Effect of mitochondria and microsomal fractions on glycolytic activity of rat brain and liver tissues under hypoxia

It is found that acute hypoxia inhibits the glycolytic activity of postmitochondrial fraction in the liver, activates in the brain, but has no effect on glycolysis under conditions of a preliminary administration of diethylaminoethylamide of parachlorophenoxyacetic acid--antihypoxic preparation. In the processes of two- and four-week interrupted training of adaptation to hypoxia the activity of the liver glycolytic system rises. Suspensions of the mitochondric and microsomal fraction added in definite ratios to the postmitochondrial fraction of the brain and liver intensify its glycolytic activity both in control and hypoxic animals. The activating effect of mitochondria is higher as compared with the control when glycolysis is decreased; when glycolysis is increased the phenomenon is not observed. A mechanism of the found changes in glycolysis and the validity of the tissue glycolysis estimation from the activity of the postmitochondrial fraction are discussed.     

Effect of altitude hypoxia on ATPase activity in the brain of rats of different ages.

In experiments on 5-day-old and adult rats of both sexes, the authors investigated Na+--K+-stimulated and Mg2+-dependent ATPase activity in the cerebral cortex, subcortical formations and the medulla oblongata. They studied the effect of 20 min acute altitude hypoxia corresponding to either 7,000 or 9,000 m, in a thermostable chamber (30 degrees C). ATPase activity was found to increase during ontogenesis -- this being the greatest in cortical tissue and the least in the medulla oblongata. Hypoxia corresponding to 7,000 m altitude significantly depressed total ATPase activity in 5-day-old rats, but significantly stimulated it in adult animals. Changes in Na+--K+-stimulated ATPase activity played the major role in these changes. Hypoxia corresponding to 9,000 m altitude likewise depressed total ATPase activity in 5-day-old rats and to practically the same extent as moderate hypoxia (7,000 m). In adult rats, marked hypoxia (9,000 m) significantly reduced only Mg2+-dependent ATPase activity. Mg2+ activity rose during ontogenesis to a lesser degree than Na+--K+-stimulated ATPase and the reciprocal ratio of these ATPase and the reciprocal ratio of these ATPase activities, in the given parts of the brain, fell progressively in adult animals to values close to 1.     

Effect of administration of 2-methyl-4-dimethylaminoazobenzene on the half-lives of rat liver mitochondria and cytochrome oxidase

The turnover of total mitochondrial proteins and cytochrome oxidase in the livers of rats administered with 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) has been determined. The incorporation of [14C]bicarbonate revealed a half-life of 3.1 days in control and 6 to 9 days in azodye administered animals for whole mitochondrial proteins. The incorporation of [35S]methionine yielded t1/2 values of 8.5 days and 15.4 days, respectively. The t1/2 of cytochrome oxidase, 10.8 days for control and 19.3 days for 2-Me-DAB-treated animals, indicated that the delay in the decay of the enzyme was of the same order as that of whole mitochondria. Short term incorporation revealed that the administration of the azodye stimulated the synthesis of the enzyme. Mitochondria isolated from azodye-administered animals appeared less susceptible to lysosomal proteolysis. Also, azodye administration seemed to impair the ability of lysosomes to degrade mitochondria.     

Influence of magnesium sulphate on evoked activity of rat brain after exposure to short-term hypoxia

Young Wistar rats (aged 12, 25 and 35 days) were exposed to short-term (60 min) hypobaric hypoxia of 41 kPa. Cortical afterdischarges (ADs) were evoked by repeated direct stimulation of the sensorimotor cortex and the duration of ADs was monitored to examine the influence of magnesium sulphate injection (0.3 g/kg b.w.). In 12-day-old hypoxia-exposed rats, an increase of the mean duration of ADs after the repeated stimulation appeared. This effect was prevented by magnesium administration. In 25- and 35-day-old rats exposed to hypoxia a shortening of ADs was registered but no specific effect of magnesium sulphate pretreatment was observed. The brain susceptibility and ability to terminate evoked seizures is discussed.     

Brain cytochrome oxidase activity of synaptic and nonsynaptic mitochondria during aging

The activity of cytochrome c oxidase was studied in aging brain on non-synaptic and intra-synaptic mitochondria from frontal cerebral cortex, hippocampus and striatum of 4, 8, 12, 16, 20 and 24 month-old Sprague-Dawley rats. Specific activities of cytochrome oxidase were significantly higher in light synaptic mitochondria than in non-synaptic or heavy ones at all the ages examined. However, enzyme activity in light mitochondria from cerebral cortex remains unchanged during aging, being increased in hippocampus and striatum. These results indicate that aging affected not only the various cerebral area (macroheterogeneity), but also the different mitochondrial populations (subcellular heterogeneity).     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Effect of prenatal hypobaric hypoxia on activity of the rat brain phosphoinositide system

Activity of the phosphoinositide system of intracellular signalization was studied in offspring of rats exposed to severe hypobaric hypoxia at the 14–16th (group 1) or the 18–20th day (group 2) of prenatal hypoxia. At the age of 15 days, in animals of both experimental groups the basal level of triphosphoinositides in the brain cortex was shown to be elevated as compared with control. In the group 1, this parameter also remains elevated in adult animals. Application of glutamate produces a more pronounced increase of the inositephosphates in brain slices of the 15-day old rats of the group 1 than in slices of animals of the control group. In the 15-day old rats of the group 2, as compared with control, the phosphoinositide response to glutamate application was reduced. No changes in the inositephosphate levels were revealed after application of glutamate upon slices of adult (the 90-day old) control animals and of adult rats of the group 2. In slices of adult rats of the group 1, on the contrary, the glutamate application produced an increase of the inositephosphate content. The obtained data indicate essential changes of the phosphoinositide metabolism in the brain of rats exposed to action of hypoxia at the period of prenatal development. The character and the severity of these changes depend on the period of development when action of hypoxia occurs.     

Purification and characterization of cytochrome c oxidase from rat liver mitochondria.

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.     

Biosynthesis of cytochrome c oxidase by isolated rat liver mitochondria.

When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo.     

The effects of hyperoxia, hypoxia, and ischemia/reperfusion on the activity of cytochrome oxidase from the rat retina

Cytochrome oxidase activity from the retina can be enhanced or depressed by free radical-mediated reactions both in positive and negative aspect. The greatest effect was exerted by ischemia/reperfusion, which significantly increased the fluorescent products of lipid peroxidation (358 %, P < 0.01) and inhibited the enzyme activity (14%, P < 0.001). After hyperoxia the fluorescent products slightly increased (192%, P < 0.05) as well as the enzyme activity (133 %, P < 0.05). Hypoxia had no effect on any of these parameters. Specific changes in the composition of fluorophores after ischemia/reperfusion were revealed in the fluorescence spectra. The fact that increased lipid peroxidation after hyperoxia and after ischemia/reperfusion does not produce the same effect upon cytochrome oxidase activity might be explained by changes in the kinetic behavior of cytochrome oxidase. In the control enzyme preparation, two binding sites for cytochrome c were observed. One was of the low-affinity (Km = 60 microM) and the other of the high-affinity (Km = 1.12 microM). After in vitro-initiated lipid peroxidation, the low-affinity binding site was lost and the activity measured under "optimum" conditions at a single cytochrome concentration was higher than in the controls. This implies that oxidative damage to cytochrome oxidase in vivo can be site-specific and its extent should be estimated by performing detailed kinetic analysis as otherwise the results might be misleading.     

Effect of benzamide derivatives on rat brain monoamine oxidase activity in vitro

The ability of moclobamide and other benzamide derivatives to inhibit the activity of monoamine oxidase in the rat brain was studied. Distinct effects of these compounds on the deamination of serotonin and norepinephrine (MAO-A substrates); 2-phenylethylamine (selective MAO-B substrate); tyramine and dopamine (MAO-A and MAO-B substrates) are shown. It was demonstrated that among all the compounds studied moclobamide appeared to be the most active and selective inhibitor of MAO-A: at a concentration of 100 microM it caused a 100% inhibition of serotonin and norepinephrine deamination, which might be explained by the presence of C1 atom in the para-position of benzene ring in moclobamide molecule. Other benzamide derivatives were less active in inhibiting MAO-A and had but a negligible effect on dopamine- and 2-phenylethylamine deamination.     

Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro

A single intraperitoneal injection of dimethyl sulfoxide (275 mg/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2-5-fold. The enzyme activity remained at this level for as long as 5 days post-injection. There was however only 10.5% increase in the content of cytochromes a and a3 (as determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2-3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase.     

Anoxia-reoxygenation-induced cytochrome c and cardiolipin release from rat brain mitochondria

Rat brain mitochondria were successively submitted to anoxia and reoxygenation. The main mitochondrial functions were assessed at different reoxygenation times. Although the respiratory control ratio decreased, the activity for each one of the enzymes participating in the respiratory chain was not affected. However, during reoxygenation, mitochondrial membrane lipoperoxidation quickly increased and was proportional to the decrease seen in membrane fluidity. Under the same conditions, cytochrome c and cardiolipin were released from mitochondria and their rate of release increased with reoxygenation time. The release of cytochrome c and cardiolipin was followed by the collapse of the membrane potential and it was not inhibited by cyclosporin A. Addition of the antioxidant alpha-tocopherol abolished all these reoxygenation-induced changes. These data indicate that, in this model, reoxygenation promotes the uncoupling of respiratory chain, and cytochrome c and cardiolipin releases. These events are not related to the membrane potential collapse but to an oxidative stress.     

Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H₂O₂ release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H₂O₂ release. H₂O₂ was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H₂O₂ production. H₂O₂ release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with -glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H₂O₂ generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H₂O₂ release in insect mitochondria supplemented with -glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H₂O₂ production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.     

Effect of neurocatin on the activity of monoamine oxidase B in rat brain synaptosomes

Neurocatin, a small (about 2,000 Dalton) neuroregulator isolated from mammalian brain, is a powerful effector of monoamine oxidase B in rat brain synaptosomes. Incubation of intact synaptosomes with neurocatin caused an inhibition of the enzyme dependent on the concentration of neurocatin. This inhibition became statistically significant at a neurocatin concentration of 10 ng/200 l and was significant at all higher neurocatin concentrations. At 40 ng/200 l, neurocatin inhibited monoamine oxidase B activity by about 60%. This inhibitory effect was almost completely abolished by breaking the synaptosomal membrane by hypotonic buffer prior to incubation with neurocatin. In addition, incubation of the synaptosomes in calcium free medium almost completely abolished the inhibitory effect of neurocatin on monoamine oxidase B. The inhibition appeared to involve covalent modification of the enzyme mediated by a neurocatin receptor(s). Measurements of the kinetic parameters of the enzyme showed that 20 ng of neurocatin caused a statistically significant decrease in V_max (by 20%) with no significant change in K_M, compared to controls. Inhibition of monoamine oxidase by neurocatin is potentially of great clinical importance because this enzyme plays a major role in catabolism of the biogenic amines and alterations in its activity is believed to contribute to several neurological disorders.