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Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart
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.
Insights
Calcium ions directly regulate the activity of pyruvate dehydrogenase (PDH) in mitochondria, influencing energy metabolism. This calcium dependence is modulated by other ions like sodium and magnesium.
Area of Science:
- Biochemistry
- Cellular Metabolism
- Mitochondrial Function
Background:
- Pyruvate dehydrogenase (PDH) is a key enzyme complex regulating glucose metabolism.
- Mitochondrial calcium (Ca2+) is a critical signaling molecule influencing various cellular processes.
- The precise mechanisms by which Ca2+ affects PDH activity are not fully elucidated.
Purpose of the Study:
- To investigate the direct effect of extramitochondrial Ca2+ concentration on the activity of pyruvate dehydrogenase (PDH).
- To explore the influence of other ions (Na+, Mg2+, P1, bicarbonate) and cellular conditions on Ca2+-mediated PDH regulation.
- To determine if changes in ATP/ADP or NADH/NAD+ ratios mediate the Ca2+ effect on PDH.
Main Methods:
- Measurement of active (dephosphorylated) pyruvate dehydrogenase (PDHA) content in isolated mitochondria under varying Ca2+ concentrations.
- Assessment of mitochondrial respiration (state-4 and state-3 conditions) and ion transport.
- Analysis of the impact of Na+, Mg2+, P1, and bicarbonate ions on PDHA content and Ca2+ handling.
- Evaluation of changes in ATP/ADP and NADH/NAD+ ratios in response to Ca2+.
Main Results:
- PDHA content significantly increased with higher extramitochondrial Ca2+ concentrations (pCa6 vs. pCa7), independent of respiratory substrate.
- This Ca2+ dependence was observed in state-4 and 50%-state-3 respiration but absent with excess ADP (state 3).
- Na+ and Mg2+ ions altered the Ca2+ sensitivity of PDHA, affecting mitochondrial Ca2+ fluxes and PDH phosphatase activity; Na+ may directly inhibit the phosphatase.
- Bicarbonate ions abolished the Ca2+ response, while physiological P1 and bicarbonate allowed for Ca2+ responsiveness.
- Ca2+ directly influenced PDHA content without significant changes in ATP/ADP or NADH/NAD+ ratios, suggesting a direct regulatory role.
Conclusions:
- Extramitochondrial Ca2+ directly regulates mitochondrial pyruvate dehydrogenase activity.
- The Ca2+ effect on PDHA is modulated by other ions and cellular energy states.
- The observed changes in PDHA content are likely a direct consequence of Ca2+ signaling, not mediated by altered energy charge or redox state.