Kynurenines Impair Energy Metabolism in Rat Cerebral Cortex

Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of l-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington’s disease, Parkinson’s disease and Alzheimer’s disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study, we investigated the in vitro effect of l-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA) on some parameters of energy metabolism, namely glucose uptake, ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate, as well as on the activities of the respiratory chain complexes I–IV and Na⁺,K⁺-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except l-kynurenine, significantly increased glucose uptake and inhibited ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by 3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I–III activities. Moreover, Na⁺,K⁺-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine intermediates provoke impairment of brain energy metabolism.     

Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

Ontogeny of Cerebral Oxidative Metabolism in the Chick Embryo

Abstract: The low cerebral energy requirements of most mammals at birth reflect an immaturity of the central nervous system, and it has been suggested that energy demands in fetuses are even less well developed than in newborns. Furthermore, fetal cerebral energy requirements are presumed to be met predominantly or exclusively by anaerobic glycolysis. To clarify these issues, we investigated cerebral oxidative metabolism in 9-, 14-, 16-, and 19-day-old chick embryos and in newly hatched peeps. Animals were decapitated and quick-frozen in liquid Freon 0-5 min post-mortem. Forebrain extracts were prepared and assayed for ATP, phosphocreatine, glucose, and lactate. Alterations in these metabolites post-decapitation were used to calculate cerebral metabolic rates (Δ∼P) and rates of maximal anaerobic glycolysis (Δ lactate). Rates of lactate accumulation during cerebral ischemia increased progressively from embryonic day 9 through hatching. Cerebral metabolic rates were not different in 9-, 14-, and 16-day-old embryos, but increased steadily thereafter. The extent to which total cerebral energy utilization could be derived from anaerobic glycolysis (Δ lactate/Δ∼ P) increased from a low at day 9 (0.29) to a maximum at day 16 (0.78). The data suggest that, despite the low cerebral metabolic activity of the chick embryo, at no time during development is anaerobic glycolysis capable of entirely supporting the energy needs of the developing brain.     

In Vivo Evidence for the Link Between l- and d-Serine Metabolism in Rat Cerebral Cortex

Abstract: To obtain an insight into the metabolic pathways of endogenous d -serine in mammalian brains, we have investigated in the infant rat the effects of systemic administration of l -serine, d -serine, and related amino acids, including glycine and threonine, on the amino acid contents in the cerebral cortex. Intraperitoneal injection of l -serine induced a rapid and transient elevation of the levels of l -serine itself in the neocortex, with its peak at 3 h post injection, and a delayed and prolonged increase in d -serine contents from 1.5 h to at least 24 h thereafter. Similarly, a significant augmentation in cerebral d -serine contents was observed 6 h after intraperitoneal administration of glycine, which also elevated the cortical l -serine levels. In contrast, l -threonine injection affected the concentrations of neither d - nor l -serine in the cortex of the pups. d -Serine given systemically, in turn, increased the neocortical contents of l -serine as well as d -serine itself, but failed to alter those of glycine and l -threonine. These in vivo data suggest the possible link between metabolic pathways of d - and l -serine in the cerebral cortex of the rat.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Rate of Protein Glycosylation in Rat Cerebral Cortex

Quantitative aspects of the pathway leading to the formation of asparagine-linked oligosaccharides were investigated in rat cerebral cortex. Steady-state labeling conditions were achieved with [2-3H]mannose by developing a micromethod of incubation of cerebral cortex particles in the presence of physiological concentrations of glucose (1 g/L). The rate of [2-3H]mannose uptake and incorporation into protein was markedly affected when the concentration of glucose was lowered to 0.05 g/L. It was found that in the presence of glucose (1 g/L), a minor fraction of the utilized [2-3H]mannose is used in glycoprotein formation and the remaining labeled sugar enters the other major metabolic pathways, generating tritiated water which is rapidly exchanged with that of the medium. Under these conditions, the intracellular isotopic dilution of [2-3H]mannose-labeled precursors was calculated to be about 11.5-fold. These data allow determination of the rate of the net transfer of mannose into proteins. Comparison of the rate of glycosylation between 5- and 30-day-old cerebral cortex revealed a striking difference: 2.1 and 0.3 ng of mannose/mg protein/h, respectively.     

Purification of Chlorpromazine-Sensitive GTPase from Rat Cerebral Cortex

Abstract

The chlorpromazine-sensitive GTPase from the cell membrane of rat cerebral cortex was purified to homogenity by using DEAE Bio-Gel A agarose, hydroxyapatite and heparin agarose chromatography. The purified chlorpromazine-sensitive GTPase was purified 370-fold to obtain a final specific activity of 40 nmol GTP hydrolyzed/min/mg protein. The purified enzyme was inhibited by chlorpromazine but not by compound 48/80. Magnesium was required for its activity instead of calcium. The purified enzyme had an apparent pH optimum of 8.0, and molecular weight was estimated to be 58,000.     

Uptake, Release, and Metabolism of D- and L-α-Aminoadipate by Rat Cerebral Cortex

Abstract: Accumulation of L-α-aminoadipate by rat cerebral cortical slices is a stereospecific and Na⁺-dependent process. The uptake of this compound is also temperature-dependent, with a K_m , of 1.6 × 10⁻⁴M for the high-affinity system. D-α-Aminoadipate has characteristics similar to those displayed by the L-isomer but to a lesser degree. L-Glutamate and L-aspartate inhibit the uptake of L-α-aminoadipate. D- and L-α-Aminoadipate are, respectively, weak uncompetitive and weak competitive inhibitors for the uptake of L-glutamate and L-aspartate. Both enantiomers inhibit GABA uptake but in quite different ways. The release of L-α-aminoadipate from the cerebral cortical slices is stimulated by a high concentration of K⁺ ions in the presence of Ca²⁺ in the perfusion buffer; the D-isomer displays this property to a lesser degree. The omission of Ca²⁺ markedly reduces the release of these two compounds. Less than 10% of the preloaded D- and L-α-aminoadipate are metabolized by the cerebral cortex during 40 min of superfusion. The possibility of L-α-aminoadipate as a neurotransmitter candidate is discussed.     

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of d-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports d-mannose by both Na⁺-dependent and Na⁺-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na⁺-independent mechanism was higher than that of the Na⁺/mannose transport system. At birth, the Na⁺-independent d-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal d-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.     

Appearance and Differentiation of NADPH-D-Neurons in Ontogeny of Cerebral Cortex in Rats

The appearance of presumptive NO-ergic nerve cells and their differentiation in the rat neocortex were studied. For this purpose, a comparative analysis of the development and differentiation of NADPH-D-positive neurons in the neocortex transplants taken from the embryos of different ages and transplanted in the occipital cortex of adult rats and in the normally developing cerebral cortex was undertaken. The nervous tissue was stained histochemically for NADPH-D. The results we obtained suggest that no NADPH-D-containing neurons were found in the transplants from 15-day embryos, while they developed in those from 18-day embryos. Hence, precursors of NO-ergic neurons were still absent in the presumptive neocortex of 15-day embryos and appeared only on day 16–18 of embryogenesis. Expression of NADPH-D begins in them only within four to five days, but the neurons are differentiated during a relatively short period of time. Most NADPH-D-positive neurons reach their structural–functional maturity already by the end of the first week of postnatal development, while their complete maturation takes place by the end of the second week of postnatal development.     

Subcellular Localization of Bioactive Cholecystokinin-Like Peptides in the Rat Cerebral Cortex

Abstract: The subcellular distribution of cholecystokinin (CCK), especially its exact localization within the synaptosome, was studied in the rat cerebral cortex. Highest CCK-like bioactivity was measured in the synaptic membrane fractions, paralleling the distribution of (Na⁺+ K⁺)-dependent ATPase. In the synaptic vesicles, which were characterized by high acetylcholine content and by the absence of (Na⁺+ K⁺)-ATPase, only minimal quantities of CCK were detected.     

On the Status of Lysolecithin in Rat Cerebral Cortex During Ischemia

Abstract: Lysolecithin (lysoglycerophosphocholine, LPC) was isolated from rat cerebral cortex and quantitatively analyzed at various times after postdecapitative ischemic treatment. In addition, different procedures for extraction and analysis of the LPC in brain were evaluated. Results indicated that LPC can be quantitatively extracted into the organic phase using the conventional extraction procedure with chloroform-methanol (2:1, vol/ vol). However, care should be taken to avoid using strong acids, which can hydrolyze the alkenylether side chain of the plasmalogens, resulting in the release of 2-acyl-phospholipids. Quantitative GLC analysis using myris-toyl-LPC as internal standard revealed a level of 1.8 nmol LPC/mg protein in brain with acyl groups comprised mainly of 16:0, 18:0, and 18:1. The acyl group profile reflects that the LPC are derived mainly from phospho-lipase A₂ action. An increase of 46% in the LPC level was observed at 1 min after ischemic treatment, but this was followed by a steady decline. Ischemia induced an increase in the LPC species that are enriched in 18:0 and 18:1 fatty acids. The transient appearance of LPC during ischemia further suggests that this phospholipid is undergoing active turnover, possibly hydrolysis by the lysophospholipase. This mechanism of action may account, at least in part, for the increase in both saturated and unsaturated fatty acids during the early phase of the ischemic treatment.     

High-Affinity Uptake of Spermine by Slices of Rat Cerebral Cortex

Abstract: The accumulation of the polyamine spermine into 0.1-mm prisms of rat cerebral cortex has been investigated at both 37°C and at 4°C. Kinetic analysis of the temperature-sensitive portion of uptake indicates two high-aftinity saturable components together with an unsaturable component at high concentrations. The 'very high'– affinity saturable system ( K _m= 3.8 nM) was temperature- and sodium-dependent, and significantly reduced by metabolic inhibitors, findings that are consistent with an active transport system for spermine into brain tissue. The 'high'– affinity saturable component ( K _m= 0.44 μM) was sodium-dependent and inhibited by ouabain, but only partially susceptible to inhibition by 2,4-dinitrophenol and sodium cyanide. The significance of these results with respect to the function of spermine in the central nervous system is discussed.     

Cerebral Polyamine Metabolism in Reversible Hypoglycemia of Rat: Relationship to Energy Metabolites and Calcium

Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-microns thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p less than or equal to 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p less than or equal to 0.01 and p less than or equal to 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamine Synthetase-Containing Cells of the Dorsal Root Ganglion at Different Stages of Rat Ontogeny

In the present study, formation, location, and morphological features of glutamine synthetaseimmunopositive cells of the dorsal root ganglion (DRG) at different stages of prenatal and postnatal development of the rat was examined. It was demonstrated that small differentiating satellite cells containing glutamine synthetase were observed in the DRG close to sensory neurons on embryonic day 18. On embryonic day 19, the forming immunopositive glial cells were located around developing neurons of the DRG in accordance with topography, which is observed in newborn and adult animals. The averaged number of satellite cells per sensory neuron in mature and aging rats was calculated and it was found that this index did not change during aging.     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

Asymmetry of Diacylglycerol Metabolism in Rat Cerebral Hemispheres

Diacylglycerols (DGs) were found to be asymmetrically distributed between the two cerebral hemispheres of rat brain. The left cerebral hemisphere (LCH) contained 100% more DG than the right cerebral hemisphere (RCH). The lateralization was enhanced in animals subjected to depolarization induced by a single electroconvulsive shock (ECS). During the acute phase of the convulsion, the DG pool increased in both hemispheres, with the LCH attaining a concentration 180% higher than the RCH. Stearate and arachidonate were the principal DG-acyl groups accumulated in the RCH, whereas in the LCH stearate and palmitate were mainly involved. After the last of a series of five shocks (one per day) the lateralization of the "DG response" was less accentuated during the acute phase of the ECS. Whereas DG release was drastically reduced in the LCH, in the RCH it was minimally affected. The DG sidedness after five shocks was nevertheless maintained at the level of arachidonate-containing DGs, which showed a higher accumulation in the LCH than in the RCH. The kinetics of DG removal showed a rapid phase during the first minute following a single or five ECSs. Total DG levels returned to basal values in the RCH, whereas in the LCH they remained slightly increased with respect to the initial levels 1 min after the convulsive episode. Minimal changes occurred in the subsequent 4 min. Chronic ECS altered the endogenous DG content and composition. Thus, 24 h after the last of four ECSs, total levels of DGs diminished by 40% in the RCH, whereas they remained unchanged in the LCH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Uptake of Carnitine by Slices of Rat Cerebral Cortex

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[¹⁴CH₃]carnitine and 27 ± 3 for D-[¹⁴CH₃]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K _m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V _max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K _i= 3.22 mM), acetylcarnitine ( K _i= 6.36 mM), and γ-aminobutyric acid ( K _i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N₂ atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na⁺-dependent, but did not require K⁺ or Ca²⁺.     

Evidence that Lithium Alters Phosphoinositide Metabolism: Chronic Administration Elevates Primarily d-myo-Inositol-1-Phosphate in Cerebral Cortex of the Rat

The administration of LiCl (3.6 mequiv./kg/day) to adult male rats for 9 days results in an increase in the cerebral cortex level of myo-inositol-1-phosphate (M1P) to 4.43 +/- 0.52 mmol/kg (dry weight) compared with a control level of 0.24 +/- 0.02 mmol/kg. This establishes that the previously observed acute effect of lithium on M1P (Allison et al., 1976) is both prolonged and augmented by repeated doses of lithium. Larger doses of LiCl over a 3-5 day period result in even larger increases in M1P and a 35% decrease in myo-inositol. In each case, 90% of the increase is due to the D-enantiomer, evidence that lithium is largely producing this effect via phospholipase C-mediated phosphoinositide metabolism. Data are presented showing that lithium is an uncompetitive inhibitor of the hydrolysis of both D- and L-M1P by M1P'ase.