bluuebunny/crossref_metadata_2025 huggingface dataset:
Gas Chromatographic‐Mass Spectrometric Determination of myo‐Inositol in Human Cerebrospinal Fluid
Authors: Swahn Carl‐Gunnar
Published: October 2006

Abstract: The concentration of free myo‐inositol in CSF was determined with a gas chromatographic‐mass spectrometric method using deuterated myo‐inositol as an internal standard after conversion to the hexa‐O‐acetyl derivative with acetic anhydride and pyridine. Twenty microliters of CSF is sufficient for the analysis which has a coefficient of variation of 9%. Identical analytical results were obtained on two different mass numbers. Schizophrenic patients were compared with healthy control persons. In addition, patients with rheumatoid arthritis or with neurological illnesses were studied. No consistent differences related to the illness could be found. The mean concentration of myo‐inositol was about 25 μg/ml. Treatment of schizophrenic patients with chlorpromazine or sulpiride had no significant effect on the concentration of myo‐inositol in CSF.
Calcium‐Stimulated Proteolysis in Myelin: Evidence for a Ca2+‐Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS
Authors: Banik Naren L. , McAlhaney Walter W. , Hogan Edward L.
Published: October 2006

Abstract: Incubation of myelin purified from rat spinal cord with CaCl2 (1–5 mM) in 10–50 mM Tris‐HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X‐100 (0.25–0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X‐100 enhanced degradation of both PLP and MBPs. The Ca2+‐activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E‐64c. CANP is active at pH 5.5–9.0, with the optimum at 7–8. The threshold of Ca2+ activation is approximately 100 μM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+ These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin‐resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin‐associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.
Morphine‐Induced Changes in Histamine Dynamics in Mouse Brain
Authors: Nishibori Masahiro , Oishi Ryozo , Itoh Yoshinori , Saeki Kiyomi
Published: October 2006

Abstract: The effect of the acute morphine treatment on histamine (HA) pools in the brain and the spinal cord was examined in mice. Morphine (1–50 mg/kg, s.c.) administered alone caused no significant change in the steadystate levels of HA and its major metabolite, tele‐methylhistamine (t‐MH), in the brain. However, depending on the doses tested, morphine significantly enhanced the pargyline (65 mg/kg, i.p.)‐induced accumulation of t‐MH and this effect was antagonized by naloxone. A specific inhibitor of histidine decarboxylase, α‐fluoromethylhistidine (α‐FMH) (50 mg/kg, i.p.), decreased the brain HA level in consequence of the almost complete depletion of the HA pool with a rapid turnover. Morphine further decreased the brain HA level in α‐FMH‐pretreated mice. Morphine administered alone significantly reduced the HA level in the spinal cord, an area where the turnover of HA is very slow. These results suggest that the acute morphine treatment increases the turnover of neuronal HA via opioid receptors, and this opiate also releases HA from a slowly turning over pool(s).
Excitatory Amino Acids: Studies on the Biochemical and Chemical Stability of Ibotenic Acid and Related Compounds
Authors: Nielsen Elsebet Ø. , Schousboe Arne , Hansen Steen H. , Krogsgaard‐Larsen Povl
Published: October 2006

Abstract: The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)‐3‐hydroxy‐4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐7‐carboxylic acid (7‐HPCA), (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), and (RS)‐α‐amino‐3‐hydroxy‐4‐bromo‐5‐isoxazolepropionic acid (4‐Br‐homoibotenic acid) were all inhibitors of (S)‐glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal‐5‐phosphate (PALP) and was inhibited by (S)‐glutamic acid, 3‐mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allylglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min−1 mg−1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)‐glutamic acid (3.2 nmol min−1 mg−1 of protein). At pH 7.4 and 37°C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7‐HPCA, but not AMPA and 4‐Br‐homoibotenic acid, decomposed, partially by decarboxylation, at 100°C in a pH‐dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose. Although muscimol was the only detectable reaction product, mechanisms other than decarboxylation may be involved. Under these conditions, the degradation reaction or reactions were partially dependent on PALP and were inhibited by AOAA and 3MPA but not by allylglycine. The present in vitro studies indicate that ibotenic acid is likely to undergo enzyme‐catalyzed decomposition to give muscimol in brain tissue and after systemic administration to animals. These aspects must be taken into consideration in the interpretation of the pharmacological or neurotoxic effects of ibotenic acid after direct application near central neurons, after local injections into animal brains, or after systemic administration to animals.
Conversion of γ‐Hydroxybutyrate to γ‐Aminobutyrate In Vitro
Authors: Vayer Philippe , Mandel Paul , Maitre Michel
Published: October 2006

Abstract: [3H]γ‐Hydroxybutyric acid ([3H]GHB) at physiological concentration incubated with brain slices in Krebs‐Ringer medium produced [3H]γ‐aminobutyric acid ([3H]GABA). This compound was identified by its Rf values on thin‐layer chromatograms and by analysis of the dansyl derivatives of the free amino acid fraction. No labelled glutamate could be detected. Brain slices incubated with labelled glutamate and nonradioactive GHB generated labelled 2‐oxoglutarate, suggesting that γ‐aminobutyrate‐2‐oxoglutarate transaminase (GABA‐T) is involved in catalyzing this reaction. Furthermore, specific inhibitors of GABA‐T blocked the production of labelled GABA from labelled GHB and of labelled 2‐oxoglutarate from labelled glutamate. Transformation of [3H]GHB into [3H]GABA was not inhibited by malonate, demonstrating that the succinate‐linked pathway is not involved in the generation of GABA. The kinetic characteristics of the multienzyme system involved in GHB degradation studied in vitro are compatible with the production of GABA in vivo.
Corticotropin‐Peptide Regulation of Intracellular Cyclic AMP Production in Cortical Neurons in Primary Culture
Authors: Weiss Samuel , Sebben Michèle , Bockaert Joël
Published: October 2006

Abstract: Previous studies have provided evidence for adrenocorticotropic hormone (ACTH) effects on a wide variety of behaviors. However, the precise sites of action and the mechanisms by which these effects may be mediated have yet to be clearly elucidated. Although ACTH was shown to augment cyclic AMP levels in glial cells isolated from whole brain, other studies found little or no effect of ACTH peptides on cyclic nucleotide metabolism in slices of cerebral cortex or homogenates of whole brain. In the present study, our objective was to determine whether ACTH peptides regulate intracellular cyclic AMP levels in neurons of the cerebral cortex in primary culture. ACTH peptides stimulated cyclic AMP synthesis up to threefold in a dose‐dependent manner; stimulation was complete within 5–10 min of exposure to agonists. Neurohormone efficacy was augmented by 0.1 μM forskolin (which was virtually ineffective alone); potency was unaffected. The order of potency (EC50) for increasing intracellular cyclic AMP levels was as follows: ACTH (1–24), ACTH (1–17) (10 nM) > α‐melanocyte stimulating hormone, β‐melanocyte stimulating hormone (α‐MSH, β‐MSH) (100 nM) > ACTH (1–10) (1 μM) > ACTH (4–10) (5 μM). The hexapeptide ACTH (4–9) as well as ACTH (11–24) were inactive at concentrations as high as 10 μM. Other neuropeptides derived from proopiocortin, such as β‐endorphin and Met‐ and Leu‐enkephalin were without effect on basal or hormonally stimulated cyclic AMP synthesis. In order to determine whether distinct receptors for ACTH are present on cortical neurons, saturating concentrations of the peptide were coincubated with either vasoactive intestinal polypeptide (VIP) or the β‐adrenergic agonist, isoproterenol (INE). The response to combinations of ACTH and INE were clearly additive. However, neither ACTH nor INE could further augment cyclic AMP formation at saturating concentrations of VIP. Comparison of structure‐activity relationships suggest that ACTH receptors mediating the elevation of cyclic AMP formation in cortical neurons may be similar to those associated with the peptide actions on arousal rather than conditioned behavior.
Guinea Pig Striatum as a Model of Human Dopamine Deamination: The Role of Monoamine Oxidase Isozyme Ratio, Localization, and Affinity for Substrate in Synaptic Dopamine Metabolism
Authors: Azzaro A. J. , King J. , Kotzuk J. , Schoepp D. D. , Frost J. , Schochet S.
Published: October 2006

Abstract: The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4‐dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO isozyme toward DA. Endogenous concentrations of DA, 3‐methoxytyramine, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline‐treated) and type B (deprenyl‐treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.
AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase
Authors: Sandberg K. , Schnaar R. L. , McKinney M. , Hanin I. , Fisher A. , Coyle J. T.
Published: October 2006

Abstract: Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuronspecific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma × glioma cell line, NG‐108‐15, with 100 μM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60–85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration‐ and time‐dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl‐coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.
Muscarinic Receptors and Hydrolysis of Inositol Phospholipids in Rat Cerebral Cortex and Parotid Gland
Authors: Jacobson Michael D. , Wusteman Monica , Downes C. Peter
Published: October 2006

Abstract: Exposure of rat brain or parotid gland slices to muscarinic receptor agonists stimulates a phospholipase C that degrades inositol phospholipids. When tissue slices were labelled in vitro with [3H]inositol, this response could be monitored by measuring the formation of [3H]inositol phosphates. Accumulation of inositol 1,4‐biphosphate in stimulated brain slices suggests that polyphosphonositides are the primary targets for phospholipase C activity. Li+ (10 mM) in the medium completely blocked the hydrolysis of inositol 1‐phosphate, partially inhibited inositol 1,4bisphosphate hydrolysis, but had no effect on the hydrolysis of inositol 1,4,5‐trisphosphate by endogenous phosphatases. Muscarinic receptor pharmacology was studied by measuring the accumulation of [3H]inositol 1‐phosphate in the presence of 10 mM Li+. In experiments on brain slices, the response to carbachol was antagonised by atropine with an affinity constant of approximately 8.79 ± 0.12. Dose‐response curves to several muscarinic agonists were constructed using brain and parotid gland slices. The results are consistent with relatively direct coupling of low‐affinity muscarinic receptors to inositol phospholipid breakdown in brain slices; full agonists were relatively more potent in the parotid gland compared with the brain. Explanations for these differences are suggested.
Changes in Brain Energy Metabolism and Protein Synthesis Following Transient Bilateral Ischemia in the Gerbil
Authors: Nowak Thaddeus S. , Fried Richard L. , Lust W. David , Passonneau Janet V.
Published: October 2006

Abstract: The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1–2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.
Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens
Authors: Missale Cristina , Castelletti Laura , Govoni Stefano , Spano Pier Franco , Trabucchi Marco , Hanbauer Ingeborg
Published: October 2006

Abstract: Active uptake of 3,4‐dihydroxyphenylethylamine (dopamine) is sodium‐ and temperature‐dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+‐dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low‐ and high‐affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.
Abnormal Expression of the Myelin‐Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice
Authors: Frail Donald E. , Braun Peter E.
Published: October 2006

Abstract: Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin‐associated glycoprotein (MAG)‐related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal litter mates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five‐ to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypep tides. The quantity of MAG synthesized by 15‐day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthe sized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated develop mental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.
Fidelity of DNA Polymerase‐β in Neurons from Young and Very Aged Mice
Authors: Rao Kalluri Subba , Martin George M. , Loeb Lawrence A.
Published: October 2006

Abstract: Neurons do not divide during adult life and thus they provide a unique system to study the effects of age‐accumulated damage to DNA in the absence of DNA replication. We have analyzed DNA polymerase activity in neurons isolated from young adult and very aged mice. The predominant catalytic activity is DNA polymerase‐β and it is present in similar amounts in neurons from young and old mice. This polymerase is highly errorprone in copying φX174 DNA, the error frequency being about 1/7,000 and not significantly different when obtained from young and old animals. This high infidelity is considered with respect to DNA repair and the protein synthesis error catastrophe theory of aging.
Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro
Authors: Cheung Marshall K. , Verity M. Anthony
Published: October 2006

Brain cell‐free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the postmitochondrial supernatant (PMS) harvested from the brain of young (10‐20‐day‐old) rats. No mercurial‐induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid‐directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl‐tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl‐tRNAPhe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg‐poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.
Elevated Density of [3H]Imipramine Binding in Aged Human Brain
Authors: Severson James A. , Marcusson Jan O. , Osterburg Heinz H. , Finch Caleb E. , Winblad Bengt
Published: October 2006

Abstract: Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5‐hydroxytryptamine (serotonin) and 5‐hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age‐related changes in indole metabolism. The age‐related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.
γ‐Aminobutyric Acid and Benzodiazepine Receptor Changes Induced by Unilateral 6‐Hydroxydopamine Lesions of the Medial Forebrain Bundle
Authors: Pan Helen S. , Penney John B. , Young Anne B.
Published: October 2006

Abstract: Quantitative autoradiography was used to ascertain alterations in [3H]muscimol, [3H]flunitrazepam (FLU), [3H]naloxone, [3H]d‐alanine‐d‐leucine‐enkephalin (DADL), and [3H]spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6‐hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, [3H]spiroperidol binding increased 33% in striatum. At 5 months, [3H]spiroperidol was only nonsignificantly increased above control. At 1 week, [3H]muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, [3H]muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in [3H]muscimol binding persisted at 5 months. [3H]FLU binding was altered in the same direction as [3H]muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in [3H]naloxone and [3H]DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr.
Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108‐15 Cells
Authors: Griffin Michael T. , Law Ping‐Yee , Loh Horace H.
Published: October 2006

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108‐15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108‐15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (Ns) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor‐mediated activation of Ns, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of Ns activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.
Two Acidic Proteins Associated with Brain Development in Chick Embryo
Authors: Shirao Tomoaki , Obata Kunihiko
Published: October 2006

Abstract: The developmental changes in protein composition of the chick optic tectum were analyzed by twodimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9–18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE‐Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.
Increased Activity of Ca2+‐Dependent Enzymes of Membrane Lipid Metabolism in Synaptosomal Preparations from Ethanol‐Dependent Rats
Authors: John G. R. , Littleton J. M. , Nhamburo P. T.
Published: October 2006

Abstract: In synaptosomal fractions of rat brain the activities of phospholipase A2 and the phospholipid base exchange enzymes are highly dependent on external Ca2+ concentrations. Their activity is inhibited by the presence of 50 mM ethanol in vitro. Administration of ethanol to rats by inhalation causes a progressive increase in the activity of these enzymes in synaptosomal preparations at all Ca2+ concentrations studied. The increased activity of these enzymes persists in preparations from rats undergoing a physical syndrome of withdrawal from ethanol. The addition of ethanol in vitro to preparations from animals that had received ethanol in vivo had no significant effect on enzyme activity. The results are discussed in relation to the possible roles of membrane lipid metabolism and synaptic Ca2+ sensitivity in ethanol tolerance and physical dependence.
Intracellular Translocation of Myelin Proteolipid Protein
Authors: Konat G. , Divakaran P. , Samorajski T. , Wiggins R. C.
Published: October 2006

Abstract: Brainstem slices prepared from 22‐day‐old rats, were employed to study the intracellular translocation of radioactively labeled myelin proteolipid protein (PLP). Double‐isotope and short pulse‐chase procedures allowed us to demonstrate the flux of PLP through nine different subcellular membrane fractions that were isolated on the basis of their particle size and buoyant density. Tagged PLP was rapidly depleted from microsomes, showed transient passage through a number of presumably intermediate membranous pools, and accumulated in myelin. On the basis of the kinetics of PLP labeling and isotope ratios, the membranes can be arranged as they participate in the intracellular translocation of PLP and consistently show a pattern indicating possible precursor‐product relationships.
Regional Distribution in Rat Brain of 1‐Pyrroline‐5‐Carboxylate Dehydrogenase and Its Localization to Specific Glial Cells
Authors: Thompson S. G. , Wong P. T.‐H. , Leong S. F. , McGeer E. G.
Published: October 2006

Abstract: A possible alternative route for production of a small glutamate pool in brain is from proline or ornithine to 1‐pyrroline‐5‐carboxylate (P5C) and thence to glutamate. The conversion from ornithine to P5C is catalyzed by ornithine δ‐aminotransferase (OrnT) whereas that from proline is catalyzed by proline oxidase (PrO). The conversion of P5C to glutamate is catalyzed by 1‐pyrroline‐5‐carboxylate dehydrogenase (PDH). Biochemical assays of PDH and PrO in various rat brain regions indicate no positive correlation between the two enzymes nor between either activity and high‐affinity glutamate uptake or the regional distribution of OrnT. We have localized PDH and PrO histochemically by modifications of the Van Gelder [J. Neurochem.12, 231–237, (1965)] method for γ‐aminobutyric acid (GABA) transaminase. The enzymes were found only in certain types of glial cells; the best stained were the Bergmann glial cells of the cerebellum but, for PDH, there was also good staining of astrocytes in the dentate area of the hippocampus. Since both these areas are believed to have heavy glutamate innervation and numerous GABA interneurons, these findings may reflect an alternative route of glutamate production in glial cells near some glutamate and/or GABA tracts but they do not support this as a possible route for glutamate formation in most brain regions. The findings do, however, provide further evidence for chemical specialization of glial cells.
Cellular and Secreted Forms of Acetylcholinesterase in Mouse Muscle Cultures
Authors: Rubin Lee L. , Chalfin Nina A. , Adamo Anna , Klymkowsky Michael W.
Published: October 2006

Abstract: When grown in primary cell culture in the absence of neurons, muscle cells from a variety of species synthesize several forms of acetylcholinesterase (AChE), including the collagen‐tailed A12 form. A12 AChE has been the subject of much study because it is thought to be a major functional enzyme form normally found in the basal lamina at the neuromuscular junction. In this paper, we show that muscle fibers derived from mouse embryos and neonates are also able to synthesize substantial percentages of their AChE as the A12 form when grown in vitro. This synthesis is modulated by a process associated with spontaneous muscle contractile activity since both total enzyme levels and the proportion of A12 AChE expressed on the cell surface are decreased when the cells are grown in the sodium channel blocker tetrodotoxin, which blocks muscle contraction. On the other hand, when the cells are treated with veratridine, which opens sodium channels, thereby mimicking one aspect of muscle contraction, their AChE levels are comparable to those of untreated cells. Although smaller in magnitude, these changes are similar to those seen in rat muscle cultures. A novel feature of mouse muscle cultures. not seen in those from rat and chick, is the presence of a secreted enzyme form that sediments in the same position as the cellular A12 form (when separated on sucrose density gradients containing high salt) and is also collagenase sensitive.
Cerebroventricular and Intravascular Metabolism of [125I]Angiotensins in Rat
Authors: Harding Joseph W. , Yoshida Melanie S. , Dilts Roger P. , Woods Theresa M. , Wright John W.
Published: October 2006

Abstract: This study compared the metabolism of [125I]angiotensin II (AII), [125I]angiotensin III (AIII), and [125I]Sar1,Ile8‐AII (SI‐AII) in the vascular and cerebroventricular compartments. Using HPLC methods to monitor degradation the following t½, values were established in the vascular compartment: AII, 12.7 ± 1.4 s; AIII, 16.3 ± 0.7 s; and SI‐AII, 100.7 ± 7.3 s. HPLC analysis also revealed that [125I]AII is converted in an obligatory manner to [125I]AIII during its degradation sequence. Cerebrospinal fluid contained no degradative capacity for [125I]AII but exhibited a significant capacity to degrade [125I]AIII. A technique that combined the intracerebroventricular injection of [125I]angiotensins followed by focused microwave fixation to stop all peptidase activity was used to determine the half‐life of [125I]angiotensins in the ventricular space. Results indicated very rapid metabolism of angiotensins with the following t½ values: AII, 23.0 s; and AIII, 7.7 s. This extremely rapid, differential, and sequential metabolism of AII and AIII in two relevant body fluid compartments underscores the need for caution when interpreting data derived from intravascular and intracerebroventricular application of angiotensins. In addition the faster metabolism of AIII than AII in the ventricular space indicates that the actual potency of AIII at central angiotensin receptors is being underestimated.
Glutamic Acid as a Putative Transmitter of the Interhemispheric Corticocortical Connections in the Rat
Authors: Peinado Jose M. , Mora F.
Published: October 2006

Abstract The effect of hemidecortication on the endogenous levels of amino acids in medial, sulcal, and dorsal frontal cortex as well as in parietal, temporal, and occipital cortex of the rat was investigated. Under aseptic conditions, the right cerebral cortex was aspirated by suction. Then, 21 days later, the content of glutamic acid, aspartic acid, γ‐aminobutyric acid, glycine, serine, threonine, and alanine was analyzed in six areas of the intact contralateral cortex using GLC. The results demonstrated a specific decrease in the endogenous levels of glutamic acid in both parietal and temporal cortex after hemidecortication of the contralateral side. This finding suggests that glutamic acid may serve as a neurotransmitter for some of the interhemispheric corticoparietal and corticotemporal fibers. In a follow‐up experiment, the effect of a frontal lesion on the endogenous levels of the same amino acids in the striatum was also examined. In this case, the glutamic acid content exhibited a decrease of 31 % relative to the control value. This observation confirms the earlier finding of a glutamate‐containing pathway from the frontal cortex to the striatum.
Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N‐Methyl‐α‐Aminoisobutyric Acid
Authors: Zloković Berislav V. , Begley David J. , Djuričić Bogdan M. , Mitrovic Dušan M.
Published: October 2006

Abstract: A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood‐to‐brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and Pco2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min−1 g−1 in the cerebral cortex, hippocampus, and caudate‐putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D‐[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N‐methyl‐α‐aminoisobutyric acid (MeAIB), and D‐[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time‐uptake data. The Kin for MeAIB (0.75 × 10−3 ml min−1 g−1) was some three times that for mannitol. It is concluded that the technique provides a stable, well‐controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.
Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat
Authors: Chaouloff Francis , Kennett Guy A. , Serrurrier Bernard , Merino Daniele , Curzon Gerald
Published: October 2006

Abstract: Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+ 70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and γ‐aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.
Inhibition of Tyrosine Hydroxylase Activity by Serotonin in Explants of Newborn Rat Locus Ceruleus
Authors: Devau G. , Multon M. F. , Pujol J. F. , Buda M.
Published: October 2006

Abstract: The long‐term regulation of tyrosine hydroxylase (TH) by serotonin has been studied with cultures of newborn rat locus ceruleus explants. The presence of serotonin in the culture medium for a 24‐h period was followed by an inhibition of TH activity in the explants. This effect lasted several days, with a maximal effect 2 days after treatment. Moreover, the decrease was reversible and dependent on the concentration of serotonin used (from 1 μMto 1 mM).The mechanisms of this regulation have been studied using drugs such as those known to act specifically on serotoninergic receptors and those known to interfere with protein synthesis. Thus, the action of serotonin (10−5M)on TH activity was suppressed with equimolar concentrations of serotoninergic antagonists such as metergoline or methiothepin.

It was reproduced by quipazine, a drug capable of acting as a serotoninergic agonist. Inhibitors of protein synthesis acting either at the transcriptional or the translational levels can reproduce the inhibition of TH activity by serotonin alone. Furthermore, the effects of one or the other of these compounds and that of serotonin were not additive. This study confirms the hypothesis of an inhibitory control by serotonin on TH activity in the noradrenergic neurons of the locus ceruleus. Serotonin could regulate the synthesis of the enzyme through specific serotoninergic receptors.
Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites
Authors: Niddam R. , Dubois A. , Scatton B. , Arbilla S. , Langer S. Z.
Published: October 2006

Abstract: The regional distribution of [3H]zoIpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD= 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl‐β‐carboline‐3‐carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]‐ zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H‐ labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover. [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl‐β‐carboline‐3‐carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.
A Rapid HPLC Method to Separate the Triplet Proteins of Neurofilament
Authors: Karlsson J.‐E. , Rosengren L. E. , Haglid K. G.
Published: October 2006

Abstract: In this article a fast HPLC technique to separate the individual neurofilament proteins is described. Highly pure fractions of the three neurofilament proteins can be obtained. As much as 50 mg of each neurofilament poly‐peptide can be separated from a crude neurofilament protein preparation in one step in less than 2 h. The short separation time is of importance in minimizing degradation, especially of the 150‐kilodalton neurofilament poly‐peptide.
[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors
Authors: Vezzani Annamaria , Wu Hui‐Qiu , Samanin Rosario
Published: October 2006

Abstract: [3H]Norepinephrine ([3H]NE) efflux from preloaded rat Hippocampal slices was increased in a dose‐dependent manner by excitatory amino acids, with the following order of potencies: N‐methyl‐D‐aspartate (NMDA) < kainic acid (KA) < L‐glutamate ≥ D,L‐homocysteate < L‐aspartate < quinolinic acid < quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D.,L‐2‐Amino‐7‐phosphonoheptanoic acid dose‐dependently inhibited the NMDA effect (ID50= 69 μM), whereas at 1 mM it was ineffective versus KA. The release of [3H]‐NE induced by quinolinic acid was blocked by 0.1 mM D,L‐2‐amino‐7‐phosphonoheptanoic acid. γ‐D‐Glutamyl‐glycine dose‐dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 μM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.
Uncoupling of Rat Cerebral Cortical α2‐Adrenoceptors from GTP‐Binding Proteins by N‐Ethylmaleimide
Authors: Kitamura Yoshihisa , Nomura Yasuyuki
Published: October 2006

Pretreatment of membranes from rat cerebral cortex with N‐ethylmaleimide (NEM) decreased [3H]clonidine binding in a concentration‐dependent manner. The Bmax values of high‐affinity sites for [3H]clonidine were reduced by 50 μM NEM treatment. Treatment with 500 μM NEM diminished the sum of Bmax of both high‐and low‐affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM‐treated membranes. The addition of purified GTP‐binding proteins caused an increase in the binding to the membranes pretreated with 50 μM NEM, but did not increase [3H]clonidine binding in membranes treated with 500 μM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)‐catalyzed ADP ribosylation of membrane‐bound (41,000‐dalton) and purified (39,000/41,000‐dalton) GTP‐binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, α2‐adrenoceptor, and GTP‐binding protein. One is a highly sensitive site to NEM (a concentration range of 1–50 μM), which is probably a cysteine residue, IAP‐catalyzed ADP‐ribosylating site on the α‐subunit of GTP‐binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1–1 mM), and are the binding domain of agonist and/or the coupling domain of GTP‐binding protein on the α2‐adrenoceptor. In addition, Ki‐ras p21 protein may lack the capacity to couple with the α2‐adrenoceptor.