Rat Liver Mitochondria In State Research Articles

The stimulation of succinate-fueled respiration of rat liver mitochondria in state 4 by α,ω-hexadecanedioic acid without induction of proton conductivity of the inner membrane. Intrinsic uncoupling of the bc1 complex

The paper shows that natural α,ω-dioic acid, α,ω-hexadecanedioic acid (HDA), is able to stimulate the respiration of succinate-fueled rat liver mitochondria in state 4 without induction of proton conductivity of the inner membrane. This effect of HDA is less pronounced in glutamate/malate-fueled mitochondria, as well as in the case of ascorbate/TMPD or ascorbate/ferrocyanide substrate systems, which transfer electrons directly to cytochrome c. It is noted that HDA-induced stimulation of respiration is not associated with damage to the inner membrane in a part of mitochondria and with shunting of electrons through the bc1 complex. Therefore, HDA can be considered as a natural decoupling agent. Specific inhibitors of the bc1 complex (antimycin A and myxothiazole) as well as malonate and dithionitrobenzoate were used in the inhibitory analysis. These and other experiments have shown that during the oxidation of succinate in liver mitochondria, the decoupling effect of HDA is mainly carried out at the level of the bc1 complex. We hypothesized that HDA is capable of promoting the cyclic transport of protons within the bc1 complex and thus switch this complex to the idle mode of operation (intrinsic uncoupling of the bc1 complex). Induction of free respiration in liver mitochondria by HDA at the level of the bc1 complex is considered as one of the "rescue pathways" of hepatocytes in various pathological conditions, accompanied by disorders of carbohydrate and lipid metabolism and increased oxidative stress.

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC 50 = 2.4 ± 0.1 μM) which marginally affected electron transport and ATPase activity (IC 50 = 97 ± 4 μM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC 50 = 20 ± 4 μM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC 50 = 2.2 ± 0.4 μM) induced by succinate oxidation in submitochondrial particles but did not collapse the ΔpH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Δ ψ) and/or the localized transmembrane pH difference (ΔpH S) without affecting the ΔpH between the two bulk aqueous phases (ΔpH B). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

The effect of pancreatitis associated ascitic fluid on some functions of rat liver mitochondria

The damage to the liver during acute pancreatitis (AP) could be partly dependent on depressive action of pancreatitis associated ascitic fluid (PAAF) on the energy metabolism of hepatocytes. The aim of the study was to assess the effect of PAAF from dogs with acute experimental pancreatitis (AEP) and from humans with AP on the respiratory function of isolated rat liver mitochondria (RLM). The mitochondrial oxygen consumption rate in state 3 respiration (with ADP) and in state 4 (without ADP) using sodium succinate as substrate and oxygen Clark's electrode was estimated. Respiratory control ratio (RCR) and P/O ratio were calculated. PAAF was collected after 6 h of AEP induced by Elliott's method in 8 dogs, and from 4 patients with AP, intraoperatively. Both animal and human PAAFs increase the oxygen consumption rate by RLM in state 4 dose dependently (by 65% with 50 microL to 150% with 200 microL of canine PAAF). This uncoupling effect of human PAAF was twice more potent than the canine. Dialysis of PAAF reduced this effect almost completely. The mitochondrial ATPase activity in RLM treated with PAAF was stimulated and this effect was also reduced by dialysis. The conclusion was that the damage to the liver in AEP could be partly dependent on the toxicity of dializable component(s) of PAAF on the energy metabolism of mitochondria. These findings may partly explain the beneficial effects of peritoneal lavage in acute pancreatitis.

β-Oxidation of polyunsaturated fatty acids having double bonds at even-numbered positions in isolated rat liver mitochondria

β-Oxidation of polyunsaturated fatty acids was studied with isolated rat liver mitochondria in state 3 or uncoupled conditions. 1. 1. Incubation of mitochondria with docosahexaenoyl-, linolenoyl- or γ-linolenoylcarnitine resulted in an increase of the absorbance at 340 minus 385 nm. This increased absorbance was due to an accumulation of β-oxidation intermediates of the polyunsaturated fatty acids, and not to the reduction of nicotinamide nucleotides. 2. 2. Experiments carried out with soluble fractions of liver mitochondria incubated with docosahexaenoyl-CoA and γ-linolenoyl-CoA indicated that this ultraviolet light-absorption was at least partly caused by acyl-CoA esters having a 2,4(,7)-di(tri)enoyl-CoA structure. 3. 3. The addition of glutamate to mitochondria oxidizing γ-linolenoylcarnitine decreased the absorbance at 340 minus 385 nm, and simultaneously stimulated respiration. With liver mitochondria isolated from fasted rats, 6 mM glutamate increased the rate of acetoacetate production from γ-linolenoylcarnitine by 130 and 210% under state 3 and uncoupled conditions, respectively. Glutamate did not have any significant effect on the degradation of oleoylcarnitine. The proposed explanation for these findings is that the glutamate dehydrogenase reaction can function as a source of NADPH for 2,4-dienoyl-CoA reductase. 4. 4. The degradation of γ-linolenoylcarnitine to ketone bodies was augmented in mitochondria isolated from rats treated with clofibrate or partially hydrogenated marine oil. 5. 5. We conclude that 2,4-dienoyl-CoA reductase is an important auxiliary enzyme in the β-oxidation of polyunsaturated fatty acids. Induction of this enzyme by clofibrate or by certain high-fat diets increases mitochondrial capacity for the degradation of polyunsaturated fatty acids.

Competition for NADH by mitochondrial dehydrogenases in rat and guinea pig livers.

The factors responsible for regulating the NAD+/NADH ratio of guinea pig and rat liver mitochondria were studied using both isolated mitochondria and perfused livers from both species. A comparison of the rates of ketone body formation and the acetoacetatelfi-hydroxybutyrate ratio in isolated guinea pig and rat liver mitochondria in State 3 show that in the absence of added citric acid cycle anions, mitochondria from both species will synthesize predominantly acetoacetate. Rat liver mitochondria produced 4 times more total ketone bodies than guinea pig liver mitochondria. The addition of malate dramatically reduced the formation of ketone bodies by supplying oxalacetate for the subsequent synthesis of citrate by mitochondria from both species. The only experimental treatment that reduced the acetoacetatelp-hydroxybutyrate ratio during State 3 in guinea pig liver mitochondria, to levels noted in mitochondria from rat liver, was

The Inhibition of Isocitrate Oxidation by Palmitoyl‐l‐carnitine and Palmitoyl‐CoA in Rat Liver Mitochondria

Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane. From titration experiments of the rate of O 2 uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.

A comparison between the effectiveness of uncouplers of oxidative phosphorylation in mitochondria and in different artificial membrane systems

The concentration range in which uncouplers of oxidative phosphorylation stimulate the respiration of rat-liver mitochondria in State 4 was compared with that in which those compounds stimulate fluxes through black lipid membranes or liposomal membranes. A poor correlation exists between the effectiveness of uncouplers in mitochondria and in black lipid membranes. In contrast, a good correlation exists between uncoupling activity in mitochondria and stimulation of valinomycin-induced swelling of liposomes or stimulation of reduction by ascorbate/ferrocene of ferricyanide included in liposomes.

Reduction and oxidation of longer-wavelength cytochrome b ( b566) in rat liver mitochondria under the influence of external oxidants

The reduced form of longer-wavelength cytochrome b ( b 566) was identified in rat liver mitochondria in State 5 on addition of ferricyanide. Addition of menadione caused oxidation of b 566 and reduction of cytochrome c 1 (+ c). In the presence of menadione, ferricyanide induced reduction of b 566 and oxidation of cytochrome c 1 + c, and exhaustion of the added ferricyanide resulted in oxidation of b 566 and reduction of cytochrome c 1 + c. Antimycin A enhanced the extent of the ferricyanide-dependent redox change of b 566 from 20 to 68% of the total absorbance at 566 nm. Uncouplers hardly affected the change of b 566 in the presence of antimycin A, but lowered the reduced level in the absence of antimycin A.

A permeability barrier in mitochondria in ‘high-energy states’ against positively charged disulphides

1. In rat liver mitochondria in state 1 or 4 there is a permeability barrier against cystamine, probably in the inner membrane. 2. The permeability barrier was broken (a) when oxidative phosphorylation was uncoupled, (b) when the respiratory chain was inhibited or in anaerobiosis, or (c) when phosphate was added in the absence of exogenous substrate. Under these conditions increased amounts of [(35)S]cystamine residues were bound to matrix proteins. 3. It appears that the permeability barrier against cystamine in mitochondria reflects a ;high-energy state'. A gradual increase in the permeability for cystamine strikingly coincided with the loss of respiratory control induced by increasing concentrations of different uncoupling agents. 4. Cystamine caused uncoupling of oxidative phosphorylation in state 2 or 5, but not in state 1, 3 or 4. The uncoupling effect of cystamine was dependent on the phosphorylation potential. ATP counteracted, whereas ADP potentiated, the uncoupling by cystamine. 5. The variable penetration of cystamine appears to depend on its positive charge, since a dication derivative, NNN'N'-tetramethylcystamine, has a similar pattern of penetration, whereas an uncharged derivative, NN'-diacetylcystamine, penetrates rapidly into mitochondria irrespective of their metabolic state. 6. It is suggested that a charge barrier is present in or across the inner mitochondrial membrane in ;high-energy states'.