Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

Transduction Mechanisms Involved in Thrombin Receptor-Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes

Abstract: In astrocytes, thrombin and thrombin receptor-activating activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [³H]thymidine incorporation into DNA (EC₅₀ = 1 n M and +650% at 100 n M for thrombin; EC₅₀ = 3 µ M and +600% at 100 µ M for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 n M thrombin or 100 µ M TRAP-14). The [³H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as G_i or G_o, is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.     

Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

CATABOLISM OF PROSTAGLANDIN ENDOPEROXIDES INTO PROSTAGLANDIN E2 AND F2x BY THE RAT BRAIN

Abstract— Tritium labeled prostaglandin (PG) endoperoxides PGG₂ and PGH₂ were rapidly transformed (2 min, 37°C) in good yield (> 50%) by homogenates of whole rat brain into a mixture of products including prostaglandin E₂ and F_2x: under similar conditions (10min. 37°C) tritium labeled arachidonic acid remained essentially unoxidised. The ratio of PGE-like products: PGF_2x formed was approx 0.5 as determined by radio thin layer chromatography. This ratio changed to unity when homogenates of cerebral cortex or cerebral hemispheres were employed. On the other hand cerebellar homogenates formed PGF_2x in much greater amounts. The structures of the products were confirmed by mass spectrometry and were further supported by experiments using octadeuterio-endoperoxides. In the latter experiments the resulting PGE₂ and PGF_2x contained the expected seven and eight deuterium atoms respectively. Evidence for the formation of heptadeuterio PGD₂. heptadeuterio-6-keto-PGF₁, and hexadeuterio 12-hydroxyheptadecatrienoic acid was also obtained by mass spectrometry. These experiments demonstrate for the first time in brain tissue the biosynthesis of labeled prostaglandins from exogenous tritiated and deuterated precursors.     

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min⁻¹· mg⁻¹ protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min⁻¹· mg⁻¹). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.     

Partial Structure and Mapping of the Human Myelin P2 Protein Gene

Abstract: The myelin P₂ protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P₂ protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P₂ gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P₂ gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.     

DISTRIBUTION OF SERUM PROTEINS AND BETA-TRACE PROTEIN WITHIN THE NERVOUS SYSTEM

Abstract— The concentration of beta-trace protein, a low molecular weight water-soluble protein, was significantly higher in cerebral and cerebellar white matter than in grey matter. A similar distribution was found for transferrin. The concentrations of gamma-trace protein and pre-albumin were almost constant in cerebral and cerebellar white and grey matter. A different distribution was shown for albumin, beta_lc/beta_lA globulins, and the immunoglobulins G, A and M, with the highest concentrations mostly encountered in the highly vascularized basal ganglia and grey matter, and the lowest concentrations in white matter. Thus, different parameters, hitherto unknown determine the distribution within the central nervous system of various proteins-those which originate from serum, and beta-trace protein which originates mainly from the central nervous system.
The amounts of the different proteins were higher in the choroid plexus than in brain tissue, with the exception of gamma-trace protein.
Foetal brains contained increasing concentrations of beta-trace protein and of transferrin with age.
Femoral nerve contained lower concentrations of beta-trace protein and gamma-trace protein, and higher concentrations of the other proteins, than the central nervous system.     

Endothelin Induces a Calcium-Dependent Phosphorylation of PEA-15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro

Abstract: PEA-15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.     

The High-Affinity Sulphonylurea Receptor Regulates KATP Channels in Nerve Terminals of the Rat Motor Cortex

Abstract: The coexpression of sulphonylurea binding sites and ATP-sensitive K⁺ (K_ATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive K_ATP channels and charybdotoxin-sensitive, large-conductance calcium-activated K⁺ BK_Ca channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium-independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and K_ATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the K_ATP channel may serve to limit glutamate release under conditions of metabolic stress.     

P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes

Previous work has established the presence of functional P2X₇ subunits in rat cerebellar astrocytes, which after stimulation with 3'- O -(4-benzoyl)benzoyl ATP (BzATP) evoked morphological changes that were not reproduced by any other nucleotide. To further characterize the receptor(s) and signaling mechanisms involved in the action of BzATP, we have employed fura-2 microfluorometry and the patch-clamp technique. BzATP elicited intracellular calcium responses that typically exhibited two components: the first one was transient and metabotropic in nature – sensitive to phospholipase C inhibition and pertussis toxin treatment –, whereas the second one was sustained and depended on the presence of extracellular calcium. The ionotropic nature of this latter component was corroborated by measurements of Mn²⁺ entry and macroscopic non-selective cation currents evoked by either BzATP (100 μM) or ATP (1 mM). The two components of the calcium response to BzATP differed in their pharmacological sensitivity. The metabotropic component was partially sensitive to pyridoxalphosphate-5'-phosphate-6-azo-(-2-chloro-5-nitrophenyl)-2,4-disulfonate, a selective antagonist of P2Y₁₃ receptors, while the ionotropic component was modulated by external magnesium and markedly reduced by brilliant blue G and 3-(5-(2,3-dichlorophenyl)-1 H -tetrazol-1-yl)methyl pyridine (A438079), thus implying the involvement of P2X₇ purinergic receptors. It is concluded that P2Y₁₃ and P2X₇ purinergic receptors are functionally expressed in rat cerebellar astrocytes and mediate the increase in intracellular calcium elicited by BzATP in these cells.     

Ontogeny and Cellular Localization of the Pyruvate Recycling System in Rat Brain

Abstract: The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of ¹³C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-¹³C₂]acetate as a substrate, was detected by ¹³C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.     

Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC₅₀ of ∼60 µ M when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC₅₀ = 1 µ M ), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

PYRUVATE METABOLISM BY HOMOGENATES OF HUMAN BRAIN: EFFECTS OF PHENYLPYRUVATE AND IMPLICATIONS FOR THE ETIOLOGY OF THE MENTAL RETARDATION IN PHENYLKETONURIA

Abstract— The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by homogenates of human brain was investigated. In the presence of 5 mM pyruvate as substrate homogenates of human cerebral cortex fixed about 1 μmol of H¹⁴CO₃^-_- per g of tissue in 30 min. Phenylpyruvate at a concentration of 5 raw inhibited the fixation of H¹⁴ CO₃^-_- by homogenates of human brain by approximately 50 per cent, whereas 5 mM phenylalanine had no effect. The inhibition of pyruvate carboxylation by phenylpyruvate was dependent upon the concentration of the inhibitor. The activity of pyruvate carboxylase (EC 6.4.1.1) in human cerebral cortex was 02–0.4 units, with a K_m for pyruvate of about 0.2 mM. Homogenates of human cerebral cortex decarboxylated [1-¹⁴C]pyruvate to ¹⁴CO₂ at a rate of about 5 μmol per g of tissue per 15 min, with a 20–50 per cent reduction in the presence of 5 mM phenylpyruvate; phenylalanine at the same concentration had no effect. The possible toxic effect of phenylpyruvate on the metabolism of pyruvate in the brains of untreated phenylketonuric patients is discussed.     

Endogenous GABA Modulates Histamine Release from the Anterior Hypothalamus of the Rat

Abstract: Using a microdialysis method, we investigated the effects of the nipecotic acid-induced increase in content of endogenous GABA on in vivo release of histamine from the anterior hypothalamus (AHy) of urethane-anesthetized rats. Nipecotic acid (0.5 m M ), an inhibitor of GABA uptake, decreased histamine release to ∼60% of the basal level. This effect was partially antagonized by picrotoxin (0.1 m M ), an antagonist of GABA_A receptors, or phaclofen (0.1 m M ), an antagonist of GABA_B receptors. These results suggest that histamine release is modulated by endogenous GABA through both GABA_A and GABA_B receptors. When the tuberomammillary nucleus, where the cell bodies of the histaminergic neurons are localized, was stimulated electrically, the evoked release of histamine from the nerve terminals in the AHy was significantly enhanced by phaclofen, suggesting that GABA_B receptors may be located on the histaminergic nerve terminals and modulate histamine release presynaptically. On the other hand, picrotoxin caused an increase in histamine release to ∼170% of the basal level, and this increase was diminished by coinfusion with d (−)-2-amino-5-phosphonopentanoic acid (0.1 m M ), an antagonist of NMDA receptors. Previously, we demonstrated tonic control of histamine release by glutamate mediated through NMDA receptors located on the histaminergic terminals in the AHy. These results suggest the possible localization of GABA_A receptors on glutamatergic nerve terminals and that the receptors may regulate the basal release of histamine indirectly.     

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons

This study examines the role of c- jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H₂O₂ resulted in activation (phosphorylation) of JNK (mostly p46^JNK1) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E_1α subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.