On the mechanism of uncoupler-dependent inhibition of acyl-carnitine oxidation by rat liver mitochondria

Degradation of rat liver cytosolic proteins at a neutral pH in the presence of 0.1% SDS was demonstrated by SDS-acrylamide gel electrophoresis. This proteolysis in vitro mimics molecular-size-dependent proteolysis in vivo; larger proteins were degraded more rapidly than smaller ones. Evidence is presented that the proteolysis is not due to contaminating lysosomal cathepsins in the cytosol.     

Membrane potential of hepatic mitochondria after acute cocaine administration in rats--the role of mitochondrial reduced glutathione

Occurrence of the classical pathway complement proteins C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 was studied in human hippocampus and temporal cortex by immunohistochemistry and Western blotting. In Alzheimer disease (AD) cases, positive staining for all of these proteins was observed in pyramidal neurons and senile plaques. In control cases, weaker pyramidal neuron staining was observed except for C1q and C1s which were not detected. On Western blots of AD hippocampal extracts, bands corresponding to those detected in normal serum were found for each of the complement proteins. Comparable bands were also detected in normal hippocampal extracts with the exception of C1s which was not observed. The intensity of the bands was generally stronger in AD than in normal extracts, but, in the latter, there was considerable variability between cases and between bands in a single case. These data suggest that pyramidal neurons may be a source of the complement components known to be associated with Alzheimer lesions.     

Comparison of side chain oxidation of potential C27-bile acid intermediates between mitochondria and peroxisomes of the rat liver: presence of beta-oxidation activity for bile acid biosynthesis in mitochondria

The oxidation of the side chains of two potential bile acid intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA), were investigated in rat liver mitochondria and peroxisomes. Both THCA and DHCA were efficiently oxidized to yield cholic acid and chenodeoxycholic acid, along with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-enoic acid, respectively, in both the mitochondria and peroxisomes. However, the spectrum of the metabolites in the mitochondria differed greatly from those in the peroxisomes. The major products from THCA and DHCA in the mitochondria were 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid and 3 alpha,7 alpha-trihydroxy-5 beta-chol-22-enoic acid, respectively, which were tentatively identified from the mass spectral data. However, the formation of these C24-unsaturated bile acids was not observed in the peroxisomes. These results strongly suggest that the cleavage of the side chain of the C27-intermediates for bile acid biosynthesis also occurs independently in the mitochondria, not due to the contamination of peroxisomes.     

Rapid determination of the membrane potential of mitochondria in small biopsy specimens of the liver

In liver transplantation, graft viability is ideally to be determined before implantation. Integrity of mitochondria may be a prerequisite to a viable graft. A new method is presented, which allows for the determination of the membrane potential of mitochondria (MPM; mV) in state 4 respiration within 50 min in 40-mg specimens, employing rhodamine 123 as a probe. Normal control showed a MPM of 239.2 mV. Storage in saline at 37 degrees C yielded an impaired MPM of 153.5 mV within 3 h. The cold storage at 1 degree C could preserve MPM at quasi-normal after 3 h but reduced it significantly after 24 h to 222.2 mV in saline (p < 0.005 vs. control) and 231.0 mV in UW solution (p < 0.05 vs. control): the difference between the 24-hour values was significant (p < 0.05).     

Response of isolated rat liver mitochondria to variation of external osmolarity in KCl medium: regulation of matrix volume and oxidative phosphorylation

When isolated rat liver mitochondria are incubated in KCI medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K+/H+ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and deltamuH+-dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD+ ratio, state 3 respiratory rate, and deltamuH+, while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.     

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria

Administration of dehydroepiandrosterone (DHEA), a steroid hormone of the adrenal cortex which acts as a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from the brain. The effect of DHEA on rat liver mitochondrial lipid peroxidation became discernible after feeding steroid-containing diet (0.6% w/w) for 3 days, and reached maximal levels between 1 and 2 weeks. DHEA in the concentration range 0.001-0.02% did not significantly increase lipid peroxidation compared to the control. Lipid peroxidation was significantly enhanced in animals given a diet containing > or = 0.05% DHEA. The addition of DHEA in the concentration range 0.1-100 microM to mitochondria isolated from control rats had no effect on lipid peroxidation. It seems, therefore, that the steroid effect is mediated by an intracellular process. Our data indicate that induction of mitochondrial membrane lipid peroxidation is an early effect of DHEA administration at pharmacological doses.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH

In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase, S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0. 10 h-1, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1Delta mutant and unaffected in an nde2Delta mutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1Delta nde2Delta double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1Delta nde2Delta mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) of an nde1Delta nde2Delta mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.     

Monoethlenic C20 and C22 fatty acids in marine oil and rapeseed oil. Studies on their oxidation and on their relative ability to inhibit palmitate oxidation in heart and liver mitochondria

1. Carnitine esters of erucic acid (22:1 n-9 cis), cetoleic acid (22:1 n-11 cis), brassidic acid (22:1 n-9 trans), gadoleic acid (20:1 n-9 cis) and oleic acid (18:1 n-9 cis) have been compared as mitochondrial substrates and as inhibitors of palmitoylcarnitine oxidation in heart and liver mitochondria. 2. Both the rate of intramitochondrial-CoA acylation and the rate of beta-oxidation decreases as the chain length increases from C18 to C22. There are no significant differences among the three C22 isomers as oxidizable substrates. 3. All the tested acylcarnitines inhibit palmitoylcarnitine oxidation. The C18 and C20 acylcarnitines inhibit by virtue of being competing substrates; i.e. the respiration is not inhibited. The C22-isomers inhibit also respiration; this shows that the inhibition of palmitolycarnitine oxidation is not compensated for by oxidation of C22-acylcarnitines. Brassidoylcarnitine inhibits the oxidation of palmitoylcarnitine and respiration less than erucoyl-and cetoleoylcarnitine. The different behaviour of the C22-isomers is probably due to the difference in their competitive properties with respect to long-chain acyl-CoA dehydrogenase. 4. All C22 acylcarnitines seem to be relatively better oxidized in the liver than in the heart mitochondria while their inhibitory effect on the usage of the radioactive palmitoylcarnitine is very similar. 5. Palmitoylcarnitine inhibits almost completely the "endogenous" formation of acetyl-CoA presumably from malate via pyruvate in the liver mitochondria while the C22-acylcarnitines cause only a partial inhibiton of this acetyl-CaO formation.     

Inhibition of pyruvate oxidation by skeletal muscle mitochondria by phenylpyruvate

1. Phenylpyruvate inhibits pyruvate plus malate oxidation in human and rat skeletal muscle mitochondria in state 3 and in the uncoupled state, it has, however, no effect in state 4. 2. Inhibition by phenylpyruvate of pyruvate oxidation by intact uncoupled rat muscle mitochondria was competitive, with the Ki value about 0.18 mM. 3. It is suggested that the inhibition of pyruvate oxidation is due to the action of phenylpyruvate on muscle pyruvate dehydrogenase, and is the principal cause of the elevated concentration of pyruvate and lactate in blood plasma of phenylketonuric patients.     

Electrocatalytic surface for the oxidation of NADH and other anionic molecules of biological significance

A simple electrochemical treatment of a carbon fiber electrode surface has been found to dramatically improve the voltammetry of NADH and several other anionic molecules under steady-state and fast scan (100 V/s) conditions. The electrocatalytic surface is generated through the electrochemical oxidation of NADH on a carbon fiber electrode that exhibits product adsorption. The oxidative product is reacted with ascorbic acid at elevated temperatures to create a surface which has very little overpotential for the oxidation of dopamine and many metabolites such as NADH, DOPAC, uric acid, and ascorbate. The electrochemical properties of the modified surface were examined voltammetrically at both slow and fast scan rates. The surface shown in this paper shifts the oxidation overpotentials different magnitudes for each analyte tested, thus allowing discrimination between analytes of interest and their major interferences. Another benefit of this new electrocatalytic wave is that it decreases the limit of detection for NADH by approximately 1 order of magnitude. Therefore, this new carbon surface not only gives better discrimination between two analytes but also gives better detection limits for certain analytes of interest.     

Transport of ascorbate into guinea pig liver mitochondria

The amount of ascorbate associated with guinea pig liver mitochondria was estimated by high-performance liquid chromatography. Incubation of mitochondria with ascorbate revealed a time-dependent and temperature-dependent accumulation of the vitamin. A steady-state level of ascorbate was obtained in the mitochondria after about 20 min of incubation at 37 degrees C, whereas no accumulation was observed at 0 degrees C. The matrix concentration of ascorbate was highly correlated to the concentration of ascorbate in the incubation medium. The initial rate of accumulation (about 7 pmol/mg protein per min at 10 degrees C) was three orders of magnitude less than for compounds that are transported across the mitochondrial inner membrane by specific carriers. Experiments with the enzyme ascorbate oxidase demonstrated that the mitochondrial membrane is also permeable to dehydroascorbate, and that the accumulated dehydroascorbate is stable in the mitochondria. There was no effect of the energy state of the mitochondrial membrane of the initial transport rate of ascorbate. Electrostatic binding of ascorbate to the membrane was excluded from experiments performed in isosmotic potassium chloride medium. Diffusion of ascorbate across the mitochondrial inner membrane accounts for the experimental findings.     

Uptake of hydroxocobalamin by rat liver mitochondria. Binding to a mitochondrial protein

Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes. The uptake system is saturable and unidirectional, leading to inside/outside concentration ratios of 17. The process also exhibits specificity: cyano[57Co]cobalamin is taken up less rapidly and to a lesser extent than hydroxocobalamin; methylcobalamin and adenoslcobalamin inhibit hydroxocobalamin uptake markedly, while cyanocobalamin does not. The [57Co]cobalamin ([57Co]Cbl) taken up is bound to a mitochondrial protein whose apparent molecular weight is 120,000 by Sephadex G-150 chromatography. Double reciprocal plots of bound [57Co]Cbl versus medium [57Co]Cbl concentration yield estimates for bound Cblmax of 29 pmol/mg of protein and for Kd is 8.2 muM. We conclude that mitochondrial uptake of cobalamins occurs via the diffusion of free cobalamins into the mitochondria and their subsequent binding to a high affinity mitochondrial protein(s) which we propose to be the source of the unidirectional character, the saturability, and the specificity of the uptake system.