bluuebunny/crossref_metadata_2025 huggingface dataset:
Examination of Parameters Influencing [3H]MK‐801 Binding in Postmortem Human Cortex
Authors: Piggott M. A. , Perry E. K. , Sahgal A. , Perry R. H.
Published: October 2006

Abstract: [3H]MK‐801 binding was used as an index of the glutamate receptor W‐methyl‐D‐aspartate‐subtype channel to examine the influence of gender, age, mode of death (agonal status), interval between death and autopsy (postmortem delay), and time in storage at ‐70°C in well washed homogenate preparations from postmortem human frontal cortex. Basal binding and the modulatory effects of glutamate, glycine, spermidine, and zinc were examined with respect to these variables. Basal binding was sensitive to agonal status, being higher in sudden death cases. The effect of added glutamate and glycine was sensitive to age, with a trend toward lower binding with increasing age. The effect of added spermidine alone was sensitive to storage time at ‐70°C, the binding being higher with longer storage time. The effect of added zinc was also sensitive to postmortem delay, with zinc causing a greater reduction in binding with shorter postmortem delays. Thus, with the exception of gender, all variables examined influenced [3H]MK‐801 binding, highlighting the attention that should be given to these factors in postmortem studies in normal and diseased human subjects.
Target‐Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish
Authors: Hieber Virginia , Agranoff Bernard W. , Goldman Daniel
Published: October 2006

Abstract: A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in a‐tubulin RNAs (3 days),‐which preceded the increase in nicotinic acetylcholine receptor RNAs (10‐15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4‐6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.
Effects of Forskolin, Dibutyryl Cyclic AMP, and 5′N‐Ethylcarboxamide Adenosine on 22Na Uptake by Rat Brain Synaptosomes Stimulated by Veratridine
Authors: Lobo M. G. B. , Ribeiro J. A.
Published: October 2006

Abstract: The effects of forskolin, dibutyryl cyclic AMP, and 5′‐N‐ethylcarboxamide adenosine on specific 22Na uptake by synaptosomes stimulated by veratridine were investigated. All substances inhibited 22Na uptake, with forskolin more potent than 5′‐N‐ethylcarboxamide and this latter one more potent than dibutyryl cyclic AMP. In the absence of prein‐cubation with forskolin, this substance caused little or no effect on 22Na uptake by synaptosomes. In the presence of the adenosine antagonist dipropylsulfophenylxanthine, the inhibitory effect of 5′‐N‐ethylcarboxamide adenosine on 22Na uptake was consistently antagonized. The results were interpreted as forskolin and 5′‐N‐ethylcarboxamide adenosine increasing cyclic AMP accumulation, and dibutyryl cyclic AMP mimicking it, and by these mechanisms decreasing sodium uptake through the sodium channels.
Down‐Regulation of β‐Adrenergic Receptors by Pindolol in Gsα‐Transfected S49 cyc− Murine Lymphoma Cells
Authors: Gonzales Jerry M. , O'Donnell J. Kevin , Stadel Jeffrey M. , Sweet Ray W. , Molinoff Perry B.
Published: October 2006

Abstract: The role of the a subunit of the guanine nucleotide‐binding regulatory protein that stimulates adenylyl cyclase (Gsα) in the down‐regulation of β‐Adrenergic receptors by pindolol was studied in S49 cyc− cells (normally Gsα‐deficient) transfected to express functional recombinant rat Gsα. An inducible cell line (S49 GsαIND) was derived from S49 cyc− cells transfected with a vector containing the full‐length coding sequence of Gsα under the inducible control of the mouse mammary tumor virus long‐terminal repeat promoter. Gsα was not detectable in S49 GsαIND cells by immunoblot or by ADP‐ribosylation in the presence of cholera toxin and [α‐32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol‐stimulated cyclic AMP accumulation increased within 3 h. After 15 h, Gsα was present at a level 40–50% of that found in S49 wild‐type (WT) cells as measured either by immunoblot analysis or by [α‐32P]ADP‐ribosylation. Membranes prepared from GsαIND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGsα+, contained the coding sequence of Gsα under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc− cells, and the resulting cells, S49 GsαCST cells, expressed Gsα at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol‐stimulated cyclic AMP accumulation in S49 GsαCST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down‐regulated the density of 0‐adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc− or Gsα IND cells. When Gsα CST cells were exposed to 50 nM pindolol for 12 h, the density ofβ‐Adrenergic receptors was down‐regulated by the same amount as in S49 WT cells. These results suggest that Gsα is necessary to restore the ability of pindolol to down‐regulate β‐adrenergic receptors in S49 cyc~ cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.
Isolation and Identification of Methylarginines from Bovine Brain
Authors: Kotani Kazuhiko , Ueno Shu‐ichi , Sano Akira , Kakimoto Yasuo
Published: October 2006

Abstract: Methylarginines in free form were identified in bovine brain. Three compounds were isolated from the basic aliphatic amino acid fraction of bovine brain with several ion‐exchange chromatographies. They showed the same Rf values in paper and thin‐layer chromatographies as those of authentic NG‐monomethylarginine, NG‐dimethylargi‐nine, and NG‐dimethylarginine. The migration distance of the isolated compounds in high‐voltage paper electropho‐resis and the retention times in ion‐exchange HPLC were also identical to those of the above authentic methylarginines. We concluded that these three compounds are the methyl derivatives of arginine described above. The amount of these three compounds isolated from 1,090 g of bovine brain was 0.3 μmol of NG‐monomethylarginine, 0.1 μmol of NG,NG‐dimethylarginine, and 0.5 μmol of NG,NG‐dimethylarginine. The occurrence of these free methylarginines may have an important role in regulating the signal transduction through the nitric oxide system.
Hydroxylated Analogues of 5‐Aminovaleric Acid as 4‐Aminobutyric AcidB Receptor Antagonists: Stereostructure‐Activity Relationships
Authors: Kristiansen Uffe , Hedegaard Anette , Herdeis Claus , Lund Trine M. , Nielsen Birgitte , Hansen Jan J. , Falch Erik , Hjeds Hans , Krogsgaard‐Larsen Povl
Published: October 2006

Abstract: The (R) and (S) forms of 5‐amino‐2‐hydroxyvaleric acid (2‐OH‐DAVA) and 5‐amino‐4‐hydroxyvaleric acid (4‐OH‐DAVA) were designed as structural hybrids of the 4‐aminobutyric acidB (GABAB) agonist (R)‐(–)‐4‐amino‐3‐hydroxybutyric acid [(R)‐(–)‐3‐OH‐GABA] and the GABAB antagonist 5‐aminovaleric acid (DAVA). (S)‐(–)‐2‐OH‐DAVA and (R)‐(−)‐4‐OH‐DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileurn preparation. The respective enantiomers, (R)‐(+)‐2‐OH‐DAVA and (S)‐ (+)‐4‐OH‐DAVA, were markedly weaker in both test systems. AH four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low‐energy conformations of (R)‐(–)‐3‐OH‐GABA, (S)(–)‐2‐OH‐DAVA, and (R)‐(–)4‐OH‐DAVA can be superimposed. These conformations may reflect the shapes adopted by these conforma‐tionally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.
Regulation of Cyclic AMP Response Element‐Binding Protein (CREB) Phosphorylation by Acute and Chronic Morphine in the Rat Locus Coeruleus
Authors: Guitart Xavier , Thompson Margaret A. , Mirante Cindy K. , Greenberg Michael E. , Nestler Eric J.
Published: October 2006

Abstract: Previous studies have implicated adaptations in the cyclic AMP system in mechanisms of opiate tolerance, dependence, and withdrawal in the rat locus coeruleus. It has been speculated that such adaptations may occur at the level of gene expression. To understand better the mechanism by which opiates produce these in‐tracellular adaptations, we studied morphine regulation of the state of phosphorylation of cyclic AMP response element‐binding protein (CREB), a transcription factor that mediates some of the effects of the cyclic AMP system on gene expression. We show here, by use of a back phosphorylation and immunoprecipitation procedure, that acute morphine decreases the state of phosphorylation of CREB, an effect that becomes completely attenuated after chronic morphine administration. In contrast, acute precipitation of opiate withdrawal, via administration of an opiate receptor antagonist, increases the phosphorylation state of CREB. Such regulation of CREB phosphorylation could be part of the molecular pathway by which opiates produce changes in gene expression that lead to addiction.
DM‐20 mRNA Is Expressed During the Embryonic Development of the Nervous System of the Mouse
Authors: Timsit S. G. , Bally‐Cuif L. , Colman D. R. , Zalc B.
Published: October 2006

Abstract: We used both the polymerase chain reaction (PCR) and in situ hybridization to search for the presence of proteolipid protein (PLP) gene transcripts in the developing mouse. Total brain RNA extracted from 13‐19‐day embryos, analyzed by PCR, demonstrated the presence of a single transcript that was unambiguously identified with the DM‐20 mRNA. RNA samples from postnatal day 2 animals also showed a signal corresponding to the PLP transcript, in addition to the DM‐20 message. By in situ hybridization of 10‐day embryos using a DM‐20 antisense cRNA probe, we showed that the localization of the DM‐20 message was restricted to the diencephalic basal plate. On the same embryo sections, in addition to the brain localization, an intense hybridizing signal was also detected in the trigeminal and spinal ganglia, the vagal glossopharyngeal ganglion, and the sympathetic ganglion chain. The demonstration of transcription of the PLP gene, long before the beginning of the myelination process, suggests that in addition to a structural function in myelin compaction, some of the products of the PLP gene (DM‐20) may have a role during the compartmentalization and differentiation of the neural tube.
Expression of Olfactory Receptors in Xenopus Oocytes
Authors: Dahmen Norbert , Wang Hung‐Li , Margolis Frank L.
Published: October 2006

Abstract: The rat olfactory epithelium and the amino acid‐sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two‐electrode voltage clamp, activates stimulus‐dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand‐activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca2+‐activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.
Inhibition of Cyclic AMP Formation by a Selective Metabotropic Glutamate Receptor Agonist
Authors: Schoepp Darryle D. , Johnson Bryan G. , Monn James A.
Published: October 2006

Abstract: It is well documented that the effects of excitatory ammo acid (EAA) agonists on phosphoinositide hydrolysis involve a GTP‐binding protein‐linked or “metabotropic’ receptor mechanism. The mechanisms by which EAAs alter cyclic AMP levels in brain slices, however, are not yet clear. In this study, the selective metabotropic EAA agonist trans‐(±)‐l‐aminocyclopentane‐l,3‐dicarboxylic acid and its isomers were examined for effects on basal and forskolin‐stimulated cyclic AMP formation in slices of the rat hippocampus. Trans‐(±)‐l‐Aminocyclopentane‐l,3‐dicarboxylic acid had little effect on basal cyclic AMP but inhibited forskolin‐stimulated cyclic AMP formation in a biphasic manner. The 1S,3R isomer of 1‐aminocy‐clopentane‐l,3‐dicarboxylic acid produced potent but only partial (~50%) inhibition of forskolin‐stimulated cyclic AMP formation. 1R,3S–l‐Aminocyclopentane‐l,3‐dicarboxylic acid fully inhibited forskolin‐stimulated cyclic AMP but with lower potency than the IS,3R isomer. These results show that in addition to the formation of phosphoinositide‐derived second messengers, the cellular consequences of selectively activating hippocampal metabotropic EAA receptors include an alteration of cellular cyclic AMP levels.
Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron‐Rich Striatal Cultures
Authors: Brass B. J. , Nonner D. , Barrett J. N.
Published: October 2006

Abstract: We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron‐rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 μg/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800‐8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of >4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase‐positive neurons was not reduced by insulin, suggesting that insulin acts by down‐regulating ChAT rather than by killing cholinergic neurons. Insulin‐like growth factor‐1 (IGF‐1) reduced ChAT activity at concentrations 10‐fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF‐1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.
The Adenosine Receptors Present on the Plasma Membrane of Chromaffin Cells Are of the A2b Subtype
Authors: Casadó Vicent , Casillas Teresa , Mallol Josefa , Canela Enric I. , Lluis Carmen , Franco Rafael
Published: October 2006

Abstract: The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R‐[3H]phenylisopropyladenosine (R‐PIA) and [3H]8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]‐DPCPX) binding. The binding of the specific A2a. ligand CGS‐21680 was low. In contrast, the binding of 5′‐(N‐[3H]‐ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non‐adenosine receptor NECA binding sites because the specific [3H]‐NECA binding was similar when unlabeled adenosine, NECA, or R‐PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX > NECA > chloroadenosine > R‐PIA > theophylline = CGS‐21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.
Preincubation with Substance P Induces Substance P‐Stimulated Phosphatidylinositol Turnover in Cultured Cerebellar Astrocytes
Authors: Marriott Derek R. , Wilkin Graham P.
Published: October 2006

Abstract: In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I‐Bolton—Hunter conjugated SP (125I‐BHSP) receptor binding, and SP‐stimulated phosphatidylinositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001‐1,‐nM) suffered a dose‐dependent decrease in both subsequent 125I‐BHSP and SP‐stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP‐stimulated PI turnover, with no change in 125I‐BHSP receptor binding. SP‐induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.
Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands
Authors: Rodriguez F. D. , Bardaji E. , Traynor J. R.
Published: October 2006

Abstract: The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea‐pig brain in HEPES buffer have been studied. The binding of tritiated μ‐, δ, and κ‐opioid agonists was promoted in a concentration‐dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea‐pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the μ‐opioid agonist [3H][D‐Ala2, MePhe4, Gly‐ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.
Enhancement of Endogenous Release of Glutamate and γ‐Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long‐Term Potentiation
Authors: Ghijsen Wim E. J. M. , Besselsen Elly , Geukers Vincent , Kamphuis Willem , da Silva Fernando H. Lopes
Published: October 2006

Abstract: The effect of long‐term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and γ‐aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K+‐induced Ca2+‐independent release components of these amino acids. In contrast, K+‐induced Ca2+‐dependent release of both glutamate and GABA increased ∼ 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.
γ‐Glutamylaminotransferase and Transglutaminase in Subcellular Fractions of Rat Cortex and in Cultured Astrocytes
Authors: Reichelt Karl L. , Poulsen Eystein
Published: October 2006

Abstract: The subcellular distribution of γ‐glutamylaminotransferase (EC 2.3.2.2) and transglutaminase (EC 2.3.2.1) has been investigated in rat brain tissue fractionated by a centrifugation and sedimentation technique. Neither of these enzymes was enriched in the synaptosomal fraction. Comparing the in vitro grown astrocytes with synaptosomes, we find that both of these enzymes may possibly be more important in the glial element of the synaptic region. γ‐Glutamylaminotransferase is most abundant in capillaries, confirming previous reports.
Activation of Protein Kinase C Blocks Astroglial Gap Junction Communication and Inhibits the Spread of Calcium Waves
Authors: Enkvist M. O. Kristian , McCarthy Ken D.
Published: October 2006

Abstract: The following two processes related to astrocytes are thought to depend on intercellular coupling through gap junctions: the spatial buffering of K+o and the spread of calcium waves in the astrocytic syncytium. We have used the following two independent methods to measure the open state of gap junctions: injection of lucifer yellow, and optical calcium imaging of calcium waves in response to probing the cells with a micropipette. The spread of lucifer yellow and calcium waves was inhibited if the cells were treated with either phorbol 12‐myristate 13‐acetate (PMA) or a synthetic diacylglycerol that activates protein kinase C. Down‐regulation of protein kinase C by a 24‐h treatment with PMA inhibited the uncoupling effect of PMA, supporting a direct involvement of protein kinase C in the regulation of astroglial gap junctions. Purinergic P2Y receptors, which are coupled to the inositol phospholipid pathway, are expressed by most astroglia in culture. Activation of the P2Y purinergic receptor with the selective agonist 2‐methylthio‐ATP uncoupled astroglia in a manner similar to the effect of treatment with PMA. Modulation of gap junctional conductance could isolate specific pathways within the astrocytic syncytium to form an extraneuronal information transfer network in brain.
Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol
Authors: Prado M. A. M. , Gomez M. V. , Collier B.
Published: October 2006

Abstract: The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2‐(4‐phenylpiperidino)cyclohexanol (vesamicol, AH5 183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse‐releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium‐3 did not prevent the toxin‐induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse‐releasable or toxin‐releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.
Effect of Diethyl Pyrocarbonate Modification of Benzodiazepine Receptors on [3H]Ro 15‐4513 Binding
Authors: Uusi‐Oukari Mikko
Published: October 2006

Abstract: The effects of treatment of brain membranes with diethyl pyrocarbonate (DEP), a histidine‐modifying reagent, on the binding of 3H‐labeled Ro 15‐4513 (ethyl‐8‐azido‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5‐a]‐[1,4]benzodiazepine‐3‐carboxylate) and [3H]diazepam were compared. DEP pretreatment produced a dose‐dependent decrease in [3H]diazepam binding, whereas low DEP concentrations enhanced the binding of [3H]Ro 15‐4513. These effects were reversed by incubation with hydroxylamine after the treatment. The enhancement of [3H]Ro 15‐4513 binding was due to an increase in the affinity of the binding sites (KD), without any effect on binding capacity (Bmax. The enhancement was perceived in cerebral cortical, cerebellar, and hippocampal membranes. DEP treatment decreased the displacement of [3H]Ro 15‐4513 binding by diazepam and FG 7142 (N‐niethyl‐β‐carboline‐3‐carboxamide) but not by Ro 15‐4513 and Ro 19‐4603 (tert‐butyl‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5‐a]thieno[2,3‐f][1,4]diazepine‐3‐carboxylate). Although the stimulating effect of γ‐aminobutyric acid (GABA) on [3H]‐diazepam binding was not affected by DEP treatment, such treatment reduced the inhibitory effect of GABA on [3H]Ro 15‐4513 binding. The enhancement of [3H]Ro 15‐4513 binding was observed in membranes pretreated with DEP in the presence of flunitrazepam, whereas such pretreatment reduced significantly the inhibitory effect of DEP on [3H]‐diazepam binding. The results indicate that a histidine residue in the benzodiazepine binding domain is critical for [3H]diazepam but not [3H]Ro 15‐4513 binding. They also suggest that the cerebellar granule cell‐specific “diazepam‐insensitive” [3H]Ro 15‐4513 binding sites lack this critical histidine.
High Sensitivity of Glutamate Uptake to Extracellular Free Arachidonic Acid Levels in Rat Cortical Synaptosomes and Astrocytes
Authors: Volterra Andrea , Trotti Davide , Cassutti Paola , Tromba Cinzia , Salvaggio Antonino , Melcangi Roberto C. , Racagni Giorgio
Published: October 2006

Abstract: By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high‐affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1–100 μM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2–1 mg/ml), a phospholipase A2 activator, or thimerosal (50–200 μM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate. whereas inhibition ≥60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1–10 mg/ml), a free fatty acid‐binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.
Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors
Authors: Cox Barbara A. , Henningsen Robert A. , Spanoyannis Athena , Neve Rachael L. , Neve Kim A.
Published: October 2006

Abstract: Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser‐193, Ser‐194, Ser‐197, and Ser‐391. Wild‐type and mutant receptors were expressed transiently in COS‐7 cells and stably in C6 glioma cells for analysis of ligand‐receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50‐fold by substitution of alanine for Ser‐193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation‐induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser‐193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser‐193 or Ser‐197. Mutation of Ser‐194 led to a complete loss of efficacy for dopamine and p‐tyramine, which would be consistent with an interaction between Ser‐194 and the p‐hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of β2‐adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.
The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing
Authors: Walsh Michael J. , Kuruc Norbert
Published: October 2006

Abstract: The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two‐dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N‐terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tubulin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1α, and receptor protein. The three neurofilament proteins are detected but a 58‐kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66‐kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68‐kDa protein is identified as a member of the hsp70/ BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N‐ethylmale‐imide‐sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115‐kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.
Binding and Active Transport of Large Analogues of Acetylcholine by Cholinergic Synaptic Vesicles In Vitro
Authors: Clarkson Edward D. , Rogers Gary A. , Parsons Stanley M.
Published: October 2006

Abstract: A previous structure‐activity investigation of acetylcholine (ACh) revealed a positive correlation between additional hydrophobic bulk and increased potency for inhibition of active transport of [3H]ACh by synaptic vesicles isolated from the electric organ of Torpedo. In the current study, several ACh analogues that are significantly larger than previously studied “false transmitters” were synthesized in the tritiated form by chemical means and tested for active transport. These are analogue 14 [(±)‐(cis,trans)‐1‐benzyl‐1‐methyl‐3‐acetoxypyrrolidinium iodide], analogue 15 [(±)‐1,1‐dimethyl‐3‐benzoyloxypyrrolidinium iodide], and analogue 16/17 [(±)‐(cis,trans)‐ 1‐benzyl‐1‐methyl‐3‐benzoyloxypyrrolidinium iodide]. These analogues place significant additional hydrophobic bulk on one or the other (analogues 14 and 15) or both (analogue 16/17) of the two pharmacophores of a small, conformationally constrained analogue of ACh. [3H]Analogue 14 and [3H]analogue 15 are actively transported, with Vmax values the same as or less than that of ACh, depending on the vesicle preparation. The observation that Vmax is the same for an analogue and ACh in some vesicle preparations suggests that the rate‐limiting step does not involve ACh bound to the transporter. [3H]‐Analogue 16/17 is actively transported very poorly. Km values for ACh and for transported ACh analogues vary by up to two‐ to threefold in different vesicle preparations. The ACh transporter is much less selective for transported substrates than anticipated.
Ligand Binding Kinetics of Substance P and Neurokinin A Receptors Stably Expressed in Chinese Hamster Ovary Cells and Evidence for Differential Stimulation of Inositol 1,4,5‐Trisphosphate and Cyclic AMP Second Messenger Responses
Authors: Takeda Yasuo , Blount Paul , Sachais Bruce S. , Hershey Andrew D. , Raddatz Rita , Krause James E.
Published: October 2006

Abstract: Stably transfected Chinese hamster ovary cells expressing either the substance P receptor or neurokinin A receptor were constructed, isolated, and characterized. Equilibrium ligand binding studies performed on whole cells demonstrated that cell lines expressing either of these receptors contained a single class of high‐affinity binding sites with an apparent KD of 0.16 nM for the substance P receptor and an apparent KD of 2.1 nM for the neurokinin A receptor. The higher affinity of substance P for its receptor was accounted for by both a greater association rate constant and a lesser dissociation rate constant. The time course and extent of ligand‐stimulated inositol 1,4,5‐trisphosphate mass increases in both cell lines were similar and displayed rapid and transient kinetics. Ligand‐stimulated cyclic AMP accumulation was also apparent in the cell lines, although the time course and magnitude of the responses were substantially different, with the neurokinin A receptor mediating a greater and more prolonged response. These studies establish the presence of functional substance P receptors and neurokinin A receptors in the stably transfected cell lines and provide evidence for agonist‐dependent differential stimulation of second messenger responses.
Distribution of Preproatrial Natriuretic Peptide mRNA in Rat Brain Detected by In Situ Hybridization of DNA Oligonucleotides: Enrichment in Hypothalamic and Limbic Regions
Authors: Gundlach A. L. , Knobe K. E.
Published: October 2006

Abstract:The expression and distribution of mRNA encoding pre‐proatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two35S‐labeled synthetic DNA Oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus (“anteroventral/third ventricle region”) and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CAl pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta. and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)‐like immunoreactivity, but the expression of ppANP mRNA in CAl pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.
Transsynaptic Regulation of Galanin, Neurotensin, and Substance P in the Adrenal Medulla: Combinatorial Control by Second‐Messenger Signaling Pathways
Authors: Fischer‐Colbrie R. , Eskay R. L. , Eiden L. E. , Maas D.
Published: October 2006

Abstract: The adrenomedullary content of neurotensin and substance P was examined 1, 6, and 12 days after hypoglycemic shock. The neurotensin content was increased 60‐fold within 24 h and remained elevated for up to 12 days, whereas the substance Pcontent was increased approximately sevenfold within 24 h of insulin treatment and returned to control levels by 12 days poststimulation. Because protein kinase A, protein kinase C, and calcium influx in the rat adrenal medulla are all stimulated following splanchnic nerve stimulation, the differential regulation of neurotensin and substance P biosynthesis following stimulation of these three pathways was examined in bovine chromaffin cells in vitro. Neurotensin levels were up‐regulated by elevated potassium, forskolin, and phorbol ester in bovine chromaffin cells. Substance P levels were up‐regulated by elevated potassium and forskolin but not by phorbol ester treatment. When chromaffin cells were treated with phorbol ester in combination with forskolin, neurotensin levels were increased in a synergistic fashion, whereas phorbol ester antagonized the forskolin‐induced elevation of substance P levels. Earlier, it was reported that galanin biosynthesis, like neurotensin biosynthesis, is upregulated by depolarization, phorbol ester stimulation, and forskolin treatment in chromaffin cells in vitro. Here we report that galanin is also, like neurotensin, increased >60‐fold after stimulation of the rat adrenal medulla in vivo. Neuropeptide‐specific combinatorial effects of stimulating the calcium, protein kinase A, and protein kinase C signaling pathways may underlie the quantitative differences between galanin and neurotensin compared with substance P up‐regulation in rat adrenal medulla after splanchnic nerve stimulation in vivo.
Depression by Sodium Ions of Calcium Uptake Mediated by Non‐N‐Methyl‐d‐Aspartate Receptors in Cultured Cerebellar Neurons and Correlation with Evoked d‐[3H]Aspartate Release
Authors: Gallo Vittorio , Giovannini Claudio , Levi Giulio
Published: October 2006

Abstract: In a previous study we noted that the release of d‐[3]aspartate evoked by non‐N‐methyl‐d‐aspartate (non‐NMDA) receptor agonists in cultured rat cerebellar granule cells was enhanced in the absence of extracellular Na+. To explain this apparent paradox, we tried in the present investigation to correlate the effect of Na+ removal on the kainate (KA)‐ and quisqualate (QA)‐induced d‐[3H]aspartate release with that on KA‐ and QA‐induced 45Ca2+ accumulation. The releasing activity of KA, which was only partially Ca2+ dependent in the presence of Na+, became totally Ca2+ dependent in its absence. Moreover, the releasing activity of QA, which was Ca2+ independent in the presence of Na+, became 50% Ca2+ dependent in the absence of the monovalent cation. The releasing action of both agonists was in all cases antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), and that induced by KA was also sensitive to kynurenic acid. When glutamate was tested as an agonist in the presence of Na+, it was found that its d‐[3H]aspartate releasing action was Ca2+ independent and was largely due to heteroexchange. The evoked release was Ca2+ independent, scarcely sensitive to CNQX, and insensitive to NMDA antagonists. In Na+‐free medium, the glutamate‐evoked d‐[3H]aspartate release was lower (due to the abolishment of heteroexchange), but was totally Ca2+ dependent and antagonized by CNQX and kynurenate. KA (30 μM–1 mM) stimulated the accumulation of 45Ca2+ in a dose‐dependent and CNQX‐sensitive way, the effect being progressively higher as the Na+ concentration in the medium was decreased. Li+ affected KA‐induced 45Ca2+ accumulation in a way similar to Na+, although 45Ca2+ uptake was somewhat lower in Li+‐containing medium. The voltage‐activated calcium channel antagonists La3+ and (–)‐202‐791 caused only a limited inhibition of the KA‐induced 45Ca2+ influx both in the presence and in the absence of Na+. Under all the conditions tested [presence and absence of Na+ and of (–)‐202‐791], the kainate‐induced 45Ca2+ uptake was scarcely sensitive to the NMDA antagonist 2‐amino‐5‐phosphonovalerate. QA and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid also stimulated 45Ca2+ influx in a CNQX‐sensitive way, the effect being enhanced in Na+‐free media. These agonists were, however, less effective than KA. It is concluded that in cultured cerebellar granule cells the influx of Ca2+ through non‐NMDA receptor channels is inhibited by Na+ and similar monovalent cations, and that the potentiated KA‐ and QA‐induced d‐[3H]aspartate release observed in Na+‐free medium can be accounted for by the increased accumulation of Ca2+ occurring under this condition.
Glucose, Insulin, and Insulin‐Like Growth Factor I Regulate the Glycogen Content of Astroglia‐Rich Primary Cultures
Authors: Dringen Ralf , Hamprecht Bernd
Published: October 2006

Abstract: The glycogen content of astroglia‐rich primary cultures derived from the brains of newborn rats depends on the concentration of glucose in the culture medium. After administration of culture medium lacking glucose, the glycogen content decreases with a half‐time of 7 min. Readdition of glucose results in replenishment of the glycogen stores within 2–3 h, but fully only if glucose is present in a concentration of at least 4 mM. Insulin, or the more potent insulin‐like growth factor I, increases the content of glycogen ∼1.7‐fold, with the half‐maximal effects being attained at concentrations of 10 and 0.5 nM, respectively. These results suggest that (a) glucose or a metabolite of it and (b) insulin‐like growth factor I or a closely related peptide, but not insulin, are likely to be physiological regulators of the level of glycogen in astrocytes.
Comparative Alterations of Nicotinic and Muscarinic Binding Sites in Alzheimer's and Parkinson's Diseases
Authors: Aubert I. , Araujo D. M. , Cécyre D. , Robitaille Y. , Gauthier S. , Quirion R.
Published: October 2006

Abstract:We have recently reported on the differential alterations of various cholinergic markers in cortical and subcortical regions in Alzheimer's disease (AD). The main purpose of the present study was to determine if cholinergic deficits observed in patients with AD are unique to this disorder or can be generalized to others such as idiopathic Parkinson's disease (PD) and PD with Alzheimer‐type dementia (PD/AD). Muscarinic M1, M2, and nicotinic receptor binding parameters (KdandBmax) were determined in various cortical and subcortical areas using selective radioligands ([3H]‐pirenzepine, [3H]AF‐DX 116, andN[3H]methylcarbamylcholine). Choline acetyltransferase activity was also determined as a marker of the integrity of cholinergic innervation. Alterations of cholinergic markers are comparable in cortical areas in AD, PD, and PD/AD brains. In frontal and temporal cortices, as well as in the hippocampus, choline acetyltransferase activity and binding capacities of M2and nicotinic binding sites are similarly decreased in these three disorders compared with age‐matched control values. M1receptor binding parameters are not significantly modified in cortical areas in patients with these disorders. In contrast, important differences between AD and PD brain tissues are found in subcortical areas such as the striatum and the thalamus. The density of M1sites is significantly increased in striatal areas only in patients with AD, whereas densities of nicotinic sites are decreased in thalamus and striatum in PD and PD/AD, but not AD, brain tissues. The binding capacity of M2sites is apparently unchanged in subcortical areas in all three disorders, although tendencies toward reductions are observed in the striatum of PD and PD/AD patients. Thus, although comparable alterations of various cholinergic markers are observed in cortical areas in the three neurological disorders investigated in the present study, important differences are seen in subcortical areas. This may be relevant to the respective etiological and clinical profiles of AD and PD.
Aluminum Alters the Electrophoretic Properties of Neurofilament Proteins: Role of Phosphorylation State
Authors: Shea Thomas B. , Beermann Mary Lou , Nixon Ralph A.
Published: October 2006

Abstract: Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)‐containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 μM) and was not reversed by 20% SDS, 6 M urea, freeze‐thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high‐molecular‐weight neurofilament subunit (NF‐H) entered SDS‐containing gels after exposure to aluminum lactate but migrated aberrantly as a long high‐molecular‐weight streak. Migration of the 160‐kDa α‐chymotryptic cleavage product of NF‐H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS‐containing gels by AlCl3. Dephosphorylation of NF‐H and the middle‐molecular‐weight neurofilament subunit (NF‐M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF‐H or NF‐M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C‐terminal sidearms of NFPs, resulting in intermolecular cross‐linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication.