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. 1984 Feb 15;218(1):235–247. doi: 10.1042/bj2180235

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+.

J G McCormack, R M Denton
PMCID: PMC1153329  PMID: 6424656

Abstract

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.

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Selected References

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  1. CHANCE B., WILLIAMS G. R. The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem. 1956;17:65–134. doi: 10.1002/9780470122624.ch2. [DOI] [PubMed] [Google Scholar]
  2. Carafoli E. The calcium cycle of mitochondria. FEBS Lett. 1979 Aug 1;104(1):1–5. doi: 10.1016/0014-5793(79)81073-x. [DOI] [PubMed] [Google Scholar]
  3. Chappell J. B., Robinson B. H. Penetration of the mitochondrial membrane by tricarboxylic acid anions. Biochem Soc Symp. 1968;27:123–133. [PubMed] [Google Scholar]
  4. Cohen P. The role of cyclic-AMP-dependent protein kinase in the regulation of glycogen metabolism in mammalian skeletal muscle. Curr Top Cell Regul. 1978;14:117–196. doi: 10.1016/b978-0-12-152814-0.50008-3. [DOI] [PubMed] [Google Scholar]
  5. Coll K. E., Joseph S. K., Corkey B. E., Williamson J. R. Determination of the matrix free Ca2+ concentration and kinetics of Ca2+ efflux in liver and heart mitochondria. J Biol Chem. 1982 Aug 10;257(15):8696–8704. [PubMed] [Google Scholar]
  6. Cooper R. H., Randle P. J., Denton R. M. Regulation of heart muscle pyruvate dehydrogenase kinase. Biochem J. 1974 Dec;143(3):625–641. doi: 10.1042/bj1430625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crompton M., Kessar P., Al-Nasser I. The alpha-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+ in vivo. Biochem J. 1983 Nov 15;216(2):333–342. doi: 10.1042/bj2160333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crompton M., Künzi M., Carafoli E. The calcium-induced and sodium-induced effluxes of calcium from heart mitochondria. Evidence for a sodium-calcium carrier. Eur J Biochem. 1977 Oct 3;79(2):549–558. doi: 10.1111/j.1432-1033.1977.tb11839.x. [DOI] [PubMed] [Google Scholar]
  9. Crompton M., Moser R., Lüdi H., Carafoli E. The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues. Eur J Biochem. 1978 Jan 2;82(1):25–31. doi: 10.1111/j.1432-1033.1978.tb11993.x. [DOI] [PubMed] [Google Scholar]
  10. Denton R. M., McCormack J. G. Calcium ions, hormones and mitochondrial metabolism. Clin Sci (Lond) 1981 Aug;61(2):135–140. doi: 10.1042/cs0610135. [DOI] [PubMed] [Google Scholar]
  11. Denton R. M., McCormack J. G., Edgell N. J. 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. Biochem J. 1980 Jul 15;190(1):107–117. doi: 10.1042/bj1900107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denton R. M., McCormack J. G. On the role of the calcium transport cycle in heart and other mammalian mitochondria. FEBS Lett. 1980 Sep 22;119(1):1–8. doi: 10.1016/0014-5793(80)80986-0. [DOI] [PubMed] [Google Scholar]
  13. Denton R. M., Randle P. J., Martin B. R. Stimulation by calcium ions of pyruvate dehydrogenase phosphate phosphatase. Biochem J. 1972 Jun;128(1):161–163. doi: 10.1042/bj1280161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Denton R. M., Richards D. A., Chin J. G. Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem J. 1978 Dec 15;176(3):899–906. doi: 10.1042/bj1760899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Exton J. H. Mechanisms involved in alpha-adrenergic phenomena: role of calcium ions in actions of catecholamines in liver and other tissues. Am J Physiol. 1980 Jan;238(1):E3–12. doi: 10.1152/ajpendo.1980.238.1.E3. [DOI] [PubMed] [Google Scholar]
  16. Fleckenstein A. Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. Annu Rev Pharmacol Toxicol. 1977;17:149–166. doi: 10.1146/annurev.pa.17.040177.001053. [DOI] [PubMed] [Google Scholar]
  17. Hansford R. G., Castro F. Effects of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate. Biochem J. 1981 Sep 15;198(3):525–533. doi: 10.1042/bj1980525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hansford R. G., Cohen L. Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria. Arch Biochem Biophys. 1978 Nov;191(1):65–81. doi: 10.1016/0003-9861(78)90068-1. [DOI] [PubMed] [Google Scholar]
  19. Hansford R. G. Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria. Biochem J. 1981 Mar 15;194(3):721–732. doi: 10.1042/bj1940721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hiraoka T., DeBuysere M., Olson M. S. Studies of the effects of beta-adrenergic agonists on the regulation of pyruvate dehydrogenase in the perfused rat heart. J Biol Chem. 1980 Aug 25;255(16):7604–7609. [PubMed] [Google Scholar]
  21. Joseph S. K., Coll K. E., Cooper R. H., Marks J. S., Williamson J. R. Mechanisms underlying calcium homeostasis in isolated hepatocytes. J Biol Chem. 1983 Jan 25;258(2):731–741. [PubMed] [Google Scholar]
  22. Kerbey A. L., Randle P. J., Cooper R. H., Whitehouse S., Pask H. T., Denton R. M. Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide. Biochem J. 1976 Feb 15;154(2):327–348. doi: 10.1042/bj1540327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kretsinger R. H. The informational role of calcium in the cytosol. Adv Cyclic Nucleotide Res. 1979;11:1–26. [PubMed] [Google Scholar]
  24. Linn T. C., Pettit F. H., Hucho F., Reed L. J. Alpha-keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria. Proc Natl Acad Sci U S A. 1969 Sep;64(1):227–234. doi: 10.1073/pnas.64.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marban E., Rink T. J., Tsien R. W., Tsien R. Y. Free calcium in heart muscle at rest and during contraction measured with Ca2+ -sensitive microelectrodes. Nature. 1980 Aug 28;286(5776):845–850. doi: 10.1038/286845a0. [DOI] [PubMed] [Google Scholar]
  26. Marshall S. E., McCormack J. G., Denton R. M. 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. Biochem J. 1984 Feb 15;218(1):249–260. doi: 10.1042/bj2180249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCormack J. G., Denton R. M. Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat. Biochem J. 1980 Jul 15;190(1):95–105. doi: 10.1042/bj1900095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCormack J. G., Denton R. M. The activation of pyruvate dehydrogenase in the perfused rat heart by adrenaline and other inotropic agents. Biochem J. 1981 Feb 15;194(2):639–643. doi: 10.1042/bj1940639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McCormack J. G., Denton R. M. The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J. 1979 Jun 15;180(3):533–544. doi: 10.1042/bj1800533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McCormack J. G., Edgell N. J., Denton R. M. Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes. Biochem J. 1982 Feb 15;202(2):419–427. doi: 10.1042/bj2020419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McCormack J. G., England P. J. Ruthenium Red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart. Biochem J. 1983 Aug 15;214(2):581–585. doi: 10.1042/bj2140581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moore C. L. Specific inhibition of mitochondrial Ca++ transport by ruthenium red. Biochem Biophys Res Commun. 1971 Jan 22;42(2):298–305. doi: 10.1016/0006-291x(71)90102-1. [DOI] [PubMed] [Google Scholar]
  33. Nicholls D. G., Crompton M. Mitochondrial calcium transport. FEBS Lett. 1980 Mar 10;111(2):261–268. doi: 10.1016/0014-5793(80)80806-4. [DOI] [PubMed] [Google Scholar]
  34. Rasmussen H., Goodman D. B. Relationships between calcium and cyclic nucleotides in cell activation. Physiol Rev. 1977 Jul;57(3):421–509. doi: 10.1152/physrev.1977.57.3.421. [DOI] [PubMed] [Google Scholar]
  35. Sale G. J., Randle P. J. Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria. Biochem J. 1980 May 15;188(2):409–421. doi: 10.1042/bj1880409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sale G. J., Randle P. J. Occupancy of phosphorylation sites in pyruvate dehydrogenase phosphate complex in rat heart in vivo. Relation to proportion of inactive complex and rate of re-activation by phosphatase. Biochem J. 1982 Aug 15;206(2):221–229. doi: 10.1042/bj2060221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Severson D. L., Denton R. M., Bridges B. J., Randle P. J. Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity. Biochem J. 1976 Jan 15;154(1):209–223. doi: 10.1042/bj1540209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stanley P. E., Williams S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem. 1969 Jun;29(3):381–392. doi: 10.1016/0003-2697(69)90323-6. [DOI] [PubMed] [Google Scholar]
  39. Vasington F. D., Gazzotti P., Tiozzo R., Carafoli E. The effect of ruthenium red on Ca 2+ transport and respiration in rat liver mitochondria. Biochim Biophys Acta. 1972 Jan 21;256(1):43–54. doi: 10.1016/0005-2728(72)90161-2. [DOI] [PubMed] [Google Scholar]
  40. Vághy P. L., Johnson J. D., Matlib M. A., Wang T., Schwartz A. Selective inhibition of Na+-induced Ca2+ release from heart mitochondria by diltiazem and certain other Ca2+ antagonist drugs. J Biol Chem. 1982 Jun 10;257(11):6000–6002. [PubMed] [Google Scholar]
  41. Williamson J. R., Cooper R. H. Regulation of the citric acid cycle in mammalian systems. FEBS Lett. 1980 Aug 25;117 (Suppl):K73–K85. doi: 10.1016/0014-5793(80)80572-2. [DOI] [PubMed] [Google Scholar]
  42. Wolff D. J., Brostrom C. O. Properties and functions of the calcium-dependent regulator protein. Adv Cyclic Nucleotide Res. 1979;11:27–88. [PubMed] [Google Scholar]

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