bluuebunny/crossref_metadata_2025 huggingface dataset:
The Deduced Amino Acid Sequences of Human Platelet and Frontal Cortex Monoamine Oxidase B Are Identical
Authors: Chen Kevin , Wu Hsiu‐Fei , Shih Jean C.
Published: October 2006

Abstract: Monoamine oxidases (MAOs) A and B play important roles in the metabolism of neuroactive, vasoactive amines. Human platelets contain only MAO B, often used as an indicator of brain MAO B. The validity of this model remained to be evaluated. This report describes the molecular cloning of human MAO B from frontal cortex and platelets. Two overlapping PCR‐amplified clones of human platelet MAO B and four PCR‐amplified clones of human frontal cortex MAO B covering the entire coding region were sequenced using five internal oligomers and M13 reverse and forward primers. The nucleotide sequences of human MAO B cDNA from platelet and frontal cortex were identical to that of human liver MAO B except for three nucleotides that differed in frontal cortex: nucleotides 440 A → G, 794 C → T, and 825 C → T. Whether or not these differences are artifactual, all three represent silent mutations, which would not alter the amino acid of the encoded polypeptides. Thus, the deduced amino acid sequences of MAO B from frontal cortex, platelet, and liver are identical. These findings indicate the validity of using platelet MAO B mRNA as a marker for brain MAO B and provide a new approach to study the role of brain MAO B in humans.
Glutamic Acid and γ‐Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain
Authors: Bonanno G. , Pittaluga A. , Fedele E. , Fontana G. , Raiteri M.
Published: October 2006

Abstract: The effects of γ‐aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [3H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC5017.2 μM) and Asp (EC50 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (‐)‐baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na+ dependent. Glu enhanced the release of [3H]GABA (EC50 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N‐methyl‐d‐aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D‐threo‐hydroxyaspartic acid (THA) and it was Na+ sensitive. Similarly to Glu, D‐Asp increased [3H]GABA release (EC50 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antagonist 6‐cyano‐7‐nitroquinoxaiine‐2, 3‐dione, whereas the 6‐cyano‐7‐nitroquinoxaline‐ 2, 3‐dione‐insensitive portion of the effect was prevented by THA. GABA increased the [3H]D‐Asp outflow (EC50 13.7 μM) from hippocampal synaptosomes in a muscimol‐, (‐)‐ baclofen‐, bicuculline‐, and picrotoxin‐insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na+ dependent. Glu increased the release of [3H]‐ GABA from hippocampal synaptosomes (EC50 7.1 μM) in an N‐methyl‐d‐aspartic acid‐, kainic acid‐, or quisqualic acid‐insensitive way. The effect of Glu was prevented by THA and was Na+ dependent. As in the cortex, the effect of Glu was mimicked by D‐Asp in a THA‐sensitive manner. It is proposed that high‐affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA‐ BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.
Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site‐Directed Mutagenesis
Authors: Carbini Luis A. , Hersh Louis B.
Published: October 2006

Abstract: The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl‐CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila, pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His268, His393, and His426. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His268Leu‐ChAT, HisZ68Gln‐ChAT, and His393Gln‐ChAT and inactive His393Leu‐ChAT, His426Leu‐ ChAT, and His426Gln‐ChAT. The kinetic constants Km(CoA), Km(acetyloholine). and Vmax were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His393Leu‐ChAT is disrupted but conserved in His426Leu‐ChAT and His426Gln‐ ChAT. These results suggest that His426 is an essential catalytic residue and could serve as an acid/base catalyst.
Investigations into the Adenosine Outflow from Hippocampal Slices Evoked by Ischemia‐Like Conditions
Authors: Pedata Felicita , Latini Serena , Pugliese Anna Maria , Pepeu Giancarlo
Published: October 2006

Abstract: The characteristics of adenosine and inosine outflow evoked by 5 min of ischemia‐like conditions in vitro (superfusion with glucose‐free Krebs solution gassed with 95% N2/5% CO2) were investigated on rat hippocampal slices. The viability of the slices after “ischemia” was evaluated by extracellular recording of the evoked synaptic responses in the CA1 region. The evoked dendritic field potentials were abolished after 5 min of superfusion under “ischemia” but a complete recovery occurred after 5 min of reperfusion with normal oxygenated Krebs solution. No recovery took place after 10 min of “ischemia.” The addition of the adenosine A, receptor antagonist 8‐phenylthe‐ ophylline to the superfusate antagonized the depression of the evoked field potentials caused by 5 min of “ischemia.” Five minutes of “ischemia” brought about a six‐ and fivefold increase in adenosine and inosine outflow, respectively, within 10 min. Tetrodotoxin reduced the outflow of adenosine and inosine by 42 and 33%, respectively, whereas the removal of Ca2+ caused a further increase. The NMDA receptor antagonist d(‐)‐2‐amino‐7‐ phoshonoheptanoic acid and the non‐NMDA antagonist 6,7‐dinitroquinoxaline‐2,3‐dione brought about small, not statistically significant decreases of adenosine and inosine outflow. The glutamate uptake inhibitor dihydrokainate did not affect the outflow of adenosine and inosine. Inhibition of ecto‐5′‐nucleotidase by α, β‐methylene ADP and GMP did not affect basal adenosine outflow but potentiated “ischemia”‐evoked adenosine outflow. It is concluded that ischemia‐like conditions in vitro evoke a Ca2+‐independent adenosine and inosine outflow, through a mechanism that partly depends on propagated nervous activity but does not involve excitatory amino acids. The efflux of adenosine is probably responsible for the depression of the evoked synaptic electrical activity during “ischemia” in the hippocampal slices.
Lipid and Fatty Acid Composition of Brain Tissue from Adrenoleukodystrophy Patients
Authors: Wilson Robert , Sargent John R.
Published: October 2006

Abstract: White matter and active plaque tissue from adrenoleukodystrophy (ALD) patients were analysed for lipid class and fatty acid compositions and the results compared with white matter from normal brain. ALD white matter was characterized by increased levels of cholesteryl esters and decreased levels of phosphatidylethanola‐ mine, including phosphatidylethanolamine plasmalogen, in comparison with normal brain white matter. In addition to even higher levels of cholesteryl esters, ALD plaque tissue had reduced levels of cerebrosides as well as phosphati‐dylethanolamines. The loss of phosphatidylethanolamine plasmalogen is indicative of early demyelination. Total lipid from ALD white matter and ALD plaque tissue contained nearly five times and seven times, respectively, more 26:0 than total lipid from normal brain white matter. The 26:0 in ALD white matter was elevated in all lipid classes except phosphatidylinositol, but was located mainly in cerebrosides, phosphatidylcholine, sphingomyelin, and sulfatides. Most of the 26:0 in ALD plaque tissue was present in cholesteryl esters, followed by phosphatidylcholine and sphingomyelin, with reduced amounts in cerebrosides as compared with ALD white matter. The results are consistent with an initial accumulation of very‐long‐chain fatty acids in ALD white matter, primarily in sphingolipids and phosphatidylcholine, and subsequent accumulation of very‐long‐ chain fatty acids in cholesteryl esters during demyelination. In addition, it was notable that the sphingolipids, especially sphingomyelin in ALD brain, had decreased levels of 24:1 and increased levels of 18:0, as well as increased levels of very‐long‐chain fatty acids. The extent to which the data shed light on mechanisms of demyelination in ALD is discussed.
Serotonin Modulates Nicotinic Responses of Adrenal Chromaffin Cells
Authors: Vijayaraghavan Sukumar , Schmid Herbert A. , Mapp Karen S.
Published: October 2006

Abstract: 5‐Hydroxytryptamine (5‐HT) specifically and reversibly inhibits nicotine‐induced currents and catecholamine release in bovine adrenal chromaffin cells in culture. Pharmacological analysis indicates that the inhibition is not mediated by known 5‐HT receptor subtypes. The inhibition is noncompetitive over a range of nicotine concentrations between 1 and 100 μM. Preincubation with either 5‐HT or substance P significantly protects the response from nicotine‐induced desensitization. It is concluded that 5‐HT inhibits nicotinic acetylcholine receptors on bovine adrenal chromaffin cells, probably by binding to a noncompetitive site on the receptor itself. Because both blood and the chromaffin cells contain 5‐HT, the inhibition provides an opportunity for negative control of catecholamine secretion from the adrenals.
Regulation of Melatonin Production by Pineal Photoreceptor Cells: Role of Cyclic Nucleotides in the Trout (Oncorhynchus mykiss)
Authors: Thibault Christelle , Falcón Jacky , Greenhouse Shelley S. , Lowery Christopher A. , Gern William A. , Collin Jean‐Pierre
Published: October 2006

Abstract: The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N‐acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.
Insulin Stimulates Phosphatidylinositol 3‐Kinase Activity in Rat Neuronal Primary Cultures
Authors: Patel Rajiv A. , Kurian Pawels , Raizada Mohan K. , Crews Fulton T.
Published: October 2006

Abstract: We investigated the effect of insulin on phosphatidylinositol (PtdIns) 3‐kinase (PtdIns 3‐kinase) activity in neuronal cultures to determine if this enzyme is involved with the neurotrophic actions of insulin. Insulin caused a concentration‐dependent increase in PtdIns 3‐kinase activity in anti‐phosphotyrosine immuno‐precipitates. The kinase activity was able to phosphorylate PtdIns, PtdIns 4‐phosphate, and PtdIns 4,5‐bisphosphate. In intact neurons, a 10‐min 1 mM insulin treatment in the presence of [32P]orthophosphate increased the levels of both 3‐[32P]PtdIns phosphate and 3,4‐[32P]PtdIns bisphosphate by 55 and 193%, respectively. This increase was associated with an increase in neurite outgrowth mediated by insulin. Our results indicate that insulin treatment of neuronal cells in primary culture increases PtdIns 3‐kinase activity and the formation of the unique d‐3‐phosphorylated phosphoinositides, suggesting that growth factor‐mediated neuronal growth may include the formation of novel phosphoinositide 3‐phosphate phospholipids.
Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+‐Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α‐Latrotoxin
Authors: Deri Z. , Adam‐Vizi V.
Published: October 2006

Abstract: A novel fluorescent Na+ indicator, Na+‐binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na+ concentration ([Na+]1) of synaptosomes. The dye, when loaded into synapto‐ somes in the form of its acetoxymethyl ester, was responsive to changes of [Na+]1. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na+ concentration was equilibrated with different concentrations of extracellular Na+ in the presence of 2 μM gramicidin D. The basal value of [Na+]1 in synaptosomes in the presence of 140 mM extracellular Na+ was found to be 10.9 ± 1.8 mM. Veratridine, which opens potential‐dependent Na+ channels, caused a sudden increase in [Na+]1 in a concentration‐dependent manner (1 ‐20 μM), whereas the effect of ouabain (20 and 50 μM), the inhibitor of the plasma membrane Na+,K+‐ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μM tetrodotoxin. α‐Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 nM). This report confirms our earlier finding demonstrating a Na+‐dependent component in the action of α‐Iatrotoxin, and shows that changes in [Na+]1 in synaptosomes can be followed by SBFI.
Substance P Receptors on O‐2A Progenitor Cells and Type‐2 Astrocytes In Vitro
Authors: Marriott D. R. , Wilkin G. P.
Published: October 2006

Abstract: Bradykinin‐ and substance P (SP)‐stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin‐stimulated synthesis of phospho‐ inositides (PI) in type‐1 astrocytes and oligodendrocytes. SP‐stimulated PI accumulation was restricted to oligoden‐ drocyte/type‐2 astrocyte progenitor cells and type‐2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP‐stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type‐2 astrocyte type‐2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.
N‐Acetylaspartylglutamate Inhibits Forskolin‐Stimulated Cyclic AMP Levels via a Metabotropic Glutamate Receptor in Cultured Cerebellar Granule Cells
Authors: Wroblewska Barbara , Wroblewski Jarda T. , Saab Omar H. , Neale Joseph H.
Published: October 2006

Abstract: The neuronal dipeptide N‐acetylaspartylglutamate (NAAG) fulfills several of the criteria for classification as a neurotransmitter including localization in synaptic vesicles, calcium‐dependent release after neuronal depolarization, and low potency activation of N‐methyl‐d‐aspartate receptors. In the present study, the influence of NAAG on metabotropic receptor activation in cerebellar granule cells was examined in cell culture. Stimulation of granule cell adenylate cyclase with forskolin increased cyclic AMP (cAMP) several hundredfold above basal levels within 10 min in a concentration‐dependent manner. Although gluta‐mate, NAAG, and the metabotropic receptor agonist frans‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid did not alter the low basal cAMP levels, the application of 300 μM glutamate or NAAG or trans‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid reduced forskolin‐stimulated cAMP in granule cells by 30–50% in the absence or presence of inhibitors of ionotropic acidic amino acid receptors, as well as 2‐amino‐4‐phosphonobutyrate. No additivity in the inhibition of cAMP was found when 300 μM NAAG and trans‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid were coapplied. The β‐analogue of NAAG failed to reduce cAMP levels. Similar effects of NAAG and glutamate were obtained under conditions of inhibition of phosphodiesterase activity and were prevented by pretreatment of the cells with pertussis toxin. These data are consistent with the activation by NAAG of a metabotropic acidic amino acid receptor coupled to an inhibitory G protein. In contrast, the metabotropic acidic amino acid receptor coupled to phosphoinositol turnover in these cells was not activated by NAAG. Granule cells in culture expressed very low levels of extracellular peptidase activity against NAAG, converting to glutamate <0.1% of the 10 μM through 1 mM NAAG applied to these cells during 15‐min in vitro assays.
ATP‐Sensitive Potassium Channels and Local Energy Demands in the Rat Hippocampus: An In Vivo Study
Authors: Fellows L. K. , Boutelle M. G. , Fillenz M.
Published: October 2006

Abstract: Microdialysis coupled with an enzyme‐based flow injection analysis was used to monitor brain extracellular lactate and glucose in the freely moving rat. Glucose levels reflect the balance between supply from the blood and local utilisation, and lactate efflux indicates the degree of local nonoxidative glucose metabolism. Local application of tolbutamide, a blocker of the ATP‐sensitive potassium channel, decreased extracellular glucose and lactate levels in the hippocampus but not in the striatum. The increase in glucose and lactate levels following mild behavioural stimulation was also reduced by tolbutamide in the hippocampus. Similar effects on both basal and stimulated lactate levels were obtained with local application of 10 mMglucose. These results indicate that ATP‐sensitive potassium channels are active under physiological conditions in the hippocampus and that the effects of tolbutamide can be mimicked by physiological glucose levels.
Age‐Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin
Authors: Palestini Paola , Masserini Massimo , Fiorilli Amelia , Calappi Emanuela , Tettamanti Guido
Published: October 2006

Abstract: Age‐related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal‐phase HPLC. Each ganglioside was then fractionated by reverse‐phase HPLC into the molecular species carrying a single long‐chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the “b series” than in the “a series” gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age‐related control.
Solubilization of High‐Affinity, Guanine Nucjeotide‐Sensitive μ‐Opioid Receptors from 7315c Cell Membranes
Authors: Cote Thomas E. , Gosse Marilyn E. , Weems Henri B.
Published: October 2006

Abstract: High‐affinity μ‐opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane‐associated receptors with morphine before their solubilization in the detergent 3‐[(3‐cholamidopropyl) dimethyl]‐1‐propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]‐etorphine with high affinity (KD= 0.304 ± 0.06 nM; Bmax= 154 ± 33 fmol/mg of protein). Of the membrane‐associated [3H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ‐selective and non‐selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, 5‐ and K‐opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ‐selective ligand [3H][D‐Ala2,A/‐Me‐Phe4,Gly5‐ol]enkephalin also bound with high affinity (KD= 0.79 rM; Bmax= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane‐associated [3H][D‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′‐O‐(3‐thiotriphosphate), GTP, and guanosine 5′‐O‐(2‐thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin binding in a concentration‐dependent manner. Finally, μ‐opioid receptors from rat brain membranes were also solubilized in a high‐affinity, guanine nucleotide‐sensitive state if membrane‐associated receptors were occupied with morphine before and during their solubilization with the detergent 3‐[(3‐cholamidopropyl) dimethyl]‐1‐propane sulfonate.
Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain
Authors: Lamensa John W. E. , Moscarello Mario A.
Published: October 2006

Abstract: A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca2+, a temperature optimum at ∼50°C, and two Kmvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher Kmhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C‐1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]200. At 5°C, the [θ]200of the deiminated protein was ‐70 × 103 compared with ‐30 × 103 deg cm2/dmol for the native protein. When the temperature was increased to 70°C, the [θ]200 was ‐44 × 103 for the deiminated protein and ‐20 × 107 deg cm2/ dmol for the native C‐1. When plotted as a function of temperature, [θ]200 decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C‐8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.
Biliverdin Reductase Is Heat Resistant and Coexpressed with Constitutive and Heat Shock Forms of Heme Oxygenase in Brain
Authors: Ewing James F. , Weber Colleen M. , Maines Mahin D.
Published: October 2006

Abstract: Two heme oxygenase (HO) isozymes—HO‐1, which is a heat shock protein (HSP32), and HO‐2—catalyze the isomer‐specific production of biliverdin IXα and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO‐1 and/or HO‐2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock‐inducible HO‐1 protein. Exposure of male rats to 42°C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH‐and NADH‐dependent activities at 1 and 6 h following hyperthermia. High levels of the ∼ 1.5‐kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat‐shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO‐1 and/or HO‐2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected. The same population, however, responded to heat shock by an intense increase in the level of HO‐1. We postulate that the constitutive presence of the reductase in this cell population and the overall thermal stability of the enzyme represent a safeguard mechanism in the brain for the prompt conversion of biliverdin to bilirubin under conditions when oxidation of the heme moiety of denatured hemoproteins by HO‐1 is accelerated.
Immunocytochemical Quantification of Tyrosine Hydroxylase at a Cellular Level in the Mesencephalon of Control Subjects and Patients with Parkinson's and Alzheimer's Disease
Authors: Kastner A. , Hirsch E. C. , Herrero M. T. , Javoy‐Agid F. , Agid Y.
Published: October 2006

Abstract: Parkinson's disease is characterized by massive degeneration of the melanized dopaminergic neurons in the substantia nigra. The functional capacity of the surviving nigral neurons is affected, as indicated by the subnormal levels of tyrosine hydroxylase (TH) mRNA in these neurons and the presence in the parkinsonian mesencephalon of melanized neurons lacking TH immunoreactivity. This is apparently in contradiction with the known overactivity of dopamine synthesis and release that occurs in the remaining dopaminergic terminals. To test the ability of the surviving neurons to express TH protein, a semiquantitative immunocytochemical method was developed. The relative amounts of TH were estimated with a computer‐assisted image analysis system in the dopaminergic neurons of representative mesencephalic sections of control and parkinsonian brains and for comparison in brains from patients with Alzheimer's disease. In control brains, the mean TH content per neuron differed from one subject to another and between the different dopaminergic cell groups of the mesencephalon in the same subject. Within a given dopaminergic region, the level of TH was variable among neurons. In patients with Parkinson's disease, the ratio of TH protein content per neuron in the substantia nigra by reference to that of the central gray substance was reduced. In patients with Alzheimer's disease, the amount of TH was selectively reduced in the remaining dopaminergic neurons of the ventral tegmental area, a region characterized by a loss in dopaminergic neurons. The decrease in cellular TH content might therefore be related to the presence of the neurodegenerative process in the area considered. In patients with Parkinson's disease, the incapacity of the surviving neurons to express normal TH levels may reduce the efficiency of the hyperactivity mechanisms that develop in the remaining striatal dopaminergic terminals.
Nerve Growth Factor Stimulates the Production of Inositol 1,3,4‐and 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate in PC 12 Cells
Authors: Contreras M. L.
Published: October 2006

Abstract: In PC12 cells, preincubated with [3H]inositol, nerve growth factor (NGF) stimulated an ∼ 100% increase in the levels of [3H]inositol 1,3,4‐trisphosphate {[3H]‐Ins(1,3,4)P3}, [3H]inositol 1,4,5‐trisphosphate {[3H]lns(1,4,5)P3}, and [3H]inositol 1,3,4,5‐tetrakisphosphate {[3H]‐Ins(1,3,4,5)P4} as early as 5–15 s after addition of NGF. This NGF‐mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS‐containing medium did not display NGF‐mediated increases in [3H]‐Ins(1,3,4)P3, [3H]‐Ins(1,4,5)P3, and [3H]‐Ins(1,3,4,5)P4 levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [3H]lns(1,3,4)P3, [3H]‐Ins(1,4,5)P3, and [3H]lns(1,3,4,5)P4. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF‐and NGF‐stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of lns(1,3,4)P3, lns(1,4,5)P3, and lns(1,3,4,5)P4 and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of lns(1,3,4)P3, lns(1,4,5)P3, and lns(1,3,4,5)P4 may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.
Identification and Characterization of Endothelin Receptor Subtype B in Rat Retina
Authors: de Juan J. A. , Moya F. J. , Garcia de Lacoba M. , Fernandez‐Cruz A. , Fernandez‐Durango R.
Published: October 2006

Abstract: The presence of immunoreactive (IR) endothelin (ET)‐1 and ET‐1 receptors in rat retina has been studied by radioimmunoassay and receptor assay, respectively. The specific binding of 125I‐ET‐1 to rat retinal particulate preparations was saturable. Apparent equilibrium conditions were established within 120–140 min. Scatchard analysis of binding data indicated a single class of high‐affinity binding sites with a KD of 35 ± 11 pM and a Bmax of 168 ± 60 fmol/mg of protein. 125I‐ET‐1 binding to retinal particulate preparations was not inhibited by 1 μM concentrations of somatostatin, atrial natriuretic factor, brain natriuretic peptide, thyroid‐stimulating hormone, growth hormone, or insulin. The three endothelin isoforms, ET‐1,‐2, and‐3, had similar affinity for the receptor. Cross‐linking of 125I‐ET‐1 to retinal particulate preparations with disuccinimidyl suberate resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. We have established a highly sensitive and specific radioimmunoassay for ET‐1. The concentration of IR‐ET‐1 in rat retina was 35 ± 10 fmol/g wet weight. The demonstration of specific high‐affinity ETB receptors and the presence of IR‐ET‐1 suggest that the peptide may act as a neurotransmitter or neuro‐modulator in the retina.
Cerebral Vulnerability Is Associated with Selective Increase in Extracellular Glutamate Concentration in Experimental Thiamine Deficiency
Authors: Hazell Alan S. , Butterworth Roger F. , Hakim Antoine M.
Published: October 2006

Abstract: Microdialysis in the awake, freely moving rat was used to determine the effect of pyrithiamine‐induced thiamine deficiency on the levels of amino acids in the brain. Studies were carried out on (a) presymptomatic animals immediately before the development of behavioral changes and (b) acute symptomatic animals within 6 h following loss of righting reflexes. This latter stage precedes the appearance of histological lesions. The results were compared with pair‐fed controls. Dialysis probes were implanted in one vulnerable structure [ventral posterior medial thalamus (VPMT)] and one nonvulnerable area [frontal parietal cortex (FPC)] on the contralateral side. In VPMT of acute symptomatic animals, the glutamate concentration was significantly increased (3.37 ± 0.64 μM; p < 0.005) compared with control values (0.93 ± 0.09 μM), whereas in FPC no change in glutamate content was evident. These results suggest that glutamate plays a significant role in the development of central thiamine deficiency lesions. The absence of any increase in glutamate levels in the nonvulnerable FPC suggests that a glutamate‐mediated excitotoxic mechanism may be responsible for the selective cerebral vulnerability in thiamine deficiency.
Aluminum, Iron, and Zinc Ions Promote Aggregation of Physiological Concentrations of β‐Amyloid Peptide
Authors: Mantyh Patrick W. , Ghilardi Joseph R. , Rogers Scott , DeMaster Eric , Allen Clark J. , Stimson Evelyn R. , Maggio John E.
Published: October 2006

Abstract: A major pathological feature of Alzheimer's disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide βA4, a 40‐mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human βA4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10‐10M). In physiological buffers, 10‐3M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of βA4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100–1,000‐fold). The aggregation of βA4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of βA4.
Metabolism of [U‐13C]Glutamate in Astrocytes Studied by 13C NMR Spectroscopy: Incorporation of More Label into Lactate than into Glutamine Demonstrates the Importance of the Tricarboxylic Acid Cycle
Authors: Sonnewald U. , Westergaard N. , Petersen S. B. , Unsgård G. , Schousboe A.
Published: October 2006

Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U‐13C]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C‐labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C‐3 position and two doublets in the C‐2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton‐decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C‐3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C‐4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C‐3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.
Evidence for the Existence of Differential O‐Glycosylated α5‐Subunits of the γ‐Aminobutyric AcidA Receptor in the Rat Brain
Authors: Sieghart Werner , Item Chike , Buchstaller Andrea , Fuchs Karoline , Höger Harald , Adamiker Dieter
Published: October 2006

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N‐ or C‐terminal part of the γ‐aminobutyric acidA (GABAA) receptor α5‐subunit. These anti‐peptide α5(2–10) or anti‐peptide α5(427–433) antibodies reacted specifically with GABAA receptors purified from the brains of 5–10‐day‐old rats in an enzyme‐linked immunosorbent assay and were able to dose‐dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N‐Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O‐Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti‐peptide α5(2–10) and the anti‐peptide α5(427–433) antibody. These results indicate the existence of at least two different α5‐sub‐units of the GABAA receptor that differ in their carbohydrate content. In contrast to other α‐ or β‐subunits of GABAA receptors so far investigated, at least one of these two α5‐subunits contains O‐linked carbohydrates.
Effects of Graded Levels of Tissue Oxygen Pressure on Dopamine Metabolism in the Striatum of Newborn Piglets
Authors: Pastuszko Anna , Saadat‐Lajevardi Nasser , Chen Jie , Tammela Outi , Wilson David F. , Delivoria‐Papadopoulos Maria
Published: October 2006

Abstract: The effect of graded levels of tissue hypoxia on the extracellular levels of dopamine, 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid has been monitored in vivo by microdialysis. Reproducible levels of decreased oxygen in the brain were obtained by increasing the rate of ventilation from the control value of 25/min to as high as 95/min. With increasing ventilatory rate, the oxygen pressure in the cortex decreased from ∼40 torr to 16 torr. As the oxygen pressure decreased stepwise from 40 to 27, 22, and 16 torr, the dopamine levels in the extracellular medium rose by 70, 90, and 150%, respectively, returning to baseline within a few minutes of return to control ventilation rates. Levels of the catabolic products 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid decreased with decreasing tissue oxygen. Unlike the dopamine levels, these catabolite levels continued to decrease through 30 min of recovery (to 50% of control), returning to baseline only after recovery periods of 1–2 h. These data suggest that hypoxia induces long‐term alterations in the neurotransmitter turnover. The marked effects of mild tissue hypoxia (decrease of oxygen from 40 torr to 26 torr) on both the extracellular dopamine concentration and dopamine metabolism indicate that the metabolic consequences of decreased tissue oxygen pressure extend to higher values than generally appreciated.
Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease
Authors: Lange K. W. , Wells F. R. , Jenner P. , Marsden C. D.
Published: October 2006

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [3H]quinuclidinyl benzilate, for muscarinic M1 receptors with [3H]pirenzepine, for muscarinic M2 receptors with [3H]oxotremorine‐M, and for nicotinic receptors with (–)‐[3H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [3H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [3H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients Bmax values for specific [3H]oxotremorine‐M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)‐[3H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [3H]oxotremorine‐M and (–)‐[3H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [3H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.
On the Production and Disposition of Quinolinic Acid in Rat Brain and Liver Slices
Authors: Speciale Carmela , Schwarcz Robert
Published: October 2006

Abstract: The de novo production and subsequent disposition of the endogenous excitotoxin quinolinic acid (QUIN) was investigated in vitro in tissue slices from rat brain and liver. Incubation of tissue with QUIN's immediate bioprecursor 3‐hydroxyanthranilic acid (3‐HANA) in oxygenated Krebs‐Ringer buffer yielded measurable amounts of QUIN both in the tissue and in the incubation medium. Saturation was reached between 16 and 64 μM 3‐HANA (166 pmol of QUIN formed per milligram of protein after a 60‐min incubation with 64 μM 3‐HANA). In the brain, more QUIN was recovered from the tissue than from the incubation medium at all time points examined (5 min to 4 h). In contrast, the tissue‐to‐medium ratio for QUIN in parallel experiments with hepatic slices was ≪ 1. The disposition of newly synthesized QUIN was further elaborated in tissue slices that had been preincubated for 60 min with 64 μM 3‐HANA. Subsequent incubation of brain tissue in fresh buffer revealed a steady but relatively slow efflux of QUIN from the cellular compartment, with >30% remaining in the tissue after a 90‐min incubation. Analogous experiments with liver slices showed that >93% of newly synthesized QUIN had entered the extracellular compartment within 30 min. Striatal and nigral slices obtained 7 days after an intrastriatal ibotenic acid injection showed severalfold increases in QUIN production compared with control tissues, in all likelihood due to astrogliosis and associated large increases in 3‐hydroxyanthranilic acid oxygenase activity. In addition, the apparent tissue‐to‐medium ratio was markedly reduced in striatal slices from lesioned animals. Taken together, these data indicate that both brain and liver cells have a rather limited capacity to retain QUIN, and that 3‐hydroxyanthranilic acid oxygenase activity is a critical determinant controlling extracellular QUIN concentrations in both organs. Changes in the activity of QUIN's biosynthetic enzyme in the brain can therefore be expected to influence the possible function of QUIN as an endogenous agonist at the N‐methyl‐D‐aspartate receptor in health and disease.
Heterogeneous Na+ Sensitivity of Na+,K+‐ATPase Isoenzymes in Whole Brain Membranes
Authors: Gerbi Alain , Debray Marcel , Maixent Jean‐Michel , Chanez Claude , Bourre Jean‐Marie
Published: October 2006

Abstract: The Na+ sensitivity of whole brain membrane Na+,K+‐ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10−5 and 10−7M), we were able to discriminate Na+ sensitivity of Na+, K+‐ATPase isoenzymes using nonlinear regression. The ouabain low‐affinity isoform, α1, exhibited high Na+ sensitivity [Ka of 3.88 ± 0.25 mMNa+ and a Hill coefficient (n) of 1.98 ± 0.13]; the ouabain high‐affinity isoform, α2, had two Na+ sensitivities, a high (Ka of 4.98 ± 0.2 mM Na+ and n of 1.34 ± 0.10) and a low (Ka of 28 ± 0.5 mM Na+ and an n of 1.92 ± 0.18) Na+ sensitivity activated above a thresh old (22 ± 0.3 mM Na+); and the ouabain very‐high‐affinity isoform, α3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.
Endothelins Stimulate c‐fos and Nerve Growth Factor Expression in Astrocytes and Astrocytoma
Authors: Ladenheim R. G. , Lacroix I. , Foignant‐Chaverot N. , Strosberg A. D. , Couraud P. O.
Published: October 2006

Abstract: Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c‐fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c‐fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c‐fos and nerve growth factor, and (b) phorbol esterevoked down‐regulation of protein kinase C completely abolishes the c‐fos induction by endothelin, but not that by the β‐adrenergic agonist isoproterenol, a known activator of the cyclic AMP‐dependent pathway. Our results support the hypothesis that c‐fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.