Effect of the extramitochondrial adenine nucleotide pool size on oxidative phosphorylation in isolated rat liver mitochondria

We report a long-term follow-up of abduction-extension osteotomy of the first metacarpal, performed for painful trapeziometacarpal osteoarthritis. Of a consecutive series of 50 operations, 41 thumbs (82%) were reviewed at a mean follow-up of 6.8 years. Good or excellent pain relief was achieved in 80%, and 93% considered that surgery had improved hand function, while 82% had normal grip and pinch strength, with restoration of thumb abduction. Metacarpal osteotomy was equally successful in relieving symptoms of those with early (grade 2) and moderate (grade 3) degenerative changes. This simple procedure provides lasting pain relief, corrects adduction contracture and restores grip and pinch strength, giving good results with few complications.     

Non-enzymatic peroxidation of lipids isolated from rat liver microsomes, mitochondria and nuclei

AIM: To compare lansoprazole 30 mg daily with ranitidine 150 mg b.d. in the treatment of acid-related dyspepsia in general practice. METHODS: In a double-blind, parallel group, randomized, mutlicentre study conducted in 32 general practices in the UK, 213 patients were randomized to receive lansoprazole 30 mg daily, and 219 to receive ranitidine 150 mg b.d., for 4 weeks. All patients had experienced symptoms of reflux-like or ulcer-like dyspepsia on at least 4 of the 7 days prior to the study; 75% had experienced dyspepsia in the past, and 74 of the lansoprazole patients and 77 of the ranitidine patients had documented histories of acid-related disorders, investigating by either radiology or endoscopy. RESULTS: After 2 weeks 55% of the lansoprazole patients and 33% of the ranitidine group were symptom-free (P = 0.001, chi 2 = 7.12) with corresponding 4-week figures of 69% and 44%, respectively (P = 0.001, chi 2 = 18.03). Similar figures were found at both 2 and 4 weeks for daytime and night-time heartburn and epigastric pain scores; in the lansoprazole group, at 4 weeks, 80% of patients were free of daytime heartburn and 81% of night-time epigastric pain, compared with 55% (P = 0.001, chi 2 = 15.44) and 65% (P = 0.01, chi 2 = 6.10) in the ranitidine group. CONCLUSION: Superior symptom relief for patients presenting with ulcer-like and reflux-like symptoms in general practice is provided by lansoprazole 30 mg daily compared with ranitidine 150 mg twice daily.     

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.     

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.     

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.     

An oxidative damage-specific endonuclease from rat liver mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.     

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.     

Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.     

Effect of ribosomal proteins on synthesis and assembly of preribosomal particles in isolated rat liver nuclei

Ribosomal proteins are rapidly taken up by isolated rat liver nuclei. The proteins are localized mainly in the nucleolus and are found associated with a nucleolar 80 S particle containing newly synthesized 45 S RNA. Ribosomal proteins in the nucleoplasm were associated with particles containing 18 and 28 S RNAs. In the absence of ribosomal proteins in the incubations, there was a decrease in the amount of newly synthesized 45, 18, and 28 S RNAs and an increase in low molecular weight RNA in both the nucleolus and nucleoplasm. This in vitro system appears to be useful for studies on the formation of ribosomal particles in the nucleus.     

Effect of phenobarbital treatment on characteristics determining susceptibility to oxidants of homogenates, mitochondria and microsomes from rat liver

This study was designed to investigate the possible oxidative changes associated with alterations in cytochrome P450 levels in rat liver. Accordingly, extent of peroxidative processes, cytochrome and antioxidant content, capacity to face an oxidative stress were determined in liver microsomes, mitochondria, and homogenates from normal and phenobarbital (PB)-treated rats. Liver content of microsomal and mitochondrial proteins was also determined by the values of the activities of marker enzymes (glucose-6-phosphatase and cytochrome oxidase, respectively) in liver homogenate and in two cellular fractions. The increase in the liver content of microsomal and mitochondrial proteins indicated that PB caused proliferation of both smooth endoplasmic reticulum and mitochondrial population. Treatment with PB also gave rise to a general increase in peroxidative reactions (evaluated measuring malondialdehyde and hydroperoxides (HPs)), in the different cell compartments, even though HPs were not found significantly increased in mitochondrial fraction. The increase in peroxidative processes was associated with significant decreases in antioxidant concentration (expressed in terms of equivalent concentration of an antioxidant, such as the desferrioxamine), in all preparations from PB-treated rats. The response to oxidative stress in vitro (evaluated determining the parameters characterizing light emission from preparations stressed with sodium perborate) showed a substantial PB-induced increase in the susceptibility to oxidative challenge only in liver homogenate. The lack of changes in the mitochondrial preparations is likely due to decrease in concentration of both free radical producing species and antioxidants. The lack of changes in microsomal fraction is apparently in contrast with its lower oxidant capacity and higher content of cytochromes which are able to determine sensitivity to pro-oxidants. However, it could be due to the ability of cytochrome P450 to interact with the active oxygen species formed at its active center.     

Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria

OBJECTIVE: To investigate the role of phospholipase during the activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by peritoneal dialysis effluent (PDE). DESIGN: Examine the action of 4-hour dwell PDE upon phospholipase activation in the circulating neutrophils obtained from healthy individuals. RESULTS: We have previously reported that PDE stimulated superoxide release by the NADPH oxidase of human neutrophils and primed the response to the bacterial peptide, fMLP (fMetLeuPhe). To elucidate the biochemical mechanisms underlying these observations, we have examined the roles of phospholipases (PL) C, D, and A2, whose activation causes the release of a range of intracellular secondary messengers. Following fMLP stimulation, we observed a rapid activation of both PLC and PLD as well as a small but nonsignificant increase in PLA2 activity. Peritoneal dialysis effluent alone failed to stimulate either PLC or PLD, while pre-incubation with PDE had no affect upon fMLP-induced PLC and PLD activation. However, PDE caused a small but nonsignificant increase in PLA2 activity (which was comparable to that observed with fMLP) and primed the fMLP-induced response. In common with a role for PLA2 and the subsequent release of arachidonic acid (AA), we have demonstrated dose-dependent inhibition of PDE-induced superoxide release by the PLA2 inhibitor mepacrine, as well as activation and priming of the fMLP-induced superoxide generation by AA. CONCLUSIONS: These results imply that PDE-induced NADPH-oxidase activation and priming in human neutrophils is mediated via a PLA2-dependent but PLC- and PLD-independent mechanism.