The effect of the prostaglandin analogue-misoprostol on rat liver mitochondria after chronic alcohol feeding

Rats fed ethanol (36% of total calories in a nutritionally adequate liquid diet) for 5 weeks develop functional alterations of hepatic mitochondria and steatosis of the liver. At the fatty liver stage, ADP-stimulated respiration of mitochondria was depressed in ethanol fed rats by 30% (p less than 0.001) with glutamate + malate and by 23% (p less than 0.001) with succinate as substrates. A similar decrease was noted in the respiratory control ratio (RCR) (34% and 29%, respectively). The total lipid content of the liver increased 2.6 fold (p less than 0.001). Mitochondrial dysfunction could be prevented, in part, by the treatment with a synthetic derivative of prostaglandin E1, misoprostol, at a mean daily dose of 80 micrograms/kg of body weight. The RCR with glutamate + malate as substrates was improved by 36% (p less than 0.05). We conclude that misoprostol attenuates several functional alterations in liver mitochondria during alcohol feeding.     

The effect of highly-dispersed zinc powder on the functional state of blood components and rat liver mitochondria

The functional state of blood components and liver mitochondria have been investigated by means of ESR method and atomic adsorption spectroscopy. The experimental data indicated that injection of highly dispersed Zn-powder in the concentration of 5 mg/kg of weight did not result in disturbance of mitochondria bioenergetic functions, although the functional state of blood components and the level of metal ions in it were changed.     

The role of Mg2+ in the regulation of the structural and functional steady-states in rat liver mitochondria

A possible relationship between mitochondrial Mg²⁺ levels, structural configurations, and functional steady states has been studied in rat liver mitochondria. The results show that the concentration of mitochondrial Mg²⁺ in respiratory state 4 is definitely higher than in respiratory state 3. The metabolic transition from state 3 to state 4 and vice-versa is associated with reversible influx-efflux of about 10 nmol of Mg²⁺ per mg protein. The net uptake of this aliquot of Mg²⁺ is a necessary condition in order for the metabolic transition to state 4, both structurally and functionally, to occur. This process requires a threshold concentration of external Mg²⁺ greater than 5 mM. The phosphorylative mechanism does not appear to depend on the presence or absence of external Mg²⁺. The role of Mg²⁺ on the attainment and maintenance of the structural and functional steady state 4 seems to be correlated with its regulatory effect on the concentration of the mitochondrial P_i.     

The structural organization of haem synthesis in rat liver mitochondria

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe(3+) is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the K(m) for Co(2+) is 11x10(-6)m and that for mesoporphyrin is 5x10(-6)m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.     

The effect of bile salts and calcium on isolated rat liver mitochondria

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.     

An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

The effect of choline deficiency on the outer membranes of rat liver mitochondria

The outer membranes of mitochondria prepared from the liver of rats kept 12 days on a choline-deficient diet were analyzed for changes in phospholipid and protein content. The total amount of phospholipid in the outer membranes was not affected by the deficiency. There was, however, a significant decrease in the amount of phosphatidylcholine and an increase in phosphatidylethanolamine. The alterations in the membrane phospholipids were reflected in a reduction in the fluorescence of the membrane probe, 8-anilino-1-naphthalene sulfonate. Choline deficiency also affected the protein composition of the outer membranes as judged by electrophoretic analysis; however, the activity of several enzymes which serve as markers for the outer membrane was not affected by the deficiency.     

The effect of inhibitors on the formation of phosphoenolpyruvate by rat liver mitochondria

1. Rat liver mitochondria oxidizing malate produce PEP (phosphoenolpyruvate) without the addition of ATP or other nucleotides. 2. The addition of oligomycin in the presence of 2,4-dinitrophenol did not abolish PEP formation and in some instances stimulated its formation. 3. Formation of PEP was inhibited by arsenate. 4. Arsenite decreased PEP formation and caused accumulation of pyruvate. 5. Added GTP and ITP had no effect on PEP formation. 6. PEP formed from malate in the presence of GTP and labelled P(i) had a specific radioactivity approximately the same as the P(i) with no contribution from the phosphate of the added GTP. 7. There was no parallelism between the effects of inhibitors on PEP formation from malate and their effects on the assayed activity of PEP carboxykinase. 8. In a direct comparison it was shown that the PEP carboxykinase content of mitochondria was insufficient to account for the PEP formation from malate. 9. Consideration of the kinetic characteristics of PEP carboxykinase and mitochondrial content of oxaloacetate and GTP show that this enzyme cannot account for the PEP formed from malate by mitochondria.     

The effect of oligomycin on rat liver mitochondria respiring in state 4

It has been found that oligomycin inhibits up to at least 50% state-4 mitochondrial respiration. A time dependence of oligomycin inhibition has been shown. A titration curve for state-4 respiration of sigmoidal profile has been presented. The possibility of misreading this oligomycin effect, so far never reported, has been excluded by evaluating the quality of mitochondrial preparations used in respect to their morphological, functional and electrochemical properties. The conclusion has therefore been put forward that the most part of respiration in steady-state-4 is driven by ATP synthesis.     

The effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria

Effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria is described. State 3 respiration is inhibited by acetoacetate, while state 4 respiration is not inhibited, though cytochrome c reduction was decreased. Acetoacetate is also non-competitive inhibitor of 3-hydroxybutyrateoxidase and 3-hydroxybutyrate dehydrogenase activity in frozen-thawed mitochondria. The results are discussed in terms of the thermodynamic hypothesis and control strength method.     

The effect of agaric acid on citrate transport in rat liver mitochondria

The effect of agaric acid (αcetyl citric acid) , a competitive inhibitor of the adenine nucleotide translocase, was studied on the citrate uptake in rat liver mitochondria. The experiments carried out reveal that citrate uptake is progressively inhibited by increasing concentrations of agaric acid, showing a typical competitive type of inhibition. The apparent Ki for agaric acid is 5.2 μM, a concentration lower than that which inhibits the adenine nucleotide translocase. The results also show that mersalyl diminishes the Ki to 3.4 μM; 10 mM KCl reverses the inhibitory action of agaric acid on the ADP and ATP exchange but does not affect the agaric acid induced inhibition of citrate uptake.     

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.