Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.     

Coenzymes Q9 and Q10, vitamin E and peroxidation in rat synaptic and non-synaptic occipital cerebral cortex mitochondria during ageing

Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q9 (CoQ9) and Coenzyme Q10 (CoQ10) contents in rat brain mitochondria. The trends found for CoQ9 and CoQ10 levels in synaptic and non-synaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in non-synaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.     

Effect of in vivo treatment of clonidine on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex

The effects on energy-consuming ATP-ases were studied in two types of synaptic plasma membranes from rat cerebral cortex after in vivo injection of clonidine. To study the mechanism of action of clonidine at subcellular level, the enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase, Low- and High-affinity Ca²⁺-ATP-ase, and Mg²⁺-ATP-ase were evaluated on synaptic plasma membranes of control and treated animals with clonidine (5 g · kg^–1; i.p. 30 minutes). Acute treatment with clonidine decreased the catalytic activity of Ca²⁺, Mg²⁺-ATP-ase and of low-affinity Ca²⁺-ATP-ase only in synaptic plasma membranes of II type, that is the fraction enriched in synaptic plasma membranes. The decreases of these enzymatic activities are related to the interference of the drug on Ca²⁺ homeostasis in synaptoplasm. The reductions of these enzyme-consuming ATP-ases give further evidence that clonidine has not only neuroreceptorial effects, but that the drug also affects the energy metabolism of cerebral tissue, improving the knowledges about the pharmacology of clonidine. Because the elevation of [Ca²⁺]_i, during ischemia/hypoxia contributes to cellular injury, these findings may suggest that the prevention of calcium overload may be the key mechanism of protection by ₂-agonist.     

Effect of tetanus toxin and colchicine on synaptic membranes of rat cerebral cortex]

It was shown that purified tetanus toxin did not influence the activity of the Na, K-ATP-ase fractions of the synaptic membranes of the rat cerebral cortex, it had no effect on the inhibition of Na, K,-ATP-ase under electrical stimulation of the synaptic membrane suspension, or the GABA--3H binding by the synaptosomes in vitro. Tetanus toxin (400--4000 DLM) and colchicine (1 mM) induced a decrease of osmotic sensitivity of the nerve endings. Colchicine in low concentrations (10(-5)-10(-3) M) failed to influence Mg-and Na, K-ATP-ases, but considerably inhibited both ATP-ases at higher concentrations.     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

Action ofl-acetylcarnitine on age-dependent modifications of mitochondrial membrane proteins from rat cerebellum

Protein patterns of mitochondrial outer membrane, inner membrane, and matrix from nonsynaptic (free) mitochondria from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months) were analyzed by gel electrophoresis. Acutel-acetylcarnitine treatment was per-formed by a single i.p. injection (100 mg/kg body weight) of the substance 60 min before the sacrifice of the animals. Different age-dependent changes were obtained for the proteins of the three fractions. The amount of some protein subunits increased and/or decreased after drug treatment. In particular, protein composition of the inner mitochondrial membrane showed significant age-related modifications. This result probably indicates differences in protein synthesis and/or turnover rates in the various mitochondrial compartments during aging. Acutel-acetylcarnitine treatment caused: a high increase in the amount of one inner membrane protein with Mw 16 kDa, at all the ages studied; a decrease in the amount of many other inner membrane proteins; modifications of some matrix proteins. Our results show that in vivo administration ofl-acetylcarnitine affects mainly the inner membrane protein composition of cerebellar mitochondria.     

Characterization of purified rat testicular transglutaminase and age-dependent changes of the enzyme activities

The Ca2+-dependent tissue transglutaminase is widely distributed in various tissues and has been reported to participate in many cellular growth and differentiation processes. In the past decade, tissue transglutaminase is also identified as a G protein, G(alphah), for intercellular signaling. To further characterize testicular transglutaminase, the rat testicular transglutaminase was purified by ammonium sulfate precipitation, DEAE ion-exchange, heparin-agarose, and GTP-agarose affinity chromatographies. This purification protocol resulted in a 8400-fold enrichment of the enzyme with a reproducible 15% yield. The purified enzyme showed as a single band of 78kDa on SDS-polyacrylamide gel. Western blot analysis using anti-liver tissue transglutaminase monoclonal antibody also recognized the enzyme, indicating it is a t-TGase in nature. The Km values of purified testicular transglutaminase for putrescine and N,N-dimethylcasein were determined to be 35 and 17 microM, respectively. Its transglutaminase cross-linking activity was strongly inhibited by EGTA, GTP, polyamines, and cystamine, as well as moderately by ATP and NaCl. The enzyme exhibited a magnesium-dependent GTP-hydrolyzing capacity, but its GTP-binding activity did not require magnesium. Furthermore, the enzyme activity was found to be closely related with the first wave of spermatogenesis. Thus, testicular transglutaminase is speculated to participate in the event of spermatogenesis. In conclusion, the purified testicular transglutaminase displays property of either the tissue-type transglutaminase, or the GTP-binding and hydrolyzing characteristics. The activity of testicular transglutaminase is age-dependent, greatly stimulated during the first wave of spermatogenesis.     

Heterogeneous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex

Rat brain cerebral cortex derived synaptic vesicles sedimenting on a 0.4 M sucrose solution were further fractionated according to size by column chromatography on Sephacryl-1000 and analyzed for their binding activities of antibodies directed against the vesicle-associated proteins synaptophysin, synapsin I, protein 65 and clathrin. Whereas synapsin I and particularly protein 65 and clathrin are associated with a large range of vesicle sizes, synaptophysin elutes with small vesicles only. Using monoclonal antibodies against either synaptophysin or protein 65 and polyacrylamide beads for solid matrix immunoprecipitation, significant differences could be revealed in the protein composition of the resulting vesicle populations. Whereas synapsin I is associated with both synaptophysin and protein 65 immunoprecipitated vesicle populations, synaptophysin appears to be only a minor constituent of vesicles precipitated with anti-protein 65. Vesicles precipitated with anti-synaptophysin antibodies are enriched in acetylcholine. Our results suggest that the vesicle membrane protein synaptophysin and protein 65 may not have a ubiquitous distribution among synaptic vesicles. Protein 65 containing large vesicle populations contain little synaptophysin and synaptophysin is mainly associated with synaptic vesicles of small diameter.     

Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat

Summary Glycogen synthetase and phosphorylase activities in the paraboloid glycogen of the accessory cone of the chick retina were studied electron histochemically, while the paraboloid glycogen was observed by electron microscopy.Some of the paraboloid of the accessory cone of the chick retina contained abundant glycogen granules, but some showed no glycogen granules. Some inner segments of the accessory cones were occupied by deposition of glycogen granules.Polyglucose particles synthesized by glycogen synthetase activity in the chick paraboloid were demonstrated in fine granular form with diameter from 70 to 130 Å. These particles were less stainable with lead citrate than native glycogen granules. Synthesized polyglucose particles were located in the cytoplasmic matrices and expanded them. Lamellar and membrane structures were not related to synthesized polyglucose.Polyglucose particles synthesized by phosphorylase activity in the chick paraboloid were located in the cytoplasmic matrices and expanded them widely. Tubular structure appeared to be flattened by deposition of synthesized polyglucose particles. These features showed the resemblance to the inner segment of the accessory cone filled with a great amount of glycogen granules. Synthesized polyglucose was demonstrated in macromolecular form with diameter from 200 to 500 Å. There were no relationships between lamellar or membrane structures and polyglucose.The present study suggests that the chick paraboloid not only is a field for active glycogen metabolism, but also becomes a storage of glycogen.     

Postnatal changes in dolichol-pathway enzyme activities in cerebral cortex neurons.

Neuronal perikarya were isolated from rat cerebral cortex at different stages of postnatal development. Membranes sedimenting at 100000 g were obtained from these neurons to study several glycosyltransferases of the dolichol pathway. Enzyme activities from stages before and during synapse formation were compared (days 5 and 15 respectively). Dolichyl diphosphate (Dol-P-P) N-acetylglucosamine, dolichyl phosphate mannose and dolichyl phosphate glucose synthases and the enzymes catalysing Dol-P-P-GlcNAc2Man9Glc3 formation were higher at day 15 of postnatal development. The glycosyl transfer of the latter compound to endogenous protein(s) as well as to a dinitrophenyl-heptapeptide was also measured. The activity was higher at day 15. Furthermore, the activity of dolichyl phosphate mannose synthase was also measured during the time when the number of synapses ceased to increase (day 36) and in the adult stage. The activity of dolichyl phosphate mannose synthase was higher at day 36 than at day 15, and declined in the adult stage. From these results it may be concluded that there is an increase in the glycosylation of asparagine-type glycoproteins during synapse formation in the neurons of the cerebral cortex.     

Age-dependent modifications of mitochondrial proteins in cerebral cortex and striatum of rat brain

The protein composition of free mitochondria purified from cerebral cortex and striatum during aging was analyzed by gel electrophoresis. Mitochondria were isolated from cerebral cortex and striatum of 4-, 12-, and 24-month-old rat brain. The percent amount of mitochondrial proteins after gel-electrophoretic separation was determined densitometrically. A significant decrease in the amount of two polypeptides (with molecular weights of 20 and 16 kDa, respectively) in both brain regions during aging was found. The decrease was higher in the striatum indicating a greater vulnerability of this brain area to the aging process. The age-dependent modifications of mitochondrial proteins observed may play an important role in several mitochondrial functions, such as energy transduction and transport processes as well as in structural changes occurring with age, causing altered membrane permeability and fluidity.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat.

Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.     

Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart

A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).     

Effect of hypoxia on protein composition of synaptic plasma membranes from cerebral cortex during aging

The effect of hypoxia on the protein composition of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by SDS polyacrilamide gel electrophoresis and the percent content was evaluated by measuring the optical density of the stained gels. After hypoxic treatment various proteins showed significant changes. Some proteins were only affected at 4 and 12 months of age and not at 24 months. The various modified porteins may be identified according to their molecular weight, as follows: the 18 kDa protein with calmodulin; the 23 kDa protein with D₃ subunits; the 28 kDa protein could contain the subunit of the Ca²⁺ channel. The changes in the amount of some SPM proteins during hypoxia is consistent with the alteration in membrane polarization and neurotransmission observed in this condition. The effect of aging at the synaptosomal level seems to be a selective process; after hypoxia the age-related changes of many proteins are more pronounced.Special issue dedicated to Dr. Santiago Grisolia     

Effects of piperine on enzyme activities and bioenergetic functions in isolated rat liver mitochondria and hepatocytes

The influence of piperine on the enzymes and bioenergetic functions in isolated rat liver mitochondria and hepatocytes was studied. Piperine at lower concentrations (<50 μM) did not affect the RCR and ADP:O ratios, state 4 and 3 respirations supported by site-specific substrates, viz. glutamate + malate, succinate, and ascorbate + TMPD. The site-specific effects became significantly apparent only at higher concentrations. Only the state 3 respiration supported by NAD-linked substrates was impaired equipotently in mitochondria and permeabilized hepatocytes; the effect appeared to be localized at energy-coupling site 1. In hypotonic treated mitochondria, respiration supported by three kinds of substrates was not affected. Among the respiratory chain-linked enzymes, the activity of NADH-dehydrogenase registered a significant decrease of about 25, 42, and 53% at 100, 150, and 180 μM piperine, respectively. The activity of Mg⁺⁺-ATPase, however, was stimulated at concentrations above 150 μM. Among the matrix enzymes, only malate and succinate dehydrogen-ases were studied. Malate dehydrogenase only showed a strong concentration-related inhibition in both the forward and backward directions. Enzyme kinetics indicated noncompetitive inhibition with a very low Ki of 10 μM. The presence of unsaturated double bonds in the side chain of piperine appeared essential for producing this strong inhibition. The studies suggested that piperine produces concentration related site-specific effects on mitochondrial bioenergetics and enzymes of energy metabolism.     

Effect of estrogen on lysosomal enzyme activities in rat heart

The activities per microgram DNA of five lysosomal enzymes [cathepsin D, cathepsin B, beta-N-acetylglucosaminidase (beta-NAG), beta-glucuronidase, and acid phosphatase] were measured in homogenates of female and male rat (Sprague-Dawley) hearts. Female rats were studied during stages of the estrous cycle and at 3 weeks after ovariectomy. Three-week-postovariectomized female rats and intact male rats were injected subcutaneously with 17 beta-estradiol-3-benzoate. Lysosomal enzyme activities in the male rat heart were more responsive to exogenous estradiol than were activities in the female rat heart. Cathepsin B, beta-NAG, and beta-glucuronidase were increased dramatically in the male rat heart upon short-term administration of estrogen (4 days). In both female and male rat hearts, activities of two lysosomal proteinases, cathepsins B and D, were reduced significantly (approximately 50%) by extended administration of estrogen for 10 days.