scholarly journals Electroneutral efflux of Ca2+ from liver mitochondria

1985 ◽  
Vol 225 (2) ◽  
pp. 413-419 ◽  
Author(s):  
M D Brand
Keyword(s):  
Steady State ◽  
Thermodynamic Analysis ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Ionophore A23187 ◽  
Measurable Change

Respiring liver mitochondria were allowed to export Ca2+ on the endogenous Ca2+/nH+ antiporter in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) until a steady state was reached. Addition of sufficient of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) to bring the Ca2+ and H+ gradients into equilibrium did not alter the steady state. Thermodynamic analysis showed that if a Ca2+/nH+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would have caused an easily measurable change in extramitochondrial free [Ca2+]. Therefore, the endogenous carrier of liver mitochondria catalyses electroneutral Ca2+/2H+ antiport.

scholarly journals The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter

1985 ◽  
Vol 229 (1) ◽  
pp. 161-166 ◽  
Author(s):  
M D Brand
Keyword(s):  
Steady State ◽  
Thermodynamic Analysis ◽  
Rat Heart ◽  
Ruthenium Red ◽  
Heart Mitochondria ◽  
Mitochondrial Calcium ◽  
Ionophore A23187 ◽  
Obvious Change ◽  
Rat Heart Mitochondria

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.

scholarly journals The action of Nupercaine on calcium efflux from rat liver mitochondria

1980 ◽  
Vol 188 (3) ◽  
pp. 749-755 ◽  
Author(s):  
A P Dawson ◽  
D V Fulton
Keyword(s):  
Steady State ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Calcium Efflux

1. Nupercaine inhibits the Ca2+ efflux from rat liver mitochondria observed in the presence of Ruthenium Red, 50% inhibition being obtained at 80 microM-Nupercaine. 2. Neither the Ruthenium Red-stimulated efflux nor its inhibition by Nupercaine can be directly attributed to effects on mitochondrial stability. 3. Nupercaine perturbs the steady-state external Ca2+ concentration in the absence of Ruthenium Red to an extent that is explicable in terms of the inhibition of Ca2+ efflux. 4. Various factors that are likely to be involved in determining steady-state extra-mitochondrial Ca2+ concentrations are discussed.

Kinetics of high-affinity Ca2+ sequestration in permeabilized rat pancreatic acini

1988 ◽  
Vol 254 (5) ◽  
pp. C621-C627 ◽  
Author(s):  
T. W. Hurley
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Initial Rate ◽  
Ruthenium Red ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
High Affinity ◽  
Energy Dependent ◽  
Kinetics Of

Energy-dependent subcellular Ca2+ sequestration was studied in the presence of ruthenium red using rat pancreatic acini, which had been permeabilized by exposure to medium nominally free of Ca2+. The initial rate of Ca2+ uptake (approximately 2,800 pmol.min-1.mg acinar protein-1) quickly slowed, and a mean steady-state Ca2+ content of approximately 3,000 pmol/mg was reached after 5-10 min of incubation at 37 degrees C. Ca2+ uptake was stimulated by submicromolar Ca2+ concentrations (K0.5 = 156 nM); required Mg2+-ATP (K0.5 = 0.78 mM) was greatest at a pH of 7.0 and was abolished by the Ca2+ ionophore A23187. Other nucleotide phosphates as well as p-nitrophenylphosphate were relatively poor substrates, supporting Ca2+ uptake at initial rates that were 6-14% of those measured in the presence of ATP. These results show that pancreatic acini permeabilized without detergents possess a nonmitochondrial Ca2+ transporting system not located in the plasma membrane but with the properties expected of a major regulator of acinar cytosolic Ca2+ concentration.

scholarly journals The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration

1982 ◽  
Vol 202 (3) ◽  
pp. 731-737 ◽  
Author(s):  
N G Dippenaar ◽  
M D Brand
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Mitochondrial Protein ◽  
Respiratory Control ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Mesenteric Lymph ◽  
Efflux Pathway

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2-3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.

scholarly journals Evidence that glucagon acts on the liver to decrease mitochondrial calcium stores

1983 ◽  
Vol 210 (1) ◽  
pp. 73-77 ◽  
Author(s):  
H M Baddams ◽  
L B F Chang ◽  
G J Barritt
Keyword(s):  
Steady State ◽  
Intact Cell ◽  
Isolated Hepatocytes ◽  
Ruthenium Red ◽  
Mitochondrial Calcium ◽  
Calcium Stores ◽  
Ionophore A23187 ◽  
Small Decrease ◽  
Significant Difference

1. Mitochondria isolated from rats treated with glucagon for 60 min or lives perfused in the presence of glucagon for 10 min exhibited lower rates of 45Ca2+ exchange than did control mitochondria when this was measured under steady-state conditions in the presence of Mg2+, ATP, Pi and 0.13 microM- or 0.16 microM-free Ca2+ at pH 7.4 and at 25 degrees C or 37 degrees C. Under these conditions no significant difference in the rates of Ruthenium Red-induced 45Ca2+ efflux was observed. These results contrast with earlier work in which mitochondria isolated from glucagon-treated livers were shown to exhibit faster rates of Ca2+ uptake [Yamazaki (1975) J. Biol. Chem. 250, 7924-7930] and slower rates of spontaneous Ca2+ efflux [Hughes & Barritt (1978) Biochem. J. 176, 295-304] when these parameters were measured under different incubation conditions, including supra-physiological concentrations of free Ca2+ and the absence of added Mg2+ and ATP. 2. Perfusion of livers with glucagon before the addition of adrenaline or the Ca2+-selective ionophore A23187, to release Ca2+ from intracellular stores, decreased the amount of Ca2+ released by these agents. 3. Incubation of isolated hepatocytes in the presence of glucagon at 1.3 mM extracellular Ca2+ induced a small decrease in the plateau of the 45Ca2+-exchange curve obtained under steady-state conditions. 4. It is concluded that the actions of glucagon on liver mitochondrial Ca2+ transporters lead to a decrease, rather than an increase, in mitochondrial Ca2+ stores in the intact cell.

scholarly journals On the mechanism by which hormones induce the release of Ca2+ from mitochondria in the liver cell

1982 ◽  
Vol 206 (1) ◽  
pp. 121-129 ◽  
Author(s):  
J A Whiting ◽  
G J Barritt
Keyword(s):  
Arachidonic Acid ◽  
Liver Cell ◽  
Acid Metabolism ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Arachidonic Acid Metabolism ◽  
Ruthenium Red ◽  
Ionophore A23187 ◽  
Phosphate Ions ◽  
Subsequent Addition

1. The abilities of dinitrophenol, NaCl, Ruthenium Red and the Ca2+-selective ionophore A23187 to release 45 Ca2+ from isolated hepatocytes and liver mitochondria (incubated at 37 degrees C in the presence of 0.1 microM-free Ca2+, Mg2+, ATP and phosphate ions) were compared with the action of adrenaline on 45Ca2+ release from isolated hepatocytes. The effects of adrenaline were most closely described by those of the ionophore A23187. 2. In isolated hepatocytes, a release of 45Ca2+ and stimulation of O2 utilization similar to that induced by adrenaline was observed in the presence of 500 and 20 microM-arachidonic acid respectively. The effect of arachidonic acid on 45Ca2+ release was not specific for this unsaturated fatty acid. 3. Inhibitors of arachidonic acid metabolism, including indomethacin and eicosa-5,811,14-tetraynoic acid, did not block the effects of adrenaline on 45Ca2+ or glucose release from isolated hepatocytes. 4. The ability of adrenaline to stimulate 45Ca2+ release from isolated hepatocytes was rapidly reversed after the subsequent addition of phenoxybenzamine to the cell suspension, and was completely blocked by 0.5 mM-dibucaine. 5. The results are consistent with the action of a Ca2+-selective ionophore in the mechanism by which adrenaline induces the release of Ca2+ from mitochondria in the liver cell and indicate that it is unlikely that arachidonic acid or a metabolite of arachidonic acid is involved in this process.

scholarly journals The Steady State Maintenance of Accumulated Ca++ in Rat Liver Mitochondria

1965 ◽  
Vol 240 (6) ◽  
pp. 2712-2720
Author(s):  
Zdenek Drahota ◽  
Ernesto Carafoli ◽  
Carlo S. Rossi ◽  
Robert L. Gamble ◽  
Albert L. Lehninger
Keyword(s):  
Steady State ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria
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