scholarly journals Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2

1987 ◽  
Vol 248 (2) ◽  
pp. 609-613 ◽  
Author(s):  
G L Lukács ◽  
A Kapus
Keyword(s):  
Heart Mitochondria ◽  
Fura 2 ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
Matrix Free

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.

scholarly journals Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes

1990 ◽  
Vol 271 (3) ◽  
pp. 627-634 ◽  
Author(s):  
G A Rutter ◽  
N J Osbaldeston ◽  
J G McCormack ◽  
R M Denton
Keyword(s):  
Rat Heart ◽  
Free Acid ◽  
Magnesium Content ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
Fura 2 ◽  
Kd Values ◽  
Carbonyl Cyanide ◽  
The Matrix ◽  
Rat Heart Mitochondria

1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.

scholarly journals Determination of the matrix free Ca2+ concentration and kinetics of Ca2+ efflux in liver and heart mitochondria.

1982 ◽  
Vol 257 (15) ◽  
pp. 8696-8704 ◽  
Author(s):  
K E Coll ◽  
S K Joseph ◽  
B E Corkey ◽  
J R Williamson
Keyword(s):  
Heart Mitochondria ◽  
The Matrix ◽  
Matrix Free ◽  
Kinetics Of

Regulation of cardiac mitochondrial calcium by average extramitochondrial calcium

1993 ◽  
Vol 265 (4) ◽  
pp. H1203-H1208 ◽  
Author(s):  
J. R. Leisey ◽  
L. W. Grotyohann ◽  
D. A. Scott ◽  
R. C. Scaduto
Keyword(s):  
Steady State ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
Mitochondrial Matrix ◽  
Fura 2 ◽  
Steady State Levels ◽  
Rat Heart Mitochondria ◽  
The Relationship ◽  
Matrix Free

A system to perifuse isolated rat heart mitochondria was designed to study the relationship between mitochondrial matrix free Ca2+ and extramitochondrial free Ca2+ under conditions in which the latter concentration could oscillate over a range typical of that expected in vivo. We tested the hypothesis that the level of intramitochondrial Ca2+ responds to the average extramitochondrial Ca2+ in the heart. Mitochondria were immobilized within an optical chamber for measurement of endogenous NAD(P)H and fura 2 fluorescence. NAD(P)H increased significantly on provision of substrates and decreased reversibly in the presence of ADP, indicating maintenance intact coupled respiration by this preparation. Matrix free Ca2+ was measured using fura 2-loaded mitochondria and, in parallel experiments, media free Ca2+ was measured with fura 2 in the absence of mitochondria. Oscillation of extramitochondrial Ca2+ from < 0.1 microM to approximately 2 microM at frequencies of 0.5, 1.0, and 1.25 cycles/s produced steady-state levels of matrix Ca2+ that were independent of frequency but proportional to the average media free Ca2+ concentration. Matrix Ca2+ increased to a steady state on an increase in the extramitochondrial average Ca2+ concentration with a half-time (t1/2) of approximately 2 min at 22 degrees C. Oscillation of mitochondrial Ca2+ was not observed under any conditions tested. The data are taken to indicate that in vivo, the concentration of mitochondrial matrix free Ca2+ is a steady state that is proportional to the average extramitochondrial Ca2+ concentration and that changes in the latter represent a mechanism of signal transduction from the cytosol to the mitochondrial matrix.

scholarly journals A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states

1990 ◽  
Vol 266 (1) ◽  
pp. 33-39 ◽  
Author(s):  
M Crompton ◽  
A Costi
Keyword(s):  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Ionophore A23187 ◽  
Relative Permeabilities ◽  
Pulsed Flow ◽  
Pore Closure ◽  
Open States ◽  
Matrix Free ◽  
Pore Number

The permeability properties of a putative Ca2(+)-activated pore in heart mitochondria, of possible relevance to re-perfusion-induced injury, have been investigated by a pulsed-flow solute-entrapment technique. The relative permeabilities of [14C]mannitol, [14C]sucrose and arsenazo III are consistent with permeation via a pore of about 2.3 nm diameter. Ca2+ removal with EGTA induced pore closure, and the mitochondria became ‘resealed’. The permeability of the unresealed mitochondria during resealing was markedly stimulated by 200 microM-ADP, and the relative permeabilities to solutes of different size were stimulated equally, indicating an increase in open-pore number, rather than an increase in pore dimensions. This is paradoxical, since ADP also stimulated the rate of resealing. The rate of EGTA-induced resealing was also stimulated by the Ca2+ ionophore A23187, which indicates that the rate of removal of matrix free Ca2+ is limiting for pore closure. An explanation for the paradox is suggested in which ADP facilitates pore interconversion between the closed and open states in permeabilized mitochondria, and pore closure in Ca2(+)-free mitochondria occurs much faster than previously thought.

scholarly journals MORPHOLOGY AND ATP-ASE OF ISOLATED MITOCHONDRIA

1955 ◽  
Vol 1 (2) ◽  
pp. 127-138 ◽  
Author(s):  
Robert F. Witter ◽  
Michael L. Watson ◽  
Mary A. Cottone
Keyword(s):  
Electron Microscope ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Substantial Part ◽  
Mitochondrial Matrix ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
First Time ◽  
Methods Of Isolation

Changes in the morphology of rat liver mitochondria brought about by different methods of isolation and the concomitant changes in ATP-ase activity were studied. The morphology was investigated with the electron microscope. It was found that the ATP-ase activity of the isolated mitochondria cannot be readily correlated with the morphology of the mitochondria. The ATP-ase found in these preparations was latent, resembling the enzyme described in mitochondria prepared in 0.25 M sucrose. In confirmation of earlier results the use of 0.88 M sucrose yielded preparations with a higher initial ATP-ase than did other methods. Preparation in 0.25 M sucrose resulted in round, swollen mitochondria of which 30 to 40 per cent appeared to have lost a substantial part of the mitochondrial matrix. Preparations in 0.44 to 0.88 M sucrose contained mainly rod-shaped mitochondria plus a small amount of another type of swollen mitochondria. The matrix of mitochondria isolated in 0.88 M sucrose was highly condensed. By the use of 0.44 M sucrose adjusted to pH 6.2 with citric acid, it was possible to isolate, for the first time, mitochondria closely resembling those in situ and containing latent ATP-ase.

scholarly journals Absorption and Photoluminescence Study of Cds Quantum Dots: The Role of Host Matrix and Nanocrystal Size and Density

1999 ◽  
Vol 571 ◽  
Author(s):  
Yu.P. Rakovich ◽  
A.G. Rolo ◽  
M.V. Stepikhova ◽  
M.I. Vasilevskiy ◽  
M.J.M. Gomes ◽  
...  
Keyword(s):  
Volume Fraction ◽  
Optical Transition ◽  
Spectral Position ◽  
Host Matrix ◽  
The Matrix ◽  
Mean Size ◽  
Free Films ◽  
Matrix Free ◽  
Cds Crystallites

ABSTRACTIn this paper we present results of the absorption and photoluminescence (PL) of CdSdoped Si02 films fabricated by RF co-sputtering (semiconductor volume fraction f=1–15%, nano-crystallite's mean size 5–7nm) and matrix-free films of close-packed CdS nanocrystallites (f∼30%, size 2–5nm) produced by an original chemical method. The absorption spectra have been modelled using the modified Maxwell-Garnett model. This gives the e-h pair state energies and evidence of a strong absorption in the glass matrix containing CdS. The temperature dependence of the spectral position and broadening of the PL peak is analysed. It is concluded that a photo-generated hole is captured on an acceptor-type trap before the radiative recombination with a confined electron. The excitation of this ‘band-edge’ PL occurs through some states in the matrix and directly in the CdS crystallites for the two kinds of samples, respectively. The temperature coefficients of the optical transition energies for the nearly matrix-free films are similar to those of bulk CdS, while for the CdS/glass films they are smaller. This may be because of the different boundary conditions for the thermal expansion of CdS crystallites.

scholarly journals The regulation of the oxidation of fatty acids and other substrates in rat heart mitochondria by changes in the matrix volume induced by osmotic strength, valinomycin and Ca2+

1987 ◽  
Vol 244 (1) ◽  
pp. 159-164 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Rat Heart ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Acid Oxidation ◽  
Maximal Effect ◽  
Physiological Conditions ◽  
Matrix Volume ◽  
The Matrix ◽  
Rat Heart Mitochondria

1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 microliter/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 microliter/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 microliter/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria [Halestrap & Dunlop (1986) Biochem. J. 239, 559-565]. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30 s at 30 degrees C and with K0.5 for Ca2+ of 0.3 microM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37 degrees C in the presence of 0.65 microM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181 +/- 0.029 microliter/mg of protein (n = 7, P less than 0.001), similar to the Ca2+-induced changes observed with liver mitochondria [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.

scholarly journals Celecoxib Decreases Mitochondrial Complex IV Activity and Induces Oxidative Stress in Isolated Rat Heart Mitochondria: An Analysis for its Cardiotoxic Adverse Effect

2020 ◽  
Author(s):  
Saman Atashbar ◽  
Elham Mohammad Khanlou ◽  
Saleh Khezri ◽  
Peyman Kurdpour ◽  
Ahmad Salimi
Keyword(s):  
Lipid Peroxidation ◽  
Rat Heart ◽  
Isolated Rat Heart ◽  
Mitochondrial Swelling ◽  
Heart Mitochondria ◽  
Isolated Mitochondria ◽  
Biochemical Assays ◽  
Ros Formation ◽  
Rat Heart Mitochondria ◽  
Isolated Rat

Abstract Background In spite of the cardiotoxic effect of selective cyclooxygenase-2 inhibitors, they are most widely used as anti-inflammatory and analgesic drugs. Today, valdecoxib and rofecoxib have been withdrawn on the market but celecoxib remains. In this study, we focused on an analysis of celecoxib toxic effects on isolated mitochondrial. Methods isolated rat heart mitochondria were obtained using differential centrifugation. Using flowcytometry and biochemical assays we searched succinate dehydrogenases (SDH), mitochondrial membrane potential (MMP), reactive oxygen species (ROS) formation, mitochondrial swelling, lipid peroxidation and mitochondrial complexes activity in rat heart isolated mitochondria. Results In here our results indicated a significant decrease in activity of complexes IV after exposure with celecoxib (16 µg/ml). This decrease in activity of complexes IV is paralleled by the MMP collapse, ROS formation, mitochondrial swelling and lipid peroxidation. Conclusion For the first time, this introductory study has showed a significant decrease in activity of complexes IV and mitochondrial dysfunction after exposure with celecoxib in rat heart isolated mitochondria.

scholarly journals The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria

1986 ◽  
Vol 239 (1) ◽  
pp. 31-40 ◽  
Author(s):  
I Al-Nasser ◽  
M Crompton
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Arsenazo Iii ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix ◽  
Matrix Compartment ◽  
Matrix Free ◽  
Regulatory Sites

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.

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