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 Role of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes

1985 ◽  
Vol 227 (1) ◽  
pp. 129-136 ◽  
Author(s):  
R G Hansford ◽  
F Castro
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Pyruvate Dehydrogenase ◽  
Maximal Activity ◽  
Brain Mitochondria ◽  
Ruthenium Red ◽  
Ion Concentration ◽  
Maximal Effect ◽  
State Content

The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.

Cooling and pH jump-induced calcium release from isolated cardiac sarcoplasmic reticulum

1994 ◽  
Vol 267 (3) ◽  
pp. H962-H969
Author(s):  
J. J. Feher ◽  
I. M. Rebeyka
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Calcium Release ◽  
Order Rate Constant ◽  
Ruthenium Red ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Rapid Cooling ◽  
Ph Jump ◽  
Steady State Levels ◽  
Efflux Pathway

Rapid-cooling contracture in cardiac muscle preparations is thought to be caused by the release of Ca2+ from the sarcoplasmic reticulum (SR), but the mechanism of this release is unknown. Cooling of isolated canine cardiac SR from 37 to 4 degrees C resulted in the net release of enough Ca2+ to account for rapid-cooling contracture. The release of Ca2+ on cooling appeared to be a relaxation between different steady-state levels of Ca2+ uptake. Cooling release of Ca2+ was also observed in the presence of ryanodine or ruthenium red to block the ryanodine-sensitive Ca2+ efflux pathway. In the presence of ryanodine, the extent and initial rate of rapid-cooling release were increased due to increased steady-state uptake of Ca2+ by the SR. The first-order rate constant of rapid-cooling Ca2+ release was unchanged by ryanodine or ruthenium red, suggesting that the rapid-cooling release does not occur through the ryanodine-sensitive pathway. The alkalinization on cooling was not the major cause of the Ca2+ release, as comparable Ca2+ release was observed with cooling and no alkalinization. However, alkalinization without cooling produced a rapid net Ca2+ release, which was also observed in the presence of ryanodine.

Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells

1997 ◽  
Vol 272 (2) ◽  
pp. C601-C614 ◽  
Author(s):  
I. N. Pessah ◽  
T. F. Molinski ◽  
T. D. Meloy ◽  
P. Wong ◽  
E. D. Buck ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Ruthenium Red ◽  
Loading Capacity ◽  
Fk 506 ◽  
High Affinity ◽  
Plasmic Reticulum ◽  
Efflux Pathway ◽  
The Relationship ◽  
Ca2 Channels

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

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 Preparation of Rat Gingival Mitochondria with an Improved Isolation Method

2010 ◽  
Vol 2010 ◽  
pp. 1-6
Author(s):  
Noriaki Kaneko ◽  
Tetsuya Rikimaru ◽  
Tetsuyuki Fujimura ◽  
Shigeyasu Mori ◽  
Saburo Hidaka ◽  
...  
Keyword(s):  
Optimal Condition ◽  
Respiratory Control ◽  
Adenosine Diphosphate ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Isolation Method ◽  
High Quality

In order to establish a method of obtaining rat gingival mitochondria (Mt), Mt fractions were prepared in various combinations of homogenizing time with collagenase concentration. Rat gingival tissues were excised, minced, treated with collagenase, homogenized, and subjected to differential centrifugation rates. Both the respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio of the Mt fraction prepared in a combination of 40, 50, or 60 sec homogenization with collagenase in a concentration range of 0.115%–0.130% (w/v) were measured. The values for the RCR and ADP/O ratio of the Mt fraction obtained in an optimal condition was and , respectively. These results suggest that Mt of fairly high quality can be obtained through this refined combination of the homogenizing time and collagenase concentration.

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.

scholarly journals Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37° C

1984 ◽  
Vol 224 (2) ◽  
pp. 423-430 ◽  
Author(s):  
S Dalton ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Intact Cells ◽  
Phosphate Ions ◽  
Low Concentrations ◽  
Significant Impairment

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly 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.

OXIDATIVE PHOSPHORYLATION BY MUSCLE MITOCHONDRIA OF DYSTROPHIC MICE

1967 ◽  
Vol 45 (8) ◽  
pp. 1271-1278 ◽  
Author(s):  
Klaus Wrogemann ◽  
M. C. Blanchaer
Keyword(s):  
Skeletal Muscle ◽  
Oxidative Phosphorylation ◽  
Respiratory Control ◽  
Laboratory Strain ◽  
Adenosine Diphosphate ◽  
Isolation Procedure ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Dystrophic Mice ◽  
Phosphorylation Rate

Oxidative phosphorylation was studied in mitochondria isolated from the skeletal muscle of control and dystrophic mice of the Jackson Laboratory strain 129/Re, aged 32–104 days. The isolation procedure included a preliminary incubation of the muscle minced in a medium containing a proteinase (Nagarse) followed by gentle homogenization and differential centrifugation. Polarographic estimations in the presence and absence of adenosine diphosphate (ADP) indicated that the rate of oxygen uptake, ADP/0 ratio, respiratory control ratio, and phosphorylation rate were not significantly different in the mitochondria isolated from control and dystrophic mice. Bovine serum albumin increased the ADP/0 and respiratory control ratios, but the values for the control and dystrophic preparations again did not differ significantly in the presence of albumin.

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