Skip to main content
SpringerLink
Log in

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium, phosphate, and ATP at 37°C

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Summary

The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a45Ca exchange technique. The amounts of40Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of45Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 μm, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 μm. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The45Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the45Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK 0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and45Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akerman, K.E.O., 1980. Control of Ca2+ influx and efflux in liver mitochondria.Biochem. Soc. Trans. 8:262–264

    Google Scholar 

  • Andia-Waltenbaugh, A.M., Kimura, S., Wood, J., Divakaran, P., Friedmann, N. 1978. Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.Life Sci. 23:2437–2443

    Google Scholar 

  • Babcock, D.F., Chen, J-L.J., Yip, B.P., Lardy, H.A. 1979. Evidence for mitochondrial localization of the hormone-responsive pool of Ca2+ in isolated hepatocytes.J. Biol. Chem. 254:8117–8120

    Google Scholar 

  • Baker, P.F. 1978. The regulation of intracellular calcium in giant axons ofLoligo andMyxicola.Ann. N.Y. Acad. Sci. 307:250–268

    Google Scholar 

  • Barritt, G.J. 1980. Analysis of the effects of adrenaline and glucagon on calcium ion movement in liver cells by using a simple compartmental model.Biochem. Soc. Trans. 8:144–145

    Google Scholar 

  • Barritt, G.J., Parker, J.C., Wadsworth, J.C. 1981. A kinetic analysis of the effects of adrenaline on calcium distribution in isolated rat liver parenchymal cells.J. Physiol. (London) 312:29–55

    Google Scholar 

  • Becker, G.L. 1980. Steady state regulation of extramitochondrial Ca2+ by rat liver mitochondria. Effects of Mg2+ and ATP.Biochim. Biophys. Acta 591:234–239

    Google Scholar 

  • Becker, G.L., Fiskum, G., Lehninger, A.L. 1980. Regulation of free Ca2+ by liver mitochondria and endoplasmic reticulum.J. Biol. Chem. 255:9009–9012

    Google Scholar 

  • Berman, M. 1965. Compartmental analysis in kinetics.In: Computers in Biomedical Research. R. Stacy and B. Waxman, editors. Vol. 2, pp. 173–201. Academic Press, New York

    Google Scholar 

  • Binet, A., Volfin, P. 1975. Regulation by Mg2+ and Ca2+ of mitochondrial membrane integrity: Study of the effects of a cytosolic molecule and Ca2+ antagonists.Arch. Biochem. Biophys. 170:576–586

    Google Scholar 

  • Blackmore, P.F., Brumley, F.T., Marks, J.L., Exton, J.H. 1978. Studies on α-adrenergic activation of hepatic glucose output. Relationship between α-adrenergic stimulation of calcium efflux and activation of phosphorylase in isolated rat liver parenchymal cells.J. Biol. Chem. 253:4851–4858

    Google Scholar 

  • Blackmore, P.F., Dehaye, J-P., Exton, J.H. 1979. Studies on α-adrenergic activation of hepatic glucose output. The role of mitochondrial calcium release in α-adrenergic activation of phosphorylase in perfused rat liver.J. Biol. Chem. 254:6945–6950

    Google Scholar 

  • Borle, A.B. 1974. Cyclic AMP stimulation of calcium efflux from kidney, liver, and heart mitochondria.J. Membrane Biol. 16:221–236

    Google Scholar 

  • Borle, A.B., Uchikawa, T. 1979. Effects of adenosine 3′,5′-monophosphate, dibutyryl adenosine 3′,5′-monophosphate, aminophylline, and imidazole on renal cellular calcium metabolism.Endocrinology 104:122–129

    Google Scholar 

  • Boxenbaum, H.G., Riegelman, S., Elashoff, R.M. 1974. Statistical estimations in pharmacokinetics.J. Pharmacokinet. Biopharm. 2:123–148

    Google Scholar 

  • Brand, M.D., De Selincourt, C. 1980. Effects of glucagon and Na+ on the control of extramitochondrial free Ca2+ concentration by mitochondria from liver and heart.Biochem. Biophys. Res. Commun. 92:1377–1382

    Google Scholar 

  • Bygrave, F.L. 1978. Mitochondria and the control of intracellular calcium.Biol. Rev. 53:43–79

    Google Scholar 

  • Carafoli, E. 1967.In vivo effect of uncoupling agents on the incorporation of calcium and strontium into mitochondria and other subcellular fractions of rat liver.J. Gen. Physiol. 50:1849–1864

    Google Scholar 

  • Carafoli, E. 1979. The calcium cycle of mitochondria.FEBS Lett. 104:1–5

    Google Scholar 

  • Chan, T.M., Bacon, C.B., Hill, S.A. 1979. Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.J. Biol. Chem. 254:8730–8732

    Google Scholar 

  • Claret-Berthon B., Claret, M., Mazet, J.L. 1977. Fluxes and distribution of calcium in rat liver cells: Kinetic analysis and identification of pools.J. Physiol. (London) 272:529–552

    Google Scholar 

  • Czech, M.P. 1977. Molecular basis of insulin action.Annu. Rev. Biochem. 46:359–384

    Google Scholar 

  • Dawson, A.P., Selwyn, M.J., Fulton, D.V. 1979. Inhibition of Ca2+ efflux from mitochondria by nupercaine and tetracaine.Nature (London) 277:484–486

    Google Scholar 

  • Denton, R.M., McCormack, J.G., Edgell, N.J. 1980. 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. 190:107–117

    Google Scholar 

  • Fiskum, G., Lehninger, A.L. 1980. The mechanisms and regulation of mitochondrial Ca2+ transport.Fed. Proc. 39:2432–2436

    Google Scholar 

  • Foldes, M., Barritt, G.J. 1977. Regulation by calcium ions of pyruvate carboxylation, pyruvate transport and adenine nucleotide transport in isolated rat liver mitochondria.J. Biol. Chem. 252:5372–5380

    Google Scholar 

  • Friedmann, N., Rasmussen, H. 1970. Calcium manganese and hepatic gluconeogenesis.Biochim. Biophys. Acta 222:41–52

    Google Scholar 

  • Harris, E.J., Al-Shaikhaly, M., Baum, H. 1979. Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability.Biochem. J. 182:455–464

    Google Scholar 

  • Haworth, R.A., Hunter, D.R., Berkoff, H.A. 1980. Na+ releases Ca2+ from liver, kidney and lung mitochondria.FEBS Lett. 110:216–218

    Google Scholar 

  • Heaton, G.M., Nicholls, D.G. 1976. The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient.Biochem. J. 156:635–646

    Google Scholar 

  • Hughes, B.P., Barritt, G.J. 1978. Effects of glucagon and N6O2′-dibutyryladenosine 3′:5′-cyclic monophosphate on calcium transport in isolated rat liver mitochondria.Biochem. J. 176:295–304

    Google Scholar 

  • Juzu, H.A., Holdworth, E.S. 1980. New evidence for the role of cyclic AMP in the release of mitochondrial calcium.J. Membrane Biol. 52:185–186

    Google Scholar 

  • Kimura, S., Rasmussen, H. 1977. Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.J. Biol. Chem. 252:1217–1225

    Google Scholar 

  • Lawson, J.W.R., Guynn, R.W., Cornell, N., Veech, R.L. 1976. A possible role for pyrophosphate in control of hepatic glucose metabolism.In: Gluconeogenesis: Its Regulation in Mammalian Species. R.W. Hanson and M.A. Mehlman, editors. pp. 481–512. John Wiley & Sons, New York

    Google Scholar 

  • Lehninger, A.L., Carafoli, E., Rossi, C.S. 1967. Energy-linked ion movements in mitochondria systems.Adv. Enzymol. 29:259–320

    Google Scholar 

  • Lehninger, A.L., Vercesi, A., Bababunmi, E.A. 1978. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.Proc. Nat. Acad. Sci. USA 75:1690–1694

    Google Scholar 

  • Lötscher, H.R., Winterhalter, K.H., Carafoli, E., Richter, C. 1979. Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria.Proc. Nat. Acad. Sci. USA 76:4340–4344

    Google Scholar 

  • Mikkelsen, R.B., Schmidt-Ullrich, R. 1980. Concanavalin A induces the release of intracellular Ca2+ in intact rabbit thymocytes.J. Biol. Chem. 255:5177–5183

    Google Scholar 

  • Nicholls, D.G. 1978. The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria.Biochem. J. 176:463–474

    Google Scholar 

  • Nicholls, D.G., Brand, M.D. 1980. The nature of the calcium ion efflux induced in rat liver mitochondria by the oxidation of endogenous nicotinamide nucleotides.Biochem. J. 188:113–118

    Google Scholar 

  • Nicholls, D.G., Crompton, M. 1980. Mitochondrial calcium transport.FEBS Lett. 111:261–268

    Google Scholar 

  • Nicholls, D.G., Scott, I.D. 1980. The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms.Biochem. J. 186:833–839

    Google Scholar 

  • Peng, C.F., Price, D.W., Bhuvaneswaran, C., Wadkins, C.L. 1974. Factors that influence phosphoenolpyruvate-induced calcium efflux from rat liver mitochondria.Biochem. Biophys. Res. Commun. 56:134–141

    Google Scholar 

  • Perrin, D.D., Sayce, I.G. 1967. Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species.Talanta 14:833–842

    Google Scholar 

  • Prpić, V., Spencer, T.L., Bygrave, F.L. 1978. Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal.Biochem. J. 176:705–714

    Google Scholar 

  • Rasmussen, H., Gustin, M.C. 1978. Some aspects of hormonal control of cellular calcium metabolism.Ann. N.Y. Acad. Sci. 307:391–401

    Google Scholar 

  • Reed, K.C., Bygrave, F.L. 1975. Methodology forin vitro studies of Ca2+ transport.Anal. Biochem. 67:44–54

    Google Scholar 

  • Roman, I., Gmaj, P., Nowicka, C., Angielski, S. 1979. Regulation of Ca2+ efflux from kidneyand liver mitochondria by unsaturated fatty acids and Na+ ions.Eur. J. Biochem. 102:615–623

    Google Scholar 

  • Schotland, J., Mela, L. 1977. Role of cyclic nucleotides in the regulation of mitochondrial calcium uptake and efflux kinetics.Biochem. Biophys. Res. Commun. 75:920–924

    Google Scholar 

  • Shipley, R.A., Clark, R.E. 1972. Tracer Methods forin vivo Kinetics: Theory and Applications. Chap. 8. Academic Press. New York

    Google Scholar 

  • Siess, E.A., Brocks, D.G., Lattke, H.K., Wieland, O.H. 1977. Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate.Biochem. J. 166:225–235

    Google Scholar 

  • Sillen, L.G., Martell, A.E. 1971. Stability constants. Supplement 1. Special Publication 25. The Chemical Society, London

    Google Scholar 

  • Studer, R.K., Borle, A.B. 1980. The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria.Arch. Biochem. Biophys. 203:707–718

    Google Scholar 

  • Taylor, W.M., Prpić, V., Exton, J.H., Bygrave, F.L. 1980. Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon.Biochem. J. 188:443–450

    Google Scholar 

  • Van Rossum, G.D.V. 1970. Net movements of calcium and magnesium in slices of rat liver.J. Gen. Physiol. 55:18–32

    Google Scholar 

  • Veloso, D., Guynn, R.W., Oskarsson, M., Veech, R.L. 1973. The concentrations of free and bound magnesium in rat tissues. Relative constancy of free Mg2+ concentrations.J. Biol. Chem. 248:4811–4819

    Google Scholar 

  • Yamazaki, R.K. 1975. Glucagon stimulation of mitochondrial respiration.J. Biol. Chem. 250:7924–7930

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Clinical Biochemistry Unit, Flinders University School of Medicine, Flinders Medical Centre, 5042, Bedford Park, South Australia

    Gregory J. Barritt

Authors
  1. Gregory J. Barritt
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Barritt, G.J. Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium, phosphate, and ATP at 37°C. J. Membrain Biol. 62, 53–63 (1981). https://doi.org/10.1007/BF01870199

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01870199

Key words

Search

Navigation