ions and low-molecular-mass cofactors.…”

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

“…In the presence of both Na+ and Mg2 +, half-maximal activation was observed at about 0.5 pM-Ca2 +, and thus it could be argued that changes in cytoplasmic [Ca2 + ] within the physiological range (see Marban et al, 1980) would be expected to result in changes in intramitochondrial metabolism in the intact heart . These findings led us to propose that hormones and neurotransmitters which act by increasing the concentration of cytoplasmic Ca2 + could also influence intramitochondrial oxidative metabolism through increases in the intramitochondrial concentration of Ca2 +, and hence activation of the above enzymes , 1981. Strong evidence for this view has been obtained in subsequent studies on the effects of P-adrenergic agonists on the rat heart (McCormack & England, 1983;.…”

“…Studies, mainly with rat heart, have indicated tissues (see , 1981 ,1978aMcCormack & Denton, 1979,1981b dehydrogenase activity can be demonstrated withthe concentration of effector required for a half-maximal in coupled rat heart mitochondria exposed to response.…”

“…of Ca2+ Hansford, 1981;Hansford&Castro, 1981;McCormacketal., 1982). The addition of either Mg2+, which inhibits the uptake of Ca2+ into mitochondria (Crompton et al, 1976), or Na+, which allows the efflux of Ca2 + from heart mitochondria by a Ca/Na translocase (Crompton et al, 1977(Crompton et al, , 1978, was shown to increase the concentration of extramitochondrial Ca2+ required to obtain half-maximal activation of both enzymes.…”