Properties Of Rat Liver Mitochondria Research Articles

Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl- l-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl- l-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.

Macromolecules Increase the Channeling of ADP from Externally Associated Hexokinase to the Matrix of Mitochondria

Macromolecules can restore the morphological changes in the outer mitochondrial compartment that occur upon isolation of the organelle. They decrease the volume of the intermembrane space and increase the number of intermembrane contact sites. In this study, we investigated the effects of macromolecules on one of the processes occurring in the mitochondrial outer compartment and for which the native structure might be important, i.e. the ADP supply from outer-membrane-bound hexokinase-I to oxidative phosphorylation. With the use of a reconstituted system in which rat liver mitochondria and extramitochondrial pyruvate kinase compete for ADP generated by hexokinase, it was shown that (a) part of the ADP generated by mitochondrially associated hexokinase is not accessible to pyruvate kinase and is channeled into the mitochondrion, (b) in the presence of 10% (mass/vol.) macromolecules (i.e. dextran M20 or BSA) the pyruvate kinase inaccessible fraction increases from 19% to 31% of the ADP produced by hexokinase, (c) the ADP channeling is a characteristic property of bound hexokinase, and (d) the increased channeling induced by macromolecules can neither be explained by direct effects of these macromolecules on the basic respiratory properties of rat liver mitochondria, nor by direct effects of the kinetic properties of hexokinase-I. ATP and ADP determinations were performed in hexokinase/mitochondria incubation mixtures in the presence of macromolecules. These determinations showed that an important consequence of the channeling capacity of bound hexokinase is that lower extramitochondrial ADP levels and consequently higher extramitochondrial ATP/ADP ratios are maintained than when hexokinase is not bound. The experimental data demonstrate that the ADP channeling activity associated with bound hexokinase leads to the formation of two ADP concentration gradients, one across the outer membrane and one between bound hexokinase and the bulk phase.

Some properties of rat liver mitochondria with low Ca2+ content

A role for liver mitochondria in the regulation of cytoplasmic Ca’+ concentration was first suggested by their ability to accumulate large amounts of Ca’+ (Drahota et a/.. 1965) and to maintain extramitochondrial Ca2+ at 0 . 3 2 p ~ (Becker ct a/., 1980; Brand & De Selincourt, 1980; Nicholls, 1978), described by Nicholls (1978) as the set point which may be altered by varying the rates of Ca2+ uptake and efflux. This has led Joseph et al. ( 1982) and Crompton (1 985) to argue that liver mitochondria operate to buffer cytoplasmic Ca2+ in the intact cell. Liver mitochondria studied this way have contained more than 28 nmol of Ca2+ /mg of protein, e.g. Nicholls ( 1978), yet recent electron probe micro-analyses (Somlyo et a/., 1985) of liver cells and use of special fractionation techniques (Reinhart et a/., 1984) sugqest that mitochondria in situ contain less than 2 nmol of Ca +/mg of protein. Hansford & Castro (1982) showed lack of buffering by rat heart mitochondria when they contained less than 5nmol of Ca2+/mg of protein and concluded that heart mitochondria act to vary matrix Ca” and so operate in the mode of dehydrogenase control (Denton & McCormack, 1985). The purpose of the present work is to examine the behaviour of liver mitochondria with low, probably physiological, Ca2+ content towards external Ca2+ at concentrations found in hepatocyte cytoplasm. Mitochondria were prepared by the method of Chappell & Hansford ( 1972) in 250 mM-sucrose/ 10 mM-Hepes/l mMEGTA/pH 7.4, with the exception that the third centrifugation at 10000 rev./min was carried out in 250 mM-sucrose/ 5 mM-Hepes/Io mM-NTA/pH 7.4. The pellet was then resuspended in 50ml of the latter medium and left to stand on ice for 25--30 min before being centrifuged at 10 000 rev./min for 10 min. The resulting mitochondrial suspension contains 0.9 nmol of Ca” /mg of protein and 35 nmol of Mg2+/mg of protein corrected for medium Ca2+ and Mg2+ content. Ca2+ content was uninfluenced by alterations in pH (6. I-7.9), NTA concentration (1-17 mM), or by substituting 250mMsucrose with 125 mM-KCI. pCa was determined in mitochondrial incubations using a Ca” -sensitive electrode (Radiometer type F2002 with a KCI reference electrode Radiometer type K801) and calibrated using Ca-NTA buffers of known pCa (Martell & Smith. 1977; Fabiato & Fabiato, 1979). Medium Ca’+ content and mitochondrial total Ca2+ content were measured with a Perkin-Elmer atomic absorption spectrophotometer Model 380 after mitochondrial Ca” had been extracted with 5% (w/v) trichloroacetic acid/l.5% (w/v) LaCI, (final concentration) and the denatured protein removed by centrifugation. One may calculate the buffer capacity ( B ) for Ca2+ of mitochondria (B,,, ,) by comparing the rise in free external Ca” on addition of Ca2+ aliquots to 10mM-NTA plus mitochondria (B,,,,,) and on addition to IOmM-NTA alone ( B , m M N T A ) . Buffer capacity is measured by the amount of Ca + required to alter pCa by one unit. 4 -

Structural and functional properties of rat liver mitochondria in hexachlorobenzene induced experimental porphyria

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.

Evidence of changes, induced by HeNe laser irradiation, in the optical and biochemical properties of rat liver mitochondria

Photoacoustic spectroscopy measurements reveal that HeNe low-power irradiation causes changes in mitochondrial optical properties. However, it has been found that this process damages neither the membrane of the mitochondria nor some of their enzymatic activities. The HeNe laser irradiation effect was investigated both with regard to the redox state change of intramitochondrial pyridine nucleotides, induced by addition to the mitochondria of oxaloacetate, oxoglutarate plus ammonia and cis-aconitate, and to the permeability of rat liver mitochondria to the same substrates. It was found that laser irradiation modifies some NADH-linked dehydrogenase reactions of the mitochondria.

Evidence of changes, induced by HeNe laser irradiation, in the optical and biochemical properties of rat liver mitochondria

Abstract Photoacoustic spectroscopy measurements reveal that HeNe low-power irradiation causes changes in mitochondrial optical properties. However, it has been found that this process damages neither the membrane of the mitochondria nor some of their enzymatic activities. The HeNe laser irradiation effect was investigated both with regard to the redox state change of intramitochondrial pyridine nucleotides, induced by addition to the mitochondria of oxaloacetate, oxoglutarate plus ammonia and cis-aconitate, and to the permeability of rat liver mitochondria to the same substrates. It was found that laser irradiation modifies some NADH-linked dehydrogenase reactions of the mitochondria.

Respiratory properties of rat liver mitochondria immobilized on an alkylsilylated glass surface.

Rat liver mitochondria are shown to adhere to the alkylsilylated glass beads in essentially a monolayer. The amount of mitochondria bound to the beads reaches a maximum where the length of the alkyl groups covalently linked to the beads exceeds eight carbons. Mitochondria immobilized on the beads and placed in a flow system exhibit normal: (a) respiratory control, (b) phosphate to oxygen ratio, (c) uncoupling by 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, and (d) inhibition by cyanide, azide, rotenone, oligomycin, and antimycin. Reversibility of the effects of 2,4-dinitrophenol, cyanide, and azide was rapid and complete. Inhibition by rotenone, oligomycin was essentially irreversible. Mitochondria have been maintained in a viable state on the beads at 27 degrees for periods up to 4 hours. The use of immobilized organelles appears to offer a new technique for the study of membrane-bound particles whereby substances can be rapidly added and removed while monitoring the composition of solution flowing over the particles.

The Effect of Cations on Membrane Surface Properties of Rat Liver Mitochondria as Determined by Microelectrophoresis

Bacteria (5.0mg cell dry wt./ml) were incubated under anaerobic conditions at 30°C in ~ ~ O ~ M K C ~ ~ ~ M T ~ ~ S H C I in the initial pH range 6.5-6.6. O2 (24ng-atoms) was added as air-saturated medium at the arrows. Valinomycin (2Smg/g cell dry wt.) was in (a), valinomycin (2.5 mg/g cell dry wt.) and 2.5p~-carbonyl cyanide phenylhydrazone in (b), 2.5p~-carbonyl cyanide phenylhydrazone in (d). The quantity of H+ ions equivalent to the pH changes are indicated for the 4ml of medium. A decrease in the pH of the suspension is upwards.

The correlation between in vivo mitochondrial changes and tryptophan pyrrolase activity

A correlation between changes in osmotic properties of rat liver mitochondria and increased activity of tryptophan pyrrolase following the in vivo injection of a number of compounds which produce a rise in tryptophan pyrrolase activity has been shown. Compounds tested which do not significantly affect tryptophan pyrrolase activity have no sensible effect on the osmotic properties of the rat liver mitochondria. Both changes occur concomitantly; however, no associated activity change in dinitrophenol or magnesium-ion activated adenosinetriphosphatase was detected. The rate of uptake of radioactive tryptophan by isolated mitochondria was also unaffected.