Lipoperoxidation in hepatic subcellular compartments of diabetic rats

It is known that an accumulation of lipoperoxidative aldehydes malondialdehyde (MDA) and 4-hydroxynonenal (HNE) takes place in liver mitochondria during aging. The existence and role of an increased extra- and intra-cellular oxidative stress in diabetes, an aging-accelerating disease, is currently under discussion. This report offers evidence that lipoperoxidative aldehydes accumulate in liver microsomes and mitochondria at a higher rate in spontaneously diabetic BB/WOR rats than in control non-diabetic animals (HNE content, diabetes vs. control: microsomes 80.6+/-19.9 vs. 25.75+/-3.6 pmol/mg prot, p = .024; mitochondria 77.4+/-15.4 vs. 26.5+/-3.5 pmol/mg prot, p = .0103). Liver subcellular fractions from diabetic rats, when exposed to the peroxidative stimulus ADP/Fe, developed more lipoperoxidative aldehydes than those from non diabetic rats (HNE amount, diabetes vs. control: microsomes 3.60+/-0.37 vs. 2.33+/-0.22 nmol/mg prot, p = .014; mitochondria 3.62+/-0.26 vs. 2.30+/-0.17 nmol/mg prot, p = .0009). Liver subcellular fractions of diabetic rats developed more fluorescent chromolipids related to HNE-phospholipid adducts, either after in vitro peroxidation (microsomes: p = .0045; mitochondria: p = .0023) or by exposure to exogenous HNE (microsomes: p = .049; mitochondria: p = .0338). This higher susceptibility of diabetic liver membranes to the non-enzymatic attack of HNE may be due to an altered phospholipid composition. Moreover, a decreased activity of the HNE-metabolizing systems can be involved: diabetic liver mitochondria and microsomes were unable to consume exogenous HNE at the same rate as non-diabetic membranes; the difference was already significant after 5' incubation (microsomes p<.001; mitochondria p<.001). These data show an increased oxidative stress inside the hepatocytes of diabetic rats; the impairment of the HNE-metabolizing systems can play a key role in the maintenance and propagation of the damage.     

Factors influencing the establishment of the normal values of the respiratory activities of human liver mitochondria.

Some factors that influence the values of respiratory activities of liver mitochondria isolated from surgical biopsy specimens have been studied. By sedimentating of mitochondria at a lower centrifugal force (5,500 g) than usually used for rat liver mitochondria, and washing the mitochondrial pellet twice, the contamination with lysosomes and microsomes was lowered. At 37 degrees C, and in the presence of hexokinase and glucose, the oxygen uptake was greater than at 25 degrees C and in their absence. The respiratory control was good and the respiratory activities were rather stable during the first 3-4 h after isolation. The respiratory activities of mitochondria isolated from patients with duodenal or gastric ulcers, biliary diseases, and subjects with no digestive diseases (all having normal liver) were compared. Differences in oxygen uptake and acceptor control index values with some substrates were noted. The conditions for selection of controls in studies on subcellular fractions of human liver include: absence of any hepatic antecedents; no clinical evidence of liver involvement; no abnormality in routine liver function tests; a histologic aspect free of pathological conditions, and a normal aspect of the tissue during the homogenization and the fractionation procedure (absence of steatosis or fibrosis). These data provide a basis for the standardization of methods in establishing the reference values of mitochondrial activities for the modifications in a variety of diseases.     

Deficiency of the complex I of the mitochondrial respiratory chain but improved adenylate control over succinate-dependent respiration are human gastric cancer-specific phenomena

The purpose of study was to comparatively characterize the oxidative phosphorylation (OXPHOS) and function of respiratory chain in mitochondria in human gastric corpus mucosa undergoing transition from normal to cancer states and in human gastric cancer cell lines, MKN28 and MKN45. The tissue samples taken by endobiopsy and the cells were permeabilized by saponin treatment to assess mitochondrial function in situ by high-resolution oxygraphy. Compared to the control group of endobiopsy samples, the maximal capacity of OXPHOS in the cancer group was almost twice lower. The respiratory chain complex I-dependent respiration, normalized to complex II-dependent respiration, was reduced that suggests deficiency of complex I, but the respiratory control by ADP in the presence of succinate was increased. Similar changes were observed also in mucosa adjacent to cancer tissue. The respiratory capacity of MKN45 cells was higher than that of MKN28 cells, but both types of cells exhibited a deficiency of complex I of the respiratory chain which appears to be an intrinsic property of the cancer cells. In conclusion, human gastric cancer is associated with decreased respiratory capacity, deficiency of the respiratory complex I of mitochondria, and improved coupling of succinate oxidation to phosphorylation in tumor tissue and adjacent atrophic mucosa. Detection of these changes in endobiopsy samples may be of diagnostic value.     

Heterogeneity of Maize Root Mitochondria from Plants Grown in the Presence of Ammonium

Mitochondria isolated from root tissue of maize plants grown on a modified Knop solution containing 10.9 mM nitrate ± 7.2 mM ammonium were purified on the discontinuous Percoll density gradient with polyvinylpyrrolidone (PVP) added. The presence of PVP allowed separation of several mitochondrial fractions of a different density. Contrary to mitochondria isolated from plants grown in the presence of nitrate alone, revealing only two fractions, the mitochondria from NH₄ ⁺/NO₃ ^–-plants were distributed in four fractions. Total amount of mitochondria, as well as specific activities of some nitrogen metabolism enzymes and tricarboxylic acid (TCA) cycle enzymes of all mitochondrial fractions, and respiratory activities of two lower density fractions isolated from plants grown on mixed nitrogen were higher in comparison to mitochondria from nitrate-grown plants.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.     

A combined experimental and quantum chemical study on the putative protonophoric activity of thiocyanate

Inhibition of gastric acid secretion by thiocyanate is explained by a protonophoric mechanism assuming that thiocyanate induces a H(+) back flux from the acidic gastric lumen into the parietal cells of gastric mucosa. Protonophoric activity of thiocyanate was examined by swelling measurements using rat liver mitochondria and theoretically by quantum chemical methods. Mitochondria suspended in K-thiocyanate medium plus nigericin (an H/K-exchanger) swelled when the medium pH was acidic, indicating that SCN(-) initiates a transfer of H(+) across the inner membrane. To rationalize the protonophoric activity of thiocyanate, we considered the dehydration of SCN(-) to be critical for transmembranal H(+) transfer. For modeling this process, various hydrate clusters of SCN(-) and Cl(-) were generated and optimized by density functional theory (DFT) at the B3-LYP/6-311++G(d,p) level. The cluster hydration energy was lower for SCN(-) than for Cl(-). The total Gibbs free energies of hydration of the ions were estimated by a hybrid supermolecule-continuum approach based on DFT. The calculated hydration energies also led to the conclusion that SCN(-) is less efficiently solvated than Cl(-). Due to the easier removal of the hydration shell of SCN(-) relative to Cl(-), SCN(-) is favored in going across the membrane, giving rise to the protonophoric activity.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Low concentrations of zinc in gastric mucosa are associated with increased severity of Helicobacter pylori-induced inflammation

BACKGROUND: Chronic Helicobacter pylori infection is the most common cause of gastric cancer. H. pylori induces oxidative stress while zinc deficiency results in increased sensitivity to it. In Ecuador, the prevalence of gastric cancer and zinc deficiency are high. We hypothesized that zinc deficiency in Ecuadorian people would cause increased H. pylori-induced inflammation in the gastric mucosa associated with lower tissue zinc concentrations. METHODS: Three hundred and fifty-two patients with dyspepsia underwent endoscopy to obtain gastric mucosa biopsies. Diagnosis of H. pylori infection and its severity, histopathology, mucosal zinc concentration, and inflammation intensity were determined. RESULTS: H. pylori-infected patients with non-atrophic chronic gastritis had lower concentrations of zinc in gastric mucosa than uninfected patients with the same type of gastritis (251.3 +/- 225.3 vs. 426.2 +/- 279.9 ng/mg of protein; p = .016). Considering all patients, the more severe the H. pylori infection, the higher the percentage of subjects with infiltration by polymorphonuclear (PMN) cells (p = .0001). Patients with high PMN infiltration had lower mucosal zinc concentrations than patients with low PMN infiltration (35.2 +/- 20.7 vs. 242.9 +/- 191.8 ng/mg of protein; p = .021). CONCLUSIONS: The degree of inflammation in H. pylori-induced gastritis appears to be modulated by gastric tissue zinc concentrations.     

Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata.

The midpoint potentials of the mitochondrial respiratory chain cytochromes of the protozoan Crithidia fasciculata at pH 7.2, Em7.2, show great similarity to those measured in higher organisms. Values of Em7.2 for cytochromes a and a3 are +165 and +340 mV. Both c cytochromes have Em7.2 = +230 mV. There are two b cytochromes with the same spectral characteristics with Em7.2 = -20 and -135 mV. These values are compatible with two sites of energy conservation for oxidative phosphorylation in these mitochondria. All cytochrome components show potentiometric titrations with n = 1. There is a fluorescent flavoprotein in these mitochondria with Em7.2 = -40 mV and n =2, whose function is not known.     

Histamine interaction on surface recognition sites of H2-type in parietal and non-parietal cells isolated from the guinea pig stomach

In gastric cells isolated by pronase digestion from the guinea pig, histamine stimulated cAMP production in 3 fundic cell fractions (EC50 = 1.6--2 x 10(-4) M) enriched in parietal (94%), peptic (63%) and mucous cells (87%) as well as in antral cells (EC50 = 4 x 10(-4) M) that are devoid of parietal cells. Histamine stimulations were completely inhibited by the H2 antagonist cimetidine (Ki = 0.27--0.57 x 10(-6) M) or by the H1 antagonist diphenhydramine, but at 100-times lower potency (Ki = 22--45.7 x 10(-6) M), indicating the presence of histamine H2 receptors in parietal and nonparietal cells of the guinea pig gastric mucosa.     

In vitro incorporation of ( 3 H) methyl choline into phosphatidyl choline of rat liver subcellular fractions

Incubation of a rat liver total homogenate with radioactive choline and subsequent isolation of subcellular fractions, at different times, showed similar patterns of labeling. Incubation of microsomes, mitochondria and purified nuclei isolated from rat liver, showed that all fractions were able to incorporate the precursor into phosphatidyl choline. The specific activity was higher in mitochondria and increased in all cases with added supernatant. The addition of microsomes to mitochondria diminished the incorporation of label. Contamination of mitochondria by microsomes, was negligible as shown by undetectable amounts of cytochrome P₄₅₀, while NADPH₂ cytochrome c reductase showed a 10% contamination. A certain amount of radioactivity was incorporated in the absence of ATP and oxidizable substrates due to the presence of substrates and cofactors in the fraction and/or the supernatant. Labeled fractions reincubated with unlabeled choline, showed no loss of radioactivity, proving that incorporation was not due to simple exchange processes. It is concluded that although rat liver mitochondria can acquire part of their own provision of phosphatidyl choline by transference from microsomes, all organelles and specially mitochondria, can independently synthesize this phospholipid.     

ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

Increase in hepatic mitochondria on administration of ethyl α-p-chlorophenoxyisobutyrate to the rat

1. The antihypercholesterolaemic drug ethyl alpha-p-chlorophenoxyisobutyrate when fed to the rat orally or mixed with the diet increased the content of mitochondria in the liver by 50-100%. Other subcellular fractions did not show any significant change. 2. In oxidative activity, respiratory control and phosphorylating ability no significant difference was observed between the mitochondria isolated from the livers of the drug-treated rats and those from normal animals. 3. In agreement with earlier reports, administration of the drug depressed the concentration of serum cholesterol and increased liver weight and the liver content of ubiquinone. However, the increase of ubiquinone was greater in the nuclear than in the mitochondrial protein.     

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.     

Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings

Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-α-glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an α-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the α-glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial α-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the α-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked α-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose.     

tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria.

When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration.     

Kinetic alterations of cytochrome c oxidase in carbon tetrachloride induced cirrhotic rat liver

In a study of the chronic effects of CCl4 on the respiratory activities of rat liver mitochondria, the content of cytochrome c oxidase increased from 0.077 +/- 0.010 (nmol/mg protein) for normal rats to 0.101 +/- 0.009, and its specific activity increased from Vmax = 345 +/- 24 (e-/s/cytochrome aa3) to 431 +/- 19 in mitochondria of CCl4 treated rats. There was a slight increase in Km for cytochrome c from 5.63 +/- 0.08 microM to 7.79 +/- 0.80. These results would strongly suggest that an appreciable decrease in the steady state concentration of ATP in hepatic cells of CCl4 treated rats brought about a compensatory increase in the overall activity of cytochrome c oxidase. However, when the rate of oxygen uptake by mitochondria was measured in the presence of rotenone and tetramethyl-p-phenylene-diamine with NADH as substrate, the specific activity in CCl4 treated rats was lower than that of normal rats (Vmax = 345 +/- 31 (e-/s/cytochrome aa3), as compared to Vmax = 408 +/- 21) in spite of the increased activity of cytochrome c oxidase. This phenomenon was ascribed to a decrease in the activity of NADH cytochrome b5 reductase in the mitochondrial outer membrane due to CCl4 treatment.