bluuebunny/crossref_metadata_2025 huggingface dataset:
Microheterogeneity of Micro tubule‐Associated τ Proteins Is Due to Differences in Phosphorylation
Authors: Butler M. , Shelanski M. L.
Published: October 2006

Abstract We have studied the heterogeneity of the microtu‐bule‐associated τ proteins using τ‐specific antibodies and two‐dimensional electrophoresis. Both monoclonal and polyclonal antibodies to τ proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sul‐fate (SDS)‐polyacrylamide gels, with apparent molecular weights between56,000 and66,000. Immunoblots of cow brain microtubules separated on two‐dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS‐gel electrophoresis in the second, reveal that >30 isoforms of τ exist. The T proteins vary in pI from 6.5 to 8.5, with the higher‐molecular‐weight forms being more acidic. The microheterogeneity of τ is not induced by cycling of microtubules, because two‐dimensional immunoblots of τ from total brain are almost identical to those of τ from cycled tubules. Adult rat brain τ, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does τ from 7‐day‐old rats, which appears as only one band on SDS gels. After dephos‐phorylation of cow brain τ with alkaline phosphatase, the highest‐molecular‐weight band disappears on SDS gels. On two‐dimensional gels, the number of τ variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with T labeled in vivo with 32PO4 also indicate that the more acidic τ proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of τ proteins exists at all ages and is due, at least in part, to differences in the state of phosphorylation of τ isoforms.
High‐ and Low‐Affinity Binding of [3H]Imipramine in Rat Hypothalamus
Authors: Moret C. , Briley M.
Published: October 2006

Abstract In the rat hypothalamus [3H]imipramine binding is inhibited by tricyclic and nontricyclic antidepressant drugs in a complex manner, with biphasic curves and Hill coefficients <1.0. 5‐Hydroxytryptamine (serotonin) inhibited with high affinity a decreasing proportion of the [3H]‐imipramine binding sites as the [3H]imipramine concentration was raised. In the absence of sodium ions, IC50 values for the inhibition by tricyclic and nontricyclic antidepressants were increased by approximately 1,000‐fold, and the inhibition curves became classically monophasic with Hill coefficients close to 1.0. These data are interpreted as suggesting that [3H]imipramine binds to two independent sites in the rat hypothalamus. One site is sodium‐dependent with a high affinity for the drugs tested; the other is sodium‐independent and has a low affinity for these drugs.
2‐Pyrrolidinone in Human Cerebrospinal Fluid: A Major Constituent of Total γ‐Aminobutyric Acid
Authors: Haegele K. D. , Schwartz J.‐J. , Schoun J. , Schmitt A. H. , Schechter P. J.
Published: October 2006

Abstract: 2‐Pyrrolidinone, the lactam of γ‐aminobutyric acid (GABA), is identified as the major constituent of total GABA in human CSF. Structural elucidation was done by mass spectrometry. In lumbar CSF of four patients, 2‐pyr‐rolidinone represented about 54% of GABA found after acid hydrolysis, thus accounting for essentially all of the hitherto unknown GABA fraction in CSF.
α2‐Adrenergic Regulation of Arylalkylamine N‐Acetyltransferase in Organ‐Cultured Chick Pineal Gland: Characterization with Agonists and Modulation of Experimentally Stimulated Enzyme Activity
Authors: Voisin Pierre , Martin Chantal , Collin Jean‐Pierre
Published: October 2006

Abstract: In the chicken pineal gland, norepinephrine, released at sympathetic nerve endings, plays a role in synchronizing the circadian rhythm of melatonin synthesis. This effect appears to be exerted via an adrenergic inhibition of arylalkylamine N‐acetyltransferase, the melatonin rhythm‐generating enzyme. The present study indicates that the nighttime peak of N‐acetyltransferase activity developed by organ‐cultured chick pineal glands is inhibited by adrenergic agonists with a potency order characterizing α2‐adrenergic receptors: UK 14, 304 < clonidine < α‐methylnorepinephrine = epinephrine < cirazoline < phenylephrine < isoproterenol. The mechanism of this α2‐adrenergic response was further analyzed in organ cultures, by studying the ability of clonidine to block the cyclic AMP‐dependent and the depolarization‐dependent stimulations or N‐acetyltransferase activity. Clonidine prevented the rise in N‐acetyltransferase activity evoked by the adenylate cy‐clase activators forskolin and cholera toxin or by the phosphodiesterase inhibitor Ro 20,1724. The stimulatory effect of dibutyryl cyclic AMP was also blocked by clonidine. Activation of pineal α2‐adrenergic receptors effectively prevented the stimulation of N‐acetyltransferase by depolarizing concentrations of KC1. The possibility that the α2‐adrenergic effect might be exerted at a step distal to cyclic AMP production is discussed.
Progressive Deficit of Retrograde Axonal Transport Is Associated with the Pathogenesis of Di‐n‐Butyl Dichlorvos Axonopathy
Authors: Moretto Angelo , Lotti Marcello , Sabri Mohammad I. , Spencer Peter S.
Published: October 2006

Abstract: The induction of central‐peripheral distal axonopathy in hens singly dosed with some organophosphorus (OP) compounds, such as di‐n‐butyl‐2,2‐dichlorovinyl phosphate (DBDCVP), requires greater than 80% organophosphorylation and subsequent intramolecular rearrangement (“aging”) of a protein [neuropathy target esterase (NTE)] in the axon. Suprathreshold biochemical reaction, 24 h after dosing with DBDCVP (0.75–1.00 mg/kg s.c.), is shown to be associated with progressive decrement of retrograde axonal transport in sensory and motor fibers. The maximum transport deficit (about 70% reduction) is reached 7 days after DBDCVP, prior to the appearance of axonal degeneration and the onset of clinical signs of neuropathy (day 10–11). By contrast, phenylmethylsulfonyl fluoride (30 mg/kg s.c.), an agent that prevents the development of OP neuropathy by inhibiting NTE without the “aging” reaction, had no effect on axon transport, nerve fiber integrity, or clinical status and, when administered prior to a neurotoxic dose of DBDCVP (1.00 mg/kg s.c.), prevented DBDCVP effects. Paraoxon (0.2 mg/kg s.c.) neither inhibited NTE nor caused deficits in retrograde transport or neuropathy. Taken in concert, these studies demonstrate that induced deficits in retrograde transport are associated with the pathogenesis of OP‐induced nerve‐fiber degeneration and the threshold‐initiating mechanism thereof.
Complex Carbohydrate Composition of Large Dense‐Cored Vesicles from Sympathetic Nerve
Authors: Margolis R. U. , W. Ledeen R. , Sbaschnig‐Agler M. , C. Byrne M. , Klein R. L. , Douglas B. H. , Margolis R. K.
Published: October 2006

Highly purified noradrenergic, large, dense‐cored vesicles were isolated from bovine sympathetic nerve endings by sucrose‐D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 μmol/100 mg lipid‐free dry weight, respectivly. values which are similar to those previously found in vine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N‐acetylgalactosamine‐containing O‐glycosidically‐linked oligosaccharides (present in the chromo‐granins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N‐3H‐acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approxi‐mtely two‐thirds of the oligosaccharides were of the triand tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high‐mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 μmol N‐acetylglucosamine/100 mg lipid‐free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 μmol/100 mg lipid‐free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N‐acetyl‐to N‐glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin‐layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series.
Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia
Authors: Hagberg H. , Andersson P. , Lacarewicz J. , Jacobson I. , Butcher S. , Sandberg M.
Published: October 2006

Abstract: Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three‐vessel occlusion combined with hypotension (50 mm Hg) in male Sprague‐Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nudeotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 μM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.
Dietary Folate and Biogenic Amines in the CNS
Authors: Butler Ian J. , Rothenberg Sheldon P.
Published: October 2006

Abstract: Abnormal biogenic amine biosynthesis has been observed in humans and animals with endogenous and exogenous disturbances in folate metabolism. In an attempt to study this interaction biochemically, rats were depleted or repleted with folate for 10 weeks. Folate levels in depleted animals in serum and CSF correlated with stores in liver and brain, respectively. In depleted or repleted animals, there was no significant effect on biogenic amine metabolism in the CNS, as determined by quantitation of biogenic amines in brain and their respective metabolites in brain and CSF. These results are contrary to studies by other investigators. We suspect, however, that specific genetic defects in folate metabolism do result in impaired biogenic amine metabolism and probably at the level of disturbed biopterin cofactor functions.
Aluminum Inhibits the Fast Phase of Voltage‐Dependent Calcium Influx into Synaptosomes
Authors: Koenig Michael L. , Jope Richard S.
Published: October 2006

Abstract: : Aluminum has been shown to have neurotoxic effects, but the mechanisms by which it acts are not well understood. Because it has been reported that aluminum can interact with Ca2+‐binding sites, the possibility that aluminum might interfere with Ca2+ influx into synaptosomes was examined. At concentrations of 50 μM and greater, aluminum significantly inhibited the fast phase (0–1 s) of the voltage‐dependent uptake of 45Ca2+ into synaptosomes. Higher concentrations of aluminum also reduced 45Ca2+ uptake measured at 1 s in nondepolarizing media and inhibited the slow phase of 45Ca2+ uptake into synaptosomes whether they were suspended in either low K or high K media. The possibility that aluminum competitively inhibits the fast phase of Ca2+ influx was investigated. Aluminum (250 μM) increased the apparent KT (concentration of Ca2+ at which Ca2+ transport is half maximal) for 45Ca2+ of fast phase voltage‐dependent channels and slightly decreased the maximal influx (Jmax). These effects are characteristic of a mixed type inhibitor, and the apparent Kt for A13+ is estimated to be 0.64 mM. The interaction of aluminum with the fast phase of voltage‐dependent calcium influx may disrupt intraneuronal calcium homeostasis and may also represent a means by which aluminum could accumulate intraneuronally.
Mechanism of Arachidonic Acid Liberation During Ischemia in Gerbil Cerebral Cortex
Authors: Abe Koji , Kogure Kyuya , Yamamoto Hirotaka , Imazawa Masaoki , Miyamoto Kenji
Published: October 2006

Abstract: Once brain ischemia was induced in the gerbil cerebral fronto‐parietal cortex, serial changes occurred in energy metabolites and various lipids. The amounts of inosi‐tol‐containing phospholipids began to decrease immediately after energy failure, followed by an increase in the amount of 1,2‐diacylglycerol with a subsequent liberation of arachidonic acid and other free fatty acids. The fatty acid compositions of inositol‐containing phospholipids, of 1,2‐diacylglycerols produced by ischemia, and of free fatty acids liberated during ischemia were quite similar. The amount of stearic acid liberated was much larger than that of arachidonic acid between 30 s and 1 min of ischemia. On the other hand, there was no significant decrease in the amount of the other phospholipids except for phosphatidic acid. Furthermore, there was also no change in the fatty acid composition of phosphatidylcholine or phosphatidylethanol‐amine throughout 15 min of ischemia. The amount of cytidine‐monophosphate reached a peak (36.7 nmol/g wet wt) at 2 min of ischemia. These results indicated that arachidonic acid was predominantly liberated from inositol‐containing phospholipids by phospholipase C, and by the diglyceride lipase and monoglyceride lipase system rather than from phosphatidylcholine or phosphatidylethanolam‐ine by phospholipase A2 or plasmalogenase or choline phos‐photransferase during the early period of ischemia.
Acrylamide Neuropathy: Changes in the Composition of Proteins of Fast Axonal Transport Resemble Those Observed in Regenerating Axons
Authors: Bisby M. A. , Redshaw J. D.
Published: October 2006

Abstract: Proteins conveyed by fast axonal transport along sensory and motor axons of rat sciatic nerve were labelled with l‐[35S]methionine and characterized by one‐ and two‐dimensional electrophoresis on polyacrylamide gels, followed by fluorography. Nerves from normal or bis‐acrylamide‐treated animals were compared with nerves from acrylamide‐treated animals and nerves regenerating after a crush axotomy. In both sensory and motor axons significant changes in the pattern of labelled bands on one‐dimensional gels occurred after 10 days of acrylamide treatment (50 mg/ kg daily, i.p.). These changes resembled those seen in regenerating axons, but were less pronounced. No changes were detectable after shorter periods of treatment, even though the onset of the neuropathy, assessed by a behavioral test, occurred on days 4–6 of treatment. Two‐dimensional separations of the labelled proteins revealed increased labeling of growth‐associated protein 43 in acrylamide‐treated animals, but again this was less pronounced than in regenerating nerves. Acrylamide treatment induces changes in composition of fast‐transported protein that are qualitatively similar to those seen after axotomy. Since these changes are not detectable until the neuropathy is advanced, it is unlikely that they are causative factors. Instead, they are most likely a result of the cell body reaction previously observed in acrylamide intoxication, a reaction that resembles that produced by axotomy.
Lipid Requirement for μ Opioid Receptor Binding
Authors: Hasegawa Jun‐Ichi , Loh Horace H. , Lee Nancy M.
Published: October 2006

Abstract: We have previously shown that a partially purified μ opioid receptor from bovine brain requires lipids to exhibit full binding activity. In the present report, we have determined the specificity of this lipid requirement. Lipids active in this regard were found always to contain an acidic head group and a fatty acid with two or more double bonds. Free, polyunsaturated fatty acids were also able to confer high binding activity on the partially purified opioid receptor. The possible roles lipids play in opioid binding are discussed in light of these data.
Isolation and Partial Characterization of a 56,000‐Dalton Phosphoprotein Phosphatase from the Blood‐Brain Barrier
Authors: Weber Marion , Mehler Michael , Wollny Eric
Published: October 2006

Abstract: A 56,000‐dalton protein with inherent phospho‐protein phosphatase activity was isolated from porcine brain capillaries. The enzyme is not activated by divalent metal ions but strongly inhibited by zinc ions. As phosphatase inhibitor 2 readily inhibits the enzymatic activity, the protein can be classified as a type I phosphatase. The protein is stable toward protease treatment. Limited digestion with trypsin does not convert the enzyme into an active form of lower molecular weight. The physical and enzymatical properties of the phosphatase exhibit considerable similarities to those of another 56,000‐dalton phosphatase derived from rabbit reticulocytes.
α2‐Macroglobulin Synthesis in an Astrocyte Subpopulation
Authors: Gebicke‐Haerter Peter Joachim , Bauer Joachim , Brenner Angelika , Gerok Wolfgang
Published: October 2006

Abstract: The proteinase inhibitor α2‐macroglobulin (α2‐M) is an acute phase protein in the adult rat. During inflammatory events, it is synthesized in the liver and secreted into the bloodstream to remove proteases that are released on injury. Recently, its occurrence in fetal rat brain has been reported. Its cellular origin and biological function in the developing brain, however, remained obscure. In this article, it is shown that astroglial cells cultured from newborn rat brain synthesize and secrete α2‐M. Its synthesis markedly increases with time in culture. Immunocyto‐chemical studies reveal that only a subpopulation of astrocytes is α2‐M positive. α2‐M synthesis in the developing brain by neuroectoderm‐derived cells asks for a broader definition of its function in the body. Since interactions of proteases and protease inhibitors appear to play a crucial role in cell migration and neurite outgrowth, α2‐M expression in astrocytes is discussed not only in relation to its potential role in the acute phase response to injury in the adult brain but also in regard to its possible involvement in brain development.
Identification as Synapsin of a Synaptosomal Protein Immunoreacting with Anti‐Myelin Basic Protein Antiserum
Authors: Pedraza Liliana T. , Roth German A. , Cumar Federico A.
Published: October 2006

Abstract: Rat brain proteins able to react with anti‐myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sul‐fate‐polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high‐molecular‐weight proteins are relatively concentrated in a syn‐aptosome‐enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin la and Ib. A purified synapsin preparation analyzed by immunoblot after two‐dimensional gel electrophoresis also reacted with anti‐myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein‐conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross‐reacting with extra‐myelin proteins in the process of experimental allergic encephalomyelitis is considered.
Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ‐Aminobutyric Acid Studied by Intracerebral Microdialysis
Authors: Globus Mordecai Y.‐T. , Busto Raul , Dietrich W. Dalton , Martinez Elena , Valdes Isabel , Ginsberg Myron D.
Published: October 2006

Abstract: We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (1CBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or γ‐aminobutyric acid (GABA). Control and SN‐lesioned rats were subjected to 20 min of forebrain ischemia by four‐vessel occlusion combined with systemic hypotension. Levels of 1CBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi‐ and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An ∼ 500‐fold increase in DA content, a 7‐fold increase in Giu content, and a 5‐fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN‐lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.
Mechanistic Positron Emission Tomography Studies: Demonstration of a Deuterium Isotope Effect in the Monoamine Oxidase‐Catalyzed Binding of [11C]l‐Deprenyl in Living Baboon Brain
Authors: Fowler J. S. , Wolf A. P. , MacGregor R. R. , Dewey S. L. , Logan J. , Schlyer D. J. , Langstrom B.
Published: October 2006

Abstract: The application of positron emission tomography (PET) to the study of biochemical transformations in the living human and animal body requires the development of highly selective radiotracers whose concentrations in tissue provide a record of a discrete metabolic process. l‐N‐[11C‐methyl]Dtprtnyl ([11C]l‐deprenyl), a suicide inactivator of monoamine oxidase (MAO) type B, has been developed as a radiotracer for mapping MAO B in the living human and animal brain. In this investigation, [11C]l‐deprenyl (1) and [11C]l‐deprenyl‐α,α‐2H2 (2) have been compared in three different baboons by PET measurement of carbon‐11 uptake and retention in the brain and the measurement of the amount of unchanged tracer in the arterial plasma over a 90‐min time interval. For one baboon, N‐[11C‐methyl‐2H3]h‐de‐prenyl (3) was also studied. Kinetic parameters calculated using a three‐compartment model revealed a deuterium isotope effect of 3.8 ± 1.1. Comparison of the two tracers (1 and 2) in mouse brain demonstrated that deuterium substitution significantly reduced the amount of radioactivity bound to protein. HPLC and GLC analysis of the soluble radioactivity in mouse brain after injection of [11C]l‐deprenyl showed the presence of [11C]methamphetamine as a major product along with unidentified labeled products. Sodium dodecyl sulfate‐polyacrylamide electrophoresis with carbon‐14‐labeled l‐deprenyl showed that a protein of molecular weight 58,000 was labeled. These results establish that MAO‐catalyzed cleavage of the α carbon‐hydrogen bond on the propargyl group is the rate limiting (or a major rate contributing) step in the retention of carbon‐11 in brain and that the in vivo detection of labeled products in brain after the injection of [11C]L‐deprenyl provides a record of MAO activity.
Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules
Authors: Rodriguez del Castillo A. , Torres M. , Delicado E. G. , Miras‐Portugal M. T.
Published: October 2006

Abstract: Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 ± 0.05 μmol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (∼6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 μM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 ± 0.5 and 13 ± 1 μM for Ap4A and Ap5A, respectively.
Effects of Neurotransmitters on Astrocyte Glycogen Stores In Vitro
Authors: Cambray‐Deakin Martin , Pearce Brian , Morrow Christine , Murphy Sean
Published: October 2006

Abstract: We have used receptor binding assays to determine the presence of three neurotransmitter receptors in a crude membrane fraction derived from neonatal rat cortical astrocyte cultures and subsequently determined the effects of transmitter receptor activation on astrocyte glycogen content in vitro. β‐Adrenergic (KD= 88 pM; Bmax= 51 fmol/mg of protein), serotonin (KD= 70 nM; Bmax= 44 pmol/mg of protein), and muscarinic cholinergic receptors (KD= 79 pM; Bmax= 44 fmol/mg of protein) were found to be present on astrocyte membranes using [3H]dihydroalprenolol, [3H]serotonin, and [3H]quinuclidinyl benzilate, respectively, as ligands. Astrocyte cultures exposed to noradrenaline but not specific α‐ and β‐receptor agonists contained 33% less glycogen than controls. Neither serotonin nor carbachol caused alterations in astrocyte glycogen content under normal conditions. Reserpine‐treated cultures, however, responded to serotonin with a 28% decrease in glycogen content and contained higher levels of glycogen than non‐reserpine‐treated controls (a 55% increase). These results show that both noradrenaline and serotonin can evoke astrocyte glycogenolysis and that noradrenergic control of glycogen metabolism is probably exerted through both α‐ and /S‐receptors. Neurotransmitter control of astrocyte glycogen turnover may represent a form of neuron‐astrocyte signalling in addition to that provided by changes in external potassium concentration.
Brain Quinolinic Acid in Huntington's Disease
Authors: Reynolds Gavin P. , Pearson Sally J. , Halket John , Sandier Merton
Published: October 2006

Abstract: Concentrations of the endogenous neurotoxic tryptophan metabolite, quinolinic acid (QA), were measured in postmortem brain tissue obtained from patients with Huntington's disease (HD) and matched controls, using a gas chromatography/mass spectrometry method. There was no significant difference in either the putamen or the frontal cortex between the HD and control groups. These results do not support the hypothesis that increased QA is responsible for neuronal degeneration in HD.
Regulation of γ‐Aminobutyric Acid/Barbiturate Receptor‐Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals
Authors: Schwartz Rochelle D. , Skolnick Phil , Paul Steven M.
Published: October 2006

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the γ‐aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001–0.02 U/mg protein) preincubation decreased pentobarbital‐induced 36Cl‐ efflux and musci‐mol‐induced 36Cl‐ uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital‐induced 36Cl‐ efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital‐induced 36Cl‐ efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbitu‐rate receptor function in brain.
Differentiation of Dopamine Overflow and Uptake Processes in the Extracellular Fluid of the Rat Caudate Nucleus with Fast‐Scan In Vivo Voltammetry
Authors: May Leslie J. , Kuhr Werner G. , Wightman R. Mark
Published: October 2006

Abstract: Stimulated overflow of dopamine (DA) into the extracellular fluid of the rat caudate nucleus was measured with fast‐scan cyclic voltammetry. DA concentrations were sampled in less than 10 ms at 100‐ms intervals with a Na‐fion‐coated, carbon‐fiber microelectrode. Overflow of DA was induced by electrical stimulation of the medial fore‐brain bundle with 300–μA pulses of various duration and frequency. Stimulated overflow was measured as a function of stimulus duration before and after administration of benztropine, bupropion, and amphetamine. These results were correlated with simulated curves based on a simple uptake/overflow model. The observed overflow was assumed to be a function of [DA]P, the concentration of DA which overflows per stimulus pulse, and the kinetics of cellular uptake of DA. Correlation of experimental with simulated results was obtained at the 95% confidence limit for the duration studies; however, it was not possible to distinguish between the effects of pharmacological agents on uptake and overflow. In contrast, modulation of stimulus frequency did permit such distinction. Simulations of an increase in [DA]P fit results following dihydroxyphenyl‐alanine methyl ester at 95% confidence limits, whereas an equivalent change in the apparent Km did not fit. An increase in the apparent value of Km correlated with results obtained at different frequencies following nomifensine and bupropion administration at the 95% confidence limit, whereas an equivalent increase in [DA]P did not fit. The effects of GBR 12909 best correlated with an increase in the DA available for overflow.
Relationship of Dihydropyridine Binding Sites with Calcium‐Dependent Neurotransmitter Release in Synaptosomes
Authors: Massieu Lourdes , Tapia Ricardo
Published: October 2006

Abstract: In the present work, we have studied the effect of ruthenium red (RuR), La3+, and 4‐aminopyridine (4‐AP) on the specific binding of (+)‐[3H]PN200–110 to synaptosomes, as well as the effect of nitrendipine, nifedipine, and BAY K. 8644 on γ‐[3H]aminobutyric acid ([3H]GABA) release induced by potassium depolarization and by 4‐AP in synaptosomes. Scatchard plots indicated that neither RuR nor 4‐AP modifies the KD and Bmax of [3H]PN200–110 specific binding, whereas La3+ decreased the Bmax by about 25%; when the effect of the drugs on the total binding of PN200–110 was studied, a similar inhibition by La3+ was found. The calcium antagonists, nitrendipine and nifedipine, did not affect at all the potassium‐stimulated release of [3H]GABA nor its release induced by 4‐AP. The calcium agonist BAY K 8644 failed to affect both the spontaneous and the potassium‐stimulated GABA release. Our results suggest that the binding sites of dihydropyridines in presynaptic membranes are not related to the calcium channels involved in neurotransmitter release with which RuR, La3+, and 4‐AP interact.
Some Characteristics of Threonine Transport Across the Blood‐Brain Barrier of the Rat
Authors: Tovar Armando , Tews Jean K. , Torres Nimbe , Harper Alfred E.
Published: October 2006

Abstract: Threonine entry into brain is altered by diet‐induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood‐brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2‐amino‐n‐butyrate (AABA) were stronger competitors than norleucine. 2‐Aminobicyclo[2.2.1]heptane‐2‐carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2–(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p < 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium‐dependent ASC system.
Distribution of Calmodulin‐ and Cyclic AMP‐Stimulated Protein Kinases in Synaptosomes
Authors: Dunkley P. R. , Jarvie P. E. , Rostas J. A. P.
Published: October 2006

The subcellular location of calmodulin‐ and cyclic AMP‐stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [γ‐32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin‐or cyclic AMP‐stimulated protein phosphorylation. When assessed by calmodulin‐stimulated autophosphorylation of the α subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin‐stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP‐stimulated phosphorylation of endogenous substrates, no cyclic AMP‐stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP‐stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin‐ and cyclic AMP‐stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.
H3‐Receptors Control Histamine Release in Human Brain
Authors: Arrang Jean‐Michel , Devaux Bertrand , Chodkiewicz Jean‐Paul , Schwartz Jean‐Charles
Published: October 2006

The regulation of histamine release was studied on slices prepared from pieces of human cerebral cortex removed during neurosurgery and labeled with l‐[3H]‐histidine. Depolarization by increased extracellular K+ concentration induced [3H]histamine release, although to a lesser extent than from rat brain slices. Exogenous histamine reduced by up to 60% the K+‐evoked release, with an EC50 of 3.5 ± 0.5 × 10−‐8M. The H3‐receptor antagonists impromidine and thioperamide reversed the histamine effect in an apparently competitive manner and enhanced the K+‐evoked release, indicating a participation of endogenous histamine in the release control process. The potencies of histamine and the H3‐receptor antagonists were similar to those of these agents at presynaptic H3‐autoreceptors controlling [3H]histamine release from rat brain slices. It is concluded that H3‐receptors control histamine release in the human brain.
Brain Levels of NADH and NAD+ Under Hypoxic and Hypoglycaemic Conditions In Vitro
Authors: Garofalo O. , Cox D. W. G. , Bachelard H. S.
Published: October 2006

The effects of hypoxia and hypoglycaemia on the redox state in vitro have been studied. NADH and NAD+ were extracted simultaneously from superfused cerebral cortex slices and assayed by bioluminescence. The results show a nonsignificant increase in NADH and the redox ratio in „mild hypoxia,” whereas „severe hypoxia” produced an increase of over 200% in NADH and in the NADH/NAD+ ratio. When the glucose in the incubation medium was reduced from its control value of 10 mM to 0.5 mM, significant decreases in NADH and the redox ratio to 60% of control value were observed. Further decreasing the glucose to 0.2 mM gave lower levels of NADH and the redox ratio (40% of control). The effects on the redox state of alternative substrates to glucose were also tested. Re placement of glucose by 10 mM pyruvate decreased the NADH by 77% and the NADH/NAD+ ratio by 79%. Replacement of glucose with 10 mM lactate gave decreases of 70% and 71%, respectively, whereas in the presence of 15 mM 2‐deoxyglucose and 5 mM glucose, the NADH was decreased by 56% and the ratio by 50%. The results are discussed in relation to levels of creatine phosphate and ATP, as well as evoked action potentials, observed from parallel studies.
Low [3H]Cytochalasin B Binding in the Cerebral Cortex of Newborn Rat
Authors: Morin Anne M. , Dwyer Barney E. , Fujikawa Denson G. , Wasterlain Claude G.
Published: October 2006

The concentrations of glucose transporter in the cerebral cortex and brainstem of neonatal (4–7 days old) and adult rats were measured using [3H]cytochalasin B binding. There was significantly lower binding in neonatal cortex (1.9 ± 0.7 pmol/mg protein) compared to adult (8.9 ± 2.5 pmol/mg protein). Scatchard analysis indicates this difference is due to a lower Bmax (neonate, 9.7 pmol/mg protein; adult, 18.6 ± 1.3 pmol/mg protein). Measurement of [3H]cytochalasin B binding in microvessels prepared from cortex of adult (28.1 ± 3.5 pmol/mg protein) and neonate (12.8 ± 1.9 pmol/mg protein) indicates a lower binding in the microvasculature of neonates, whereas no such difference was seen in the binding in microvessels prepared from adult and neonatal brainstem (adult, 11.8 ± 2.3 pmol/mg protein; neonate, 9.4 ± 2.7 pmol/mg protein). In both adult and neonate brain, there is an enrichment of glucose transporters in the microvasculature.
Membrane Interactions Are Altered in Myelin Isolated from Central and Peripheral Nervous System Tissues
Authors: Karthigasan Jeevajothie , Kirschner Daniel A.
Published: October 2006

Isolated myelin has been used for determinations of membrane surface charge density and topographical mapping of components in the membrane. To determine how similar such myelin is to myelin of intact tissue, we have used x‐ray diffraction to compare their intermembrane interactions. The interactions were monitored by measuring the myelin period in samples treated with distilled water, buffered saline at pH 4–9 and ionic strength 0.06–0.18, and saline containing HgCl2 or triethyl tin sulfate. Myelin was isolated from whole brains and sciatic nerves of mice by conventional methods involving sucrose gradient centrifugation and osmotic shock. Consistent with previous findings, electron microscopy showed that the multilamellar morphology, staining, and repeat periods of isolated myelin were essentially like those of intact myelin; however, the membrane stacks were less extensive than those in whole tissue. X‐ray diffraction revealed that isolated CNS myelin was like intact myelin in showing reversible compaction in acidic media and in distilled water. However, unlike the myelin in whole tissue, isolated CNS myelin did not swell in hypotonic or alkaline media, or in the presence of HgCl2‐saline or triethyl tin. The altered membrane interactions could result from an increase in adhesiveness of the apposed membrane surfaces. Reorganization of proteolipid protein and/or a reduction of surface charge could account for the change in surface properties of isolated CNS myelin. Isolated PNS myelin, like the membranes in whole tissue, showed both compaction and swelling; however, the membrane pairs were disordered in the swollen structure. This irregular membrane swelling could result from charge variation in the extracellular surfaces.
Phylogenetic Distribution of Peptides Related to Chromogranins A and B
Authors: Rieker Susanne , Fischer‐Colbrie Reiner , Eiden Lee , Winkler Hans
Published: October 2006

Abstract: The presence of chromogranin‐related peptides in a wide range of species was investigated by one‐ and two‐dimensional electrophoresis followed by immunoblotting. Antisera against bovine chromogranins A and B and the peptide WE‐14 (chromogranin A316–329) were used. Chromogranins were identified by their heat stability, by their electrophoretic behavior, and by immunological cross‐reaction with antisera. In all species investigated ranging from mammals to birds, amphibians, fish, and arthropods, chromogranin A‐ and B‐like proteins could be demonstrated. For all species, there was an immunological cross‐reaction with antisera against bovine chromogranins. The molecular sizes and isoelectric points of the chromogranins were similar in all species. The antiserum against WE‐14 cross‐reacted with pig, rat, and chicken chromogranins. It is concluded that the chromogranins A and B have a widespread phylogenetic distribution with a significant conservation of molecular size, isoelectric points, and immunological epitopes. This is consistent with the concept that these peptides have a specific function.
Participation of Lipoxygenase Products from Arachidonic Acid in the Pathogenesis of Cerebral Vasospasm
Authors: Watanabe Takashi , Asano Takao , Shimizu Takao , Seyama Yousuke , Takakura Kintomo
Published: October 2006

Abstract: To examine the possible involvement of lipoxygenase products from arachidonic acid in the pathogenesis of delayed vasospasm after subarachnoid hemorrhage (SAH), we measured the contents of hydroxyeicosatetraenoic acids (HETEs) in the subarachnoid clot, the cerebrospinal fluid, and the basilar artery, using the canine “two‐hemorrhage” model. Lipoxygenase activity in the subarachnoid clot and the basilar artery was measured, ex vivo, using samples obtained 7 days after SAH. For a quantitative analysis of HETEs, each sample was homogenized with either ice‐cold saline or methanol. The lipid extract was then submitted to reverse‐phase HPLC. The identity of each HETE was further confirmed using straight‐phase HPLC and gas chromatography‐mass spectrometry. When the basilar artery was homogenized with ice‐cold saline, a significant increase in the 5‐HETE content was observed on SAH day 8. However, when the artery was homogenized with methanol, HETEs were not detected. In the case of incubation in the presence of arachidonic acid and calcium ionophore A23187, the 5‐lipoxygenase activity was remarkably increased in the basilar artery exposed to SAH, compared to that of normal dogs. The subarachnoid clot contained a significant amount of 12‐HETE (average 1.8 nmol/g wet weight) from day 2 to day 8. The administration of 1,2‐bis(nicotinamido)propane significantly ameliorated vasospasm in the two‐hemorrhage model, simultaneously inhibiting the 5‐lipoxygenase activity of the basilar artery. Our observations show that the activities of 12‐ and 5‐lipoxygenases are significantly increased after SAH in the subarachnoid clot and the basilar artery, respectively. The generated hydroperoxides and/or leukotrienes may play some pertinent role in the occurrence of delayed vasospasm.
Blood‐Brain Barrier Transport of 1‐Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport
Authors: Aoyagi Masaki , Agranoff Bernard W. , Washburn Lee C. , Smith Quentin R.
Published: October 2006

Abstract: Regional transport of 1‐aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood‐brain barrier was studied in pentobarbital‐anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best‐fit values for the kinetic constants of the frontal cortex equaled 9.7 × 10‐−4μmol/s/g for Vmax, 0.054 μmol/ml for Km, and 1.0 × 10‐−4 ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by >85% by either l‐phenylalanine or 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one‐fifth that for the more commonly used homologue, I‐aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as l‐methionine, l‐isoleucine, and l‐tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.
A Glycine Site Associated with N‐Methyl‐d‐Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists
Authors: Kessler M. , Terramani T. , Lynch G. , Baudry M.
Published: October 2006

Abstract Membranes from rat telencephalon contain a single class of strychnine‐insensitive glycine sites. That these sites are associated with N‐methyl‐d‐aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N‐[1‐(2‐thienyl)cyclohexyl]piper‐idine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate‐type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.