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 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

1980 ◽  
Vol 190 (1) ◽  
pp. 107-117 ◽  
Author(s):  
R M Denton ◽  
J G McCormack ◽  
N J Edgell
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Ruthenium Red ◽  
Heart Mitochondria ◽  
Heart Cells ◽  
Maximal Effect ◽  
Rat Heart Mitochondria ◽  
20 Nm

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2–3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.

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 Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios

1988 ◽  
Vol 252 (1) ◽  
pp. 181-189 ◽  
Author(s):  
G A Rutter ◽  
R M Denton
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Rat Heart ◽  
Adenine Nucleotides ◽  
Heart Mitochondria ◽  
Maximal Effect ◽  
Dehydrogenase System ◽  
Rat Heart Mitochondria ◽  
Oxoglutarate Dehydrogenase ◽  
Intact Heart

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.

scholarly journals The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol

1983 ◽  
Vol 214 (2) ◽  
pp. 387-393 ◽  
Author(s):  
A P Halestrap ◽  
P T Quinlan
Keyword(s):  
Liver Mitochondria ◽  
The Other ◽  
Heart Mitochondria ◽  
Protonmotive Force ◽  
Mitochondrial Matrix ◽  
Matrix Volume ◽  
The Matrix ◽  
The Mean ◽  
Isolated Liver

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.

The direct physiological effects of mitoKATP opening on heart mitochondria

2006 ◽  
Vol 290 (1) ◽  
pp. H406-H415 ◽  
Author(s):  
Alexandre D. T. Costa ◽  
Casey L. Quinlan ◽  
Anastasia Andrukhiv ◽  
Ian C. West ◽  
Martin Jabůrek ◽  
...  
Keyword(s):  
Steady State ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
Cell Physiology ◽  
State Matrix ◽  
Matrix Volume ◽  
The Matrix ◽  
Activity Yield ◽  
Rat Heart Mitochondria ◽  
Potassium Binding

The mitochondrial ATP-sensitive K+ channel (mitoKATP) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoKATP opening that should be observable at the level of the mitochondrion. MitoKATP opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoKATP opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoKATP openers and blockers with those of valinomycin, we show that four independent assays of mitoKATP activity yield quantitatively identical results for mitoKATP-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoKATP opening for mitochondria.

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