Milk-derived bioactive peptides protect against oxidative stress in a Caco-2 cell model.

Milk and milk-derived products are a relevant source of bioactive peptides, which are also potential components of functional foods. Bioactive peptides exert multiple actions including an antioxidant role. In the present paper, four synthetic peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), corresponding to milk-derived peptides were studied. Although with different features, as revealed by RP-HPLC chromatography and MS analysis, the obtained peptides were shown to be taken up by Caco-2 cells arranged in an epithelial monolayer formation. The four peptides were all able to preserve cell viability against induced oxidative stress indicating that they might have a role in the control of oxidative stress. Therefore, an estimation of total thiols and glutathione content was performed after cell treatment with oxidants like hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (TbOOH). The peptides were able to prevent the decrease of both total thiols and glutathione induced by H2O2 or TbOOH, and, in addition, they showed a protective effect on the thiol-related antioxidant enzymes thioredoxin reductase and glutathione reductase. Finally, they caused a decrease of ROS production induced by TbOOH in Caco-2 cells. The reported results highlight the relevant antioxidant role played by bioactive peptides in cells, which adds to other previously known properties.

Effect of metal complexes on thioredoxin reductase and the regulation of mitochondrial permeability conditions.

Gold(I) compounds such as auranofin, chloro(triethylphosphine) gold(I), and aurothiomalate act on mitochondrial functional parameters by determining an extensive permeability transition and a decrease of membrane potential. On the contrary, pyridine nucleotides and glutathione are not modified, whereas a slight but significant decrease of total thiols is apparent. The effect of gold(I) compounds is essentially referable to the inhibition, in the nanomolar range, of thioredoxin reductase activity and to an increase of hydrogen peroxide production. Metal ions and metal complexes (zinc and cadmium acetate, cisplatin, tributyltin) are also good inhibitors of thioredoxin reductase, although in the micromolar range, and in addition, they act as inducers of permeability transition and of membrane potential decrease. At variance with gold(I) compounds, which appear to work almost exclusively on thioredoxin reductase, metal ions and complexes are less specific, since they are active on different mitochondrial targets, including the respiratory chain.

Distribution of protein disulphide isomerase in rat liver mitochondria.

Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol-disulphide transition.

Combined effect of propofol and GSNO on oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.

Oxidation of adrenaline and its derivatives by S-nitrosoglutathione.

An oxidizing effect of S-nitrosoglutathione toward adrenaline and its cyclic derivatives (adrenochrome and adrenolutin) is reported. The oxidation was monitored either spectrophotometrically or as oxygen uptake. Adrenaline was first oxidized to adrenochrome that, after isomerization to adrenolutin, was further oxidized to products monitored as fluorescence decrease. To occur to a significant extent, this oxidation requires copper ions that, in addition to a direct effect on the oxidation of the ortho-diphenol moiety, are also able to decompose nitrosothiols, giving rise to nitric oxide. The latter, after interaction with oxygen and superoxide, produces nitrogen oxides and peroxynitrite, respectively, that are important contributors to the oxidative process. In this context, catecholamines might act as regulatory factors toward nitric oxide and its derivatives.

The mitochondrial antioxidant defence system and its response to oxidative stress.

The antioxidant systems of mitochondria are not well known. Using a proteomics-based approach, we defined these mitochondrial antioxidant systems and analyzed their response to oxidative stress. It appears that the major mitochondrial antioxidant system is made of manganese superoxide dismutase on the one hand, and of peroxiredoxin III, mitochondrial thioredoxin and mitochondrial thioredoxin reductase on the other hand. With the exception of thioredoxin reductase, all these proteins are induced by oxidative stress. In addition, a change in the peroxiredoxin III pattern can also be observed.

Isolation, purification, and characterization of a rat liver mitochondrial protein disulfide isomerase.

The isolation and purification to electrophoretical homogeneity and characterization of a protein disulfide isomerase from rat liver mitochondria is reported. The purified enzyme exhibits a single band on sodium dodecylsulfatepolyacrylamide gel electrophoresis with an apparent molecular weight of approximately 54 kDa. Comparatively, the microsomal form shows an apparent molecular weight of 57 kDa indicating that the two forms are slightly different. The antibody raised against the microsomal isoform does not recognize the mitochondrial enzyme. To characterize the enzyme, different classical methodologies utilized for protein disulfide isomerase estimation have been adopted. The isolated enzyme is active with all of them, indicating that it comprises all the features of a typical protein disulfide isomerase. At the mitochondrial level the enzyme appears mostly localized at the membrane level. Its potential involvement in mitochondrial membrane permeability control is also discussed.

Virtual instrumentation for pH measurements in biological systems.

In the present communication a personal computer control methodology for pH data acquisition and analysis in biological systems is reported. The instrumental control, acquisition, storage, processing and presentation of the experimental data are provided by a data acquisition board, a graphical programming software and numerical analysis/graphics software. The major objective of this work is to improve the performance and flexibility of the personal computer acquisition system compared with traditional approaches depending on potentiometric recorders. In particular, virtual instruments for interfacing pH meters of different brands to a personal computer and for measuring proton changes in lightly buffered solutions during enzymatic reactions are provided.

Mitochondrial permeability transition and release of cytochrome c induced by retinoic acids.

Retinoic acids, structurally related to vitamin A, inhibit the in vitro proliferation of different types of normal and neoplastic cells. The effects of all-trans, 9-cis, and 13-cis retinoic acids were tested on mitochondria isolated from rat liver. All the compounds were able to induce the membrane permeability transition observed as swelling and decrease in membrane potential, but 13-cis retinoic acid appeared to be the most effective. The latter was also shown to stimulate the release of cytochrome c from mitochondria, suggesting a potential target of retinoids in the induction of cell apoptosis. Interestingly, EGTA and cyclosporin A, which strongly inhibit the permeability transition induced by 13-cis retinoic acid, were without effect on the release of cytochrome c from the mitochondrial intermembrane space.

Effect of 2,6-diisopropylphenol and halogenated anesthetics on tetraphenylphosphonium uptake by rat brain synaptosomes: determination of membrane potential.

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP+) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202+/-8 mV (mean +/- SD) while the synaptosomal membrane showed a potential of 78+/-8 mV (mean +/- SD). When anesthetic concentrations (0.6-1 mM propofol, 10-40 microM enflurane, 30-50 microM isoflurane, 8-15 microM halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP+ taken up was observed as the anesthetic concentration was increased.

The role of adrenochrome in stimulating the oxidation of catecholamines.

Adrenochrome, a stable oxidation product formed after oxidation of adrenaline, strongly stimulates oxygen uptake occurring during the autoxidation of adrenaline, other catecholamines and ascorbate. Oxygen consumed is converted to hydrogen peroxide suggesting the occurrence of a redox cycling process. The reduction of adrenochrome operated by adrenaline is accelerated by the exclusion of oxygen indicating that the oxidation of adrenaline occurs directly and superoxide anion does not necessarily mediate it. Oxygen consumption, observed in the catecholamine/adrenochrome and ascorbate/adrenochrome systems, is due to the autoxidation of leucoadrenochrome that, at variance with adrenaline, easily autoxidizes also at physiological pH. Therefore, in these systems, leucoadrenochrome appears to be the major determinant of the production of superoxide anion.

Antioxidant properties of clozapine and related neuroleptics.

The antioxidant properties of clozapine and other related molecules were evaluated with the crocin bleaching test both in aqueous and non-aqueous environment. The tests of microsomal lipid peroxidation and carbonyl formation were also used. In aqueous solution, chlorpromazine and trifluoperazine appear particularly effective in the bleaching of crocin, while serotonin has an efficacy intermediate between those of phenothiazines and clozapine. The latter drug, on the other hand, in a non-aqueous medium shows an antioxidant power comparable to that of butylated hydroxytoluene, indicating that its antioxidant properties are better expressed in a hydrophobic environment of the type present in a biological membrane. In fact, in lipid peroxidation induced in microsomal membranes, clozapine, chlorpromazine, trifluoperazine and serotonin act as very good antioxidants; at low concentrations, clozapine appears to be the most efficient after butylated hydroxytoluene. Similarly, all these compounds markedly inhibit protein carbonyl formation, clozapine being one of the most efficient. Thus, under different in vitro experimental conditions, the neuroleptic drugs chlorpromazine and trifluoperazine and the antipsychotic substance clozapine act as very effective antioxidants; this property might, at least in part, be responsible for the physiological and clinical effects observed in vivo.

Purification of mitochondrial thioredoxin reductase and its involvement in the redox regulation of membrane permeability.

The isolation to purity of a rat liver mitochondrial thioredoxin reductase is reported. The mitochondrial enzyme shows a chromatographic behavior different from that of the cytosolic enzyme. The purified enzyme, after sodium dodecylsulfate-polyacrylamide gel electrophoresis, yields a single band with a molecular weight of approximately 54 kDa. The apparent Km for E. coli thioredoxin is about 13 microM, while the apparent Km for 5,5'-dithiobis (2-nitrobenzoic acid) is 530 microM, values comparable to those reported for the cytosolic enzyme. Mitochondrial thioredoxin reductase, in addition to its natural substrate thioredoxin, is also able to reduce chemically unrelated compounds such as 5,5 '-dithiobis (2-nitrobenzoic acid), selenite, and alloxan; the enzyme is inhibited by classical inhibitors of the cytosolic enzyme such as 1-chloro-2,4-dinitrobenzene and 13-cis-retinoic acid. A strong inhibitory action is also elicited by Mn2+ and Zn2+ ions. Thiol status appears critically involved in the control of membrane permeability and, therefore, a thiol/disulfide transition involving reduced pyridine nucleotides, matrix soluble thiols, and inner membrane thiols appears to play a fundamental role. The potential role of thioredoxin/thioredoxin reductase system in the control and redox regulation of the mitochondrial membrane permeability, is discussed.

Mitochondrial effects of L-ropivacaine, a new local anesthetic.

The effects of the local anesthetics ropivacaine and bupivacaine were investigated on isolated rat liver mitochondria. The efficiency of oxidative phosphorylation was evaluated by measuring the rates of respiration and ATP synthesis and the magnitude of the transmembrane electrical potential (deltapsi). Bupivacaine did not alter the ADP-stimulated respiration but strongly affected the resting respiration, which was more than doubled at 0.6 mM. In addition, it decreased the transmembrane electrical potential, and the ATP synthesis rate (deltapsi was less than 100 mV at 0.6 mM). Ropivacaine did not alter the ADP-stimulated respiration, and the resting respiration seemed to be substantially unaffected up to 1.2 mM; a slight increase was observed at 1.8 and 2.4 mM. The transmembrane potential was decreased by anesthetic concentrations higher than 1.2 mM and ATP synthesis was consequently affected. The findings suggest that ropivacaine is less toxic than bupivacaine, in rat liver mitochondria.

Influence of the redox state of pyridine nucleotides on mitochondrial sulfhydryl groups and permeability transition.

This work addresses a correlation between the redox state of pyridine nucleotides and that of sulfhydryl groups of the mitochondrial membranes. Several major observations emerge: (1) Conditions leading to an oxidation of the pyridine nucleotides such as incubation with tert-butyl hydroperoxide or acetoacetate determine a decrease of total mitochondrial sulfhydryl groups. Glutathione does not follow the same pattern since it decreases in the presence of tert-butyl hydroperoxide but not in the presence of acetoacetate. In addition, only in the presence of tert-butyl hydroperoxide is the decrease of sulfhydryl groups concomitant with a membrane protein polymerization, observed by polyacrylamide gel electrophoresis. (2) Under all conditions tested, the oxidation of sulfhydryl groups is further stimulated by the presence of calcium and phosphate ions. (3) Respiratory substrates, which prevent the swelling of mitochondria, also partially prevent the decrease of sulfhydryl groups.

Effect of the intravenous anesthetic 2,6-diisopropylphenol on respiration and energy production by rat brain synaptosomes.

The sensitivity of the mitochondrial energy production system to propofol (DPP) has been investigated in rat brain synaptosomes. DPP at 0.8 mM concentration produced a partial inhibition of coupled respiration, an apparent decrease of the oxygen uptake stimulation induced by CCCP and a full inhibition of the mitochondrial ATP production by synaptosomes. Higher concentrations of DPP (1 mM) fully abolish uncoupler-dependent stimulation and at 1.3 mM DPP also coupled respiration is completely blocked. Similar results were obtained when dinitrophenol replaced CCCP and phenol or propylbenzene replaced DPP. The presence of the alkyl residues seems critical for the DPP effect. In the presence of 30 mM glutamate both respiration and ATP production are enhanced but DPP effects are similar to those obtained in the absence of glutamate.

Personal computer control of electrochemical detectors utilized for mitochondrial studies.

In the present communication a personal computer control of electrodes particularly suited for mitochondrial research such as the oxygen electrode, the pH electrode and ion-selective electrodes is described. A personal computer equipped with a data acquisition board, a color monitor, a graphical programming software and a numerical analysis/graphics software provides complete instrumental control, data storage, processing and presentation of experimental data. The major objective of this work is the analysis and utilization of a virtual instrumentation software for data acquisition and control of electrochemical detectors; this may greatly improve the performance and flexibility of the system compared to traditional approaches such as the potentiometric recorders.

Effect of polycation peptides on mitochondrial permeability transition.

Synthetic polycation peptides obtained with the basic aminoacids lysine, arginine and ornithine are able to inhibit the permeability transition induced in mitochondria by calcium ions and inorganic phosphate. At least three basic aminoacid residues must be present in the peptide in order to elicit the inhibitory effect. In the presence of synthetic polycations and similarly to spermine, a lack of correlation between inhibition of swelling and glutathione release is apparent, since glutathione release occurs before the onset of a large amplitude swelling. The same lack of correlation is observed in the presence of cyclosporin. From the results obtained with the above reported polycations, different in both aminoacid composition and length, it appears that the effect is not to be referred to the individual properties of the molecules examined but rather to their cationic character; in addition, a critical number of positive charges is necessary to elicit the effect.

Inhibitory effect of pyruvate on release of glutathione and swelling of rat heart mitochondria.

Pyruvate prevents the permeability transition of rat heart mitochondria induced by the system calcium ions + phosphate or by the dithiol reagent phenylarsenoxide and measured as swelling. Since swelling induced by the latter is relieved by the dithiol 2,3-dimercaptopropanol (BAL), it is inferred that the effect of pyruvate might be mediated by the reduction of lipoic acid. In isolated mitochondria, pyruvate also exerts a protective effect when calcium + phosphate-induced swelling is exacerbated by hypoxic conditions. These results agree with our previous observations that pyruvate markedly prevents the loss of cytosolic and mitochondrial glutathione after ischemia or ischemia followed by reperfusion.