bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Sensitivity of “Metabotropic” Glutamate Receptors After Induction of Long‐Term Potentiation in Rat Hippocampus
Authors: Aronica E. , Frey U. , Wagner M. , Schroeder H. , Krug M. , Ruthrich H. , Catania M. V. , Nicoletti F. , Reymann K. G.
Published: October 2006

Stimulation of [3H]inositol monophosphate <[3H]InsP) formation by ibotenate or trans‐1‐aminocyclopentyl‐l, 3‐dicarboxylic acid (t‐ACPD) in rat hippocampal slices was enhanced after tetanic stimulation of die Schaffer collaterals projecting to the CA1 region (in vitro) or the perforant pathway projecting to the dentate gyms (in freely moving animals). This effect was observed 5 h (but not 2 h) after long‐term potentiation (LTP) induction and was abolished if tetanic stimulation was performed in the presence of specific antagonists of N‐methyl‐D‐aspartate receptors. The delayed increase in excitatory amino acid‐induced polyphosphoinositide (PPI) hydrolysis was accompanied by an enhanced responsiveness to norepinephrine, whereas the basal and carbamylcholine‐stimulated [3H]InsP formation were unchanged. These results suggest that an increased activity of „metabotropic” glutamate receptors may contribute to the synaptic mechanisms enabling the late expression and or maintenance of LTP. Accordingly, LTP decayed more rapidly (within 5 h) in rats repeatedly injected with LiCl (60–120 mg/kg, i.p., for 10 days), a treatment that led to a reduced efficacy of ibotenate and norepinephrine in stimulating PPI hydrolysis in hippocampal slices.
Effect of Ethanol on γ‐Aminobutyric Acid and Glycine Receptor‐Coupled Cl− Fluxes in Rat Brain Synaptoneurosomes
Authors: Engblom A. C. , Akerman K. E. O.
Published: October 2006

Chloride fluxes in Synaptoneurosomes in response to additions of γ‐aminobutyric acid, glycine, and ethanol were measured using a chloride‐sensitive fluorescent probe 6‐methoxy‐N‐(3‐sulfopropyl) quinolinium (SPQ). The C1− gradient was directed outward by bathing cells in a medium low in C1− concentration. The Synaptoneurosomes responded to both γ‐aminobutyric acid and glycine by outflow of C1− ions, as judged from an increase in SPQ fluorescence. These effects were inhibited by picrotoxin and strychnine, respectively. Ethanol also produced an outflow of C1− ions from the Synaptoneurosomes. Both picrotoxin and strychnine inhibited this effect. When the antagonists were used together, the inhibiting effect was additive. These results indicate that ethanol affects both γ‐aminobutyric acid and glycine receptor‐linked chloride fluxes in the rat brain.
Endopeptidase‐24.11, a Cell‐Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System
Authors: Kioussi Chrissa , Matsas Rebecca
Published: October 2006

Endopeptidase‐24.11 is a 90‐kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase‐24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and the cervical sympathetic trunk in which >99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono‐ and polyclonal anti‐endopeptidase antibodies. Endopeptidase‐24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed I25I‐insulin B‐chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM Moreover, a 90‐kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelinand non‐myelin‐forming Schwann cells. Endopeptidase‐24. 11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase‐24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.
Effects of Surgical Sympathetic Denervation on myo‐Inositol Trisphosphate Production and Contraction in the Dilator and Sphincter Smooth Muscles of the Rabbit Iris: Evidence for Interaction Between the Cyclic AMP and Calcium Signaling Systems
Authors: Abdel‐Latif Ata A. , Zhang Yawen
Published: October 2006

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3‐isobutyl‐l‐methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE‐stimulated IP3 production and contraction are enhanced, (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation, CCh‐stimulated IP3 production and contraction are attenuated, (c) The increase in tension by a maximal effective dose of NaF (20 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation, (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX‐induced cAMP formation increased in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively, (e) Isoproterenol (5 μM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively, (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh‐stimulated IP3 production and contraction of the sphincter by ∼30% of their respective controls, (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are causal by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to tie hypothesis that there are reciprocal interactions between the cAMP and IP3‐Ca2+ signaling systems and the contractile response in the iris smooth muscle.
Ascorbic Acid in Mesencephalic Cultures: Effects on Dopaminergic Neuron Development
Authors: Kalir Henry H. , Mytilineou Catherine
Published: October 2006

Abstract: Ascorbic acid exists in high intracellular concentrations in fetal rat brain. In mesencephalic cultures the cellular ascorbic acid content drops sharply to undetectable levels when no ascorbic acid is added to the medium, thus creating a model of scorbutic neuronal tissue and affording the study of ascorbic acid's effects on mesencephalic cell development and function. Cultures treated with 0.2 mM ascorbic acid were compared with controls (scorbutic cultures) by using morphological and biochemical indices. Ascorbic acid cultures at 7 and 14 days in vitro showed a marked increase in glial proliferation on glial fibrillary acidic protein staining and increased neurite growth and number on tyrosine hydroxylase staining. Significantly higher dopamine uptake and levels of dopamine and 3, 4‐dihydroxyphenylacetic acid were also observed after 7 and 14 days of ascorbic acid treatment. The capacity to accumulate ascorbic acid and the ability to retain the intracellular ascorbic acid developed gradually as the cultures matured. Ascorbic acid reached the embryonal levels by day 14 in vitro. We conclude that although neuronal cultures can survive and grow in the absence of detectable levels of ascorbic acid, its presence exerts a broad effect on dopamine neuron morphology and biochemical functioning either directly or through increased glial proliferation, or possibly both.
Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations
Authors: Okuno Etsuo , Schmidt Werner , Parks Deborah A. , Nakamura Masayuki , Schwarcz Robert
Published: October 2006

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L‐kynurenine (KYN) and by determining the irreversible conversion of [3H] KYN to [3H] KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2‐oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT‐rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate‐specific. Thus, of the amino acids tested, only α‐aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3‐hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 μM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 μMfor KYN with 1 mM 2‐oxoglutarate, but a much higher Km(910 μM) with 1 mM pyruvate. Km values for 2‐oxoglutarate and pyruvate were 150 and 160 μM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate. At that time of almost complete neuronal destruction and pronounced astrocytic proliferation, enzyme activity in the lesioned striatum was almost twice as high as in controls, suggesting a preferential astroglial localization of brain KAT. The characteristics of KAT described here are compatible with a central role of the enzyme in brain KYNA function in vivo. Abnormal KAT activity, therefore, should be considered as an etiological factor in pathological phenomena related to dysfunction of excitatory amino acid receptors in the brain.
Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l‐Methyl‐4‐Phenyl‐1, 2, 3, 6‐Tetrahydropyridine and of In Vitro 1‐Methyl‐4‐Phenylpyridinium
Authors: Miller Diane B. , Reinhard John F. , Daniels Alejandro J. , O'Callaghan James P.
Published: October 2006

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1‐methyl‐4‐phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 rag/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte‐localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS. In cultured bovine adrenal medullary cells, MPP+ (300 μM) slightly decreased catecholamine levels, but had no effect on tyrosine hydroxylase activity or cellular protein. However, the incubation of these cells with both MPP+ and DDC (1.5 or 3.0 mM) caused large decreases in all indicators of cell viability. The enhancement of MPP+ neurotoxicity by DDC in an in vitro system, where distributional factors are limited, raises die possibility that mechanisms in addition to altered kinetics may account for DDC‐induced potentiation of MPTP neurotoxicity in vivo.
K+‐Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor‐Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine‐Sensitive Calcium Channels
Authors: Iuvone P. Michael , Gan Jiwei , Avendano Gary
Published: October 2006

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia‐free, low‐density, mono‐layer cultures of chick retinal photoreceptors and neurons. In photoreceptor‐enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K+‐evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K+‐evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine‐sensitive channels is required for depolarization‐evoked stimulation of cyclic AMP accumulation in photoreceptor‐enriched cultures.
Rapid Kinetics of Potassium‐Evoked Release of Acetylcholine from Rat Brain Synaptosomes: Analysis by Rapid Superfusion
Authors: Pearce L. Bruce , Buck Theresa , Actamec Emil
Published: October 2006

The rapid kinetics of spontaneous and evoked [3H]acetylcholine efflux from synaptosomes was investigated using the technique of rapid superfusion. Synaptosomes were isolated from whole rat brain and the intraterminal pool of acetylcholine was radiolabeled by preincubation with [3H]choline. Synaptosomes were retained within the super‐fusion system on filter disks and supervised with Krebs‐bicarbonate buffer, pH 7.4, at flow rates ofO.3‐0.5 ml/s. These experimental conditions provided a mixing half‐life of 119 ms and efficiency of superfusion of <85%. The kinetics of tritium efflux was followed on the second and subsecond time scales by collection of serial 4.8‐s and 50‐ms samples for a total of 67.2 and 1.0 s, respectively. Superfusion for 48 s with isoosmotic Krebs buffer containing 10, 20, 30, 50, 75, and 100 mM potassium ion stimulated concentration‐dependent tritium release. All of potassium‐evoked release, but only 17% of spontaneous release, was calcium‐dependent. Kinetic analysis of net (total minus spontaneous) potassium‐stimulated release revealed a single calcium‐dependent component of release that fit a single exponential function with a half‐life of 12.7 s. Analysis of the area under the tritium efflux curves observed on the millisecond time scale revealed that 0.111, 0.550, and 0.614% net tritium release was evoked by superfusion for 750 ms with isoosmotic buffer containing 20, 50, and 100 mAf KC1, respectively. Consistent with the results observed on the second time scale, a small fraction of spontaneous release and all of potassium‐evoked release observed on the millisecond time scale were calcium‐dependent. These data indicate that the technique of rapid superfusion can be utilized for the direct investigation of spontaneous and evoked [3H]acetylcholine release, as well as the factors that regulate this release from brain synaptosomes on the second and millisecond time scales.
Intragenic Sequences Affect the Expression of the Gene Encoding Glial Fibrillary Acidic Protein
Authors: Sarkar Srijata , Cowan Nicholas J.
Published: October 2006

We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three as‐acting elements. A positive regulatory element that is located between nucleotides ‐1, 631 and ‐1, 479 can confer cell type‐specific expression on a heterologous gene. A second regulatory element is located between nucleotides ‐97 and ‐80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells.
Inhibition of Glutamate Release by Arachidonic Acid in Rat Cerebrocortical Synaptosomes
Authors: Herrero Inmaculada , Miras‐Portugal Maria Teresa , Sańchez‐Prieto Jose
Published: October 2006

The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca2+‐dependent release of glutamate evoked by 4‐aminopyridine (4‐AP) was inhibited by arachidonic acid (0.5–10 μM), but the KC1‐evoked release was not modified. The Ca2+‐independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 μM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca2+‐dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4‐AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K+‐channel Mocker tetraethyl‐ammonium.
Cyclic AMP‐Dependent Protein Kinase Decreases γ‐Aminobutyric AcidA Receptor‐Mediated 36Q1− Uptake by Brain Microsacs
Authors: Leidenheimer Nancy J. , Machu Tina K. , Endo Shuichi , Olsen Richard W. , Harris R. Adron , Browning Michael D.
Published: October 2006

The effect of cyclic AMP (cAMP)‐dependent protein phosphorylation on γ‐aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol‐stimulated 36C1− uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP‐ dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol‐stimulated 36C1− uptake by ∼25%. Jn parallel experiments, PKA and [γ‐32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitatc the entire GABAA receptor complex, under nondenaturing conditions, using an anti‐α1‐subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66‐kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti‐α1 subunit antibody. Phosphopeptide mapping of the 66‐kDa polypeptide demonstrated a 14‐kDa fragment similar to that obtained with the purified, PKA‐phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain G ABAAreceptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.
A Specific Transduction Mechanism for the Glutamate Action on Phosphoinositide Metabolism via the Quisqualate Metabotropic Receptor in Rat Brain Synaptoneurosomes: II. Calcium Dependency, Cadmium Inhibition
Authors: Guiramand Janique , Vignes Michel , Récasens Max
Published: October 2006

: In this article, we demonstrate that an increase in intracellular Ca2+ concentration may represent a specific common step(s) in the mechanism(s) of action of glutamate (Glu) and depolarizing agents on formation of inositol phosphates (IPs) in 8‐day‐old rat forebrain Synaptoneurosomes. In fact, A23187, a Ca2+ ionophore, induces a dose‐dependent accumulation of IPs, which is not additive with that evoked by Glu and K+ but is slightly synergistic with that induced by carbachol. In addition, Glu and K+ augment the intracellular Ca2+ concentration in synaptoneurosome preparations as measured by the fura‐2 assay. The absence of external Ca2+ decreases basal and Glu‐, and K+‐stimulated formation of IPs. Cd2+(100 μM) fully inhibits both Gluand K+‐evoked formation of IPs without affecting the carbachol‐elicited response of IPs. Zn2+ inhibits Gluand K+‐stimulated accumulation of IPs (IC50∽ 0.4 mM) but with a lower affinity than Cd2+ (IC50∽ 0.035 mM). The organic Ca2+ channel blockers verapamil (10 μM), nifedipine (10 μM), ω‐conotoxin (2 μM), and amiloride (10 μM) as well as the inorganic blockers Co2+ (100 μM) and La3+ (100 μM) block neither Glunor K+‐evoked formation of IPs, a result suggesting that the opening of the L‐, T‐, N‐, or P‐type Ca2+ channels does not participate in these responses. All these data suggest that an increase in intracellular Ca2+ concentration resulting from an influx of Ca2+, sensitive to Cd2+ but not to other classical Ca2+ antagonists, may play a key rote in the transduction mechanism activated by Glu or depolarizing agents.
Evidence for an Insulin‐Like Growth Factor Autocrine‐Paracrine System in the Retinal Photoreceptor‐Pigment Epithelial Cell Complex
Authors: Waldbillig Robert J. , Pfeffer Bruce A. , Schoen Timothy J. , Adler Alice A. , Shen‐Orr Z. , Scavo Louis , ERoith Derek , Chader Gerald J.
Published: October 2006

: The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin‐like growth factor I (IGF‐I) immunoreactivity that co‐elutes with authentic human IGF‐I in HPLC analyses. Cultured human RPE cells synthesize and release IGF‐I, raising the possibility that the RPE serves as a source of IPM IGF‐I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF‐I receptors with α‐subunits of 120 and 138 kDa, respectively. They thus appear to be of the “brain” (in photoreceptors) and “peripheral” (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF‐BP) that specifically binds IGF‐I and IGF‐II. The IPM‐BP is visualized as a single radiographic band by both ligand blot and affinity cross‐linking procedures. With enzymes specific for removing N‐and O‐linked oligosaccharides, the IPM‐BP was found to contain O‐but not N‐linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM‐BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF‐I and IGF‐BP in the IPM, together with the presence of IGF‐I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine‐paracrine system.
Electroconvulsive Shock Increases α1b‐but Not α1a‐Adrenoceptor Binding Sites in Rat Cerebral Cortex
Authors: Blendy Julie A. , Perry David C. , Pabreza Laurie A. , Kellar Kenneth J.
Published: October 2006

: Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to α1‐adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two α1‐adrenoceptor subtypes, termed α1a and α1b, whereas [3HJWB4101 labels the α1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both α1‐adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10–12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to α1a‐adrenoceptors. Measurement of α1a and α1b receptors by competition analysis of the selective α1a antagonist 5‐methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates α1b binding sites, also indicated that the ECS‐indiced increase in α1‐adrenoceptors is confined to the α1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1‐phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1‐phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on α1‐adrenoceptor‐mediated phosphoinositide hydrolysis.
D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors
Authors: Lovenberg Timothy W. , Roth Robert H. , Nichols David E. , Mailman Richard B.
Published: October 2006

: Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two‐to threefold over baseline. The pseudo‐Km for dopamine was ⋍2 μM, whereas for dihydrexidine it was ⋍30 nM. The effects of both drugs were blocked by either the D1‐selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)‐butaclamol (Ki, 5 nM). Both (−)‐butaclamol and the D2‐selective antagonist (−)‐sulpiride were ineffective (Ki > 3 μM). Forskolin (10 μM), prostaglandin E1 (1 μM), and adenosine (10 μM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 μM). Finally, muscarinic receptor stimulation (100 μM carbachol) inhibited both D1‐and forskolin‐stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.
Transferrin and Iron Uptake by the Brain: Effects of Altered Iron Status
Authors: Taylor Eve M. , Crowe Andrew , Morgan Evan H.
Published: October 2006

: Transferrin (Tf) and iron uptake by the brain were measured in rats using 59Fe‐125I‐Tf and 131I‐albumin (to correct for the plasma content of 59Fe and 125I‐Tf in the organs). The rats were aged from 15 to 63 days and were fed (a) a low‐iron diet (iron‐deficient) or, as control, the same diet supplemented with iron, or (b) a chow diet with added carbonyl iron (iron overload), the chow diet alone acting as its control. Iron deficiency was associated with a significant decrease and iron overload with a significant increase in brain nonheme iron concentration relative to the controls, in each dietary treatment group, the uptake of Tf and iron by the brain decreased as the rats aged from 15 to 63 days. Both Tf and iron uptake were significantly greater in the iron‐deficient rats than in their controls and lower in the iron‐loaded rats than in the corresponding controls. Overall, iron deficiency produced about a doubling and iron overload a halving of the uptake values compared with the controls. In contrast to that in the brain, iron uptake by the femurs did not decrease with age and there was relatively little difference between the different dietary groups. 125I‐Tf uptake by the brains of the iron‐deficient rats increased very rapidly after injection of the labelled proteins, within 15 min reaching a plateau level which was maintained for at least 6 h. The uptake of 59Fe, however, increased rapidly for 1 h and then more slowly, and in terms of percentage of injected dose reached much higher values than did 125I‐Tf uptake. It is concluded that, after the age of 15 days in the rat, there is a decline in the rate of uptake of iron by the brain, probably attributable to a decrease in the number of Tf receptors on brain capillary endothelial cells, and that the expression of these receptors is highly responsive to the iron status of the animal.
Identification of Multiple Phosphoinositide‐Linked Receptors on Human SK‐N‐MC Neuroepithelioma Cells
Authors: Fisher Stephen K. , Landon Rachel E.
Published: October 2006

:The biochemical and pharmacological characteristics of receptor‐stimulated phosphoinositide (PPI) hydrolysis in human SK‐N‐MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., nor‐epinephrine, oxotremorine‐M, endothelin‐1, and ATP, each resulted in an increased release (three‐to eightfold) of inositol phosphates from [3H]inositol‐prelabeled cells. Agonist‐stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono‐and bis‐phosphates, inositol 1,3,4‐trisphosphate, and inositol tetra‐kisphosphate. Increases in inositol 1,4,5‐trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine‐M. Norepinephrine‐stim‐ulated PPI turnover was potently inhibited by prazosin, WB‐4101, and 5‐methylurapidil (K1, < 2.5 nM), but was relatively insensitive to chloroethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an α1A‐receptor subtype. The presence of an M1 muscannic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine‐M‐stimulated inositol phosphate release (K1= 35 nM) with a 30‐fold greater potency than the M2‐selective antagonist, AF‐DX 116. Of the three endothelins tested, only the addition of endothelin‐1 and endothelin‐2 promoted PPI hydrolysis, whereas endothelin‐3 was essentially inactive. A P2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK‐N‐MC cells express at least four PPI‐linked receptors. Because the functional coupling of three of these receptors, i.e., α1A‐adrenergic, endothelin, and P2 nucleotide, has not been extensively characterized previously in neural tissues, the SK‐N‐MC cell line may provide a useful model system for studies of these receptors and their regulation.
Iron‐Melanin Interaction and Lipid Peroxidation: Implications for Parkinson's Disease
Authors: Ben‐Shachar Dorit , Riederer P. , Youdim M. B. H.
Published: October 2006

: The vulnerability of substantia mgral (SN) melaninized dopamine neurons to neurodegeneration in Parkinson's disease and the selective increases of iron and basal lipid peroxidation in SN indicate that iron‐melanin interaction could be crucial to the pathogenesis of this disease. The present study describes, for the first time, the identification and characterization of a high‐affinity (KD= 13 nM) and a lower affinity (KD= 200 nM) binding site for iron on dopamine melanin. The binding of iron to melanin is dependent on pH and the concentration of melanin Iron dictators, U74500A, desferrioxamme, and to less extent 1,10‐phenanthroline and chlorpromazine, but not the Parkinson‐inducing neurotoxin, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, can inhibit the binding of iron to melanin and iron‐induced lipid peroxidation. Although melanin atone diminishes basal lipid peroxidation in rat cortical homogenates, it can also potentiate that initiated by iron, a reaction inhibited by desferrioxamine. In the absence of an identifiable exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron‐melanin interaction in pars compacta of SN may be a strong candidate for the cytotoxic component of oxygen radical‐induced neurodegeneration of meianinized dopamine neurons.
Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine
Authors: Moroni Flavio , Russi Patrizia , Gallo‐Mezo Miguel Angel , Moneti Gloriano , Pellicciari Roberto
Published: October 2006

: Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography‐mass spectrometry or HPLC. The enzyme indoleamine 2,3‐diox‐ygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin‐induced accumulation of quinolinic acid while potentiating the accumulation of‐kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.
Endo‐Oligopeptidase A., a Putative Enkephalin‐Generating Enzyme, in the Vertebrate Retina
Authors: Ferro E. S. , Hamassaki D. E. , Camargo A. C. M. , Britto L. R. G.
Published: October 2006

: Endo‐oligopeptidase A., EC 3.4.22.19, converts small enkephalin‐containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo‐oligopeptidase A found in pigeon retinae (30.3 ± 7.3 mU/mg, mean ± standard deviation) was four times higher than in rabbit retinae (7.0 ± 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5.5′‐dithiobis(2‐nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo‐oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double‐labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo‐oligopeptidase A‐like immune‐reactivity also contained [Leu5)‐enkephalin‐like immuno‐reactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo‐oligopeptidase A in enkephalin generation in vivo.
Hippocampal Kindling in the Rat Is Associated with Time‐Dependent Increases in the Concentration of Glial Fibrillary Acidic Protein
Authors: Hansen Anette , Jørgensen Ole Steen , Bolwig Tom G. , Barry David I.
Published: October 2006

: The effect of hippocampal kindling on the regional brain concentration of total glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, was studied in partially kindled rats, and in fully kindled rats after a post‐kindling period of 24h, 1 week, and 2 months. GFAP concentration was measured in arbitrary units by dot‐blots. In the hippocampus, dentate gyrus, basolateral amygdala, pyriform cortex, and entorhinal cortex, limbic structures which are known to be involved in the kindling process, there was an increase in GFAP concentration which was maximal in the fully kindled animals studied after 24 h. In most brain areas, GFAP concentration was still elevated 1 week post‐kindling, but had declined to control level 2 months post‐kindling. A significant increase in GFAP was also found in septum, ventral pallidum/accumbens nucleus, and primary motor cortex of kindled rats with a post‐kindling period of 24h, whereas in several other brain regions GFAP was unchanged. These results suggest that astrocyte activation, indicative of degenerative changes in nearby neurons, is a transient and regional phenomenon in kindling occurring only during the development of the kindled state.
Differential Localization of Alternative Spliced Transcripts Encoding Inositol 1,4,5‐Trisphosphate Receptors in Mouse Cerebellum and Hippocampus: In Situ Hybridization Study
Authors: Nakagawa Toshiyuki , Shiota Chiyo , Okano Hideyuki , Mikoshiba Katsuhiko
Published: October 2006

: Expression of inositol 1,4,5‐trisphosphate (InsP3) receptor subtypes was determined in mouse brain using oligonucleotide‐specific in situ hybridization. All subtypes, except one that deletes 120 bp from the full‐length InsP3 receptor cDNA, were expressed in cerebellar Purkinje cells. In hippocampus, various subtypes showed distinct expression patterns. These results suggest that regionally selective expression of InsP3 receptor subtypes may result in the generation of functionally distinct channels.
Repeated Idazoxan Increases Brain Imidazoline Receptors in Normotensive (WKY) but Not in Hypertensive (SHR) Rats
Authors: Olmos Gabriel , Miralles Antonio , Barturen Fernando , Garcìa‐Sevilla Jesús A.
Published: October 2006

: The specific binding of [3H]idazoxan in the presence of 10−6M(−)‐adrenaline was used to evaluate the density of imidazoline receptors in the brain of spontaneously hypertensive (SHR) rats and sex‐and age‐matched normotensive Wistar‐Kyoto (WKY) rats. In SHR rats the density of imidazoline receptors (cerebral cortex, hypothalamus, and medulla oblongata) was not different from that in normotensive (WKY) rats. However, repeated treatment with idazoxan consistently increased (23–80%) the density of imidazoline receptors in the various brain regions of WKY rats but not in SHR rats. In normotensive Sprague‐Dawley rats, repeated treatment with the imidazoline drugs idazoxan and cirazoline also increased (33–37%) the density of imidazoline receptors in the cerebral cortex. The lack of regulation by idazoxan of the density of imidazoline receptors in the brain of SHR rats might reflect the existence of a relevant abnormality of these receptors in this genetic model of hypertension.
Differential Responses of Expressed Recombinant Human γ‐Aminobutyric AcidA Receptors to Neurosteroids
Authors: Lan Nancy C. , Gee Kelvin W. , Bolger Michael B. , Chen Jie Sheng
Published: October 2006

: Neuroactive steroids, in particular 3α‐hydroxypregnanes, are allosteric modulators of the γ‐aminobutyric acidA (GABAA) receptor. Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and ncurosteroid modulatory effects. the effect of 5α‐pregnan‐3α‐ol‐20‐one (epiallopregnanolone) on heterotropic cooperativity on the GABAA receptor complex has been studied in three subtypes of expressed recombinant human receptors and in cat brain and spinal cord. Steroid potentiation of [3H]flunitrazepam binding was greatest for the α1β1γ2 receptor complex, whereas α1β1γ2 and α2β1γ2 complexes showed < 100% enhancement in binding. Previous studies suggest that the spinal cord is devoid of α1, whereas cerebellum is rich in α1 subunits. Correspondingly, a differential enhancement of [3H]flunitrazepam binding in spinal cord (51%) versus cerebellum (28%) was also observed. The structure of neuroactive steroids is important in determining the extent of neuromodulatory activity. The 5β‐pregnanes, 5β‐pregnan‐3α‐ol‐20‐one (epipregnanolone) and 5β‐pregnan‐3α,21‐diol‐20‐one (5β‐tetrahydrodeoxycorticosterone), were both less potent than their corresponding 5α derivatives. A 3α‐hydroxyl group is essential for neuromodulatory activity in the expressed receptors, as demonstrated by the observation that 5α‐preg‐nan‐3β‐ol‐20‐one (allopregnanolone) and 4‐pregnen‐3,20‐dione (progesterone) were both inactive. The ability to screen synthetic molecules using expressed human receptors that selectively contain individual subunit subtype combinations may prove to be a powerful tool in the development of therapeutic agents that act as allosteric modulators of the GABAA receptor and other neurotransmitter receptors as well.
Activation of a Microtubule‐Associated Protein‐2 Kinase by Insulin‐Like Growth Factor‐I in Bovine Chromaffin Cells
Authors: Cahill Anne L. , Perlman Robert L.
Published: October 2006

Treatment of bovine chromaffin cells with insulin‐like growth factor‐I (IGF‐I) caused the activation of a protein kinase that phosphorylates microtubule‐associated protein‐2 (MAP‐2) in vitro. Activation of MAP‐2 kinase by IGF‐I varied with the time of treatment (maximal at 10–15 min) and the concentration of IGF‐I (maximal at 10 nM). The IGF‐I‐activated MAP‐2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF‐I‐activated kinase also phosphorylated myelin basic protein, but had little or no activity toward his‐tones or ribosomal S6 protein. To examine the role of protein tyrosine phosphoryiation in the activation of the MAP‐2 kinase, we isolated phosphotyrosine (PTyr)‐containing proteins from chromaffin cells by immunoaffinity adsorption on anti‐PTyr‐Sepharose beads. Anti‐PTyr‐Sepharose eluates from IGF‐I‐treated cells showed increased MAP‐2 kinase activity; thus, the MAP‐2 kinase (or a closely associated protein) appears to be a PTyr‐containing protein. Treatment of anti‐PTyr‐Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphoryiation of the IGF‐I‐activated kinase is required for its activity.
Evidence for Atrial Natriuretic Peptide (ANP) Synthesis and the Presence of ANP‐Transducing Receptors in the Rat Olfactory Bulb
Authors: Gutkowska J. , Tremblay J. , Meyer R. , Marcinkiewicz M. , Nemer M.
Published: October 2006

This study demonstrates the presence of both atrial natriuretic peptide (ANP) precursor and ANP transcripts in the rat olfactory bulb (OB), a key brain structure involved in the generation of olfaction‐dependent behavior. In addition to synthesizing ANP, the OB contains ANP‐transducing receptors coupled to the guanylate cyclase system but it is devoid of ANP “clearance receptors.” The characterization of biologically active ANP receptors and the evidence for in situ ANP synthesis in this region of the CNS adds credence to the hypothesis that the peptide plays a putative role in olfaction.
Tellurium Blocks Cholesterol Synthesis by Inhibiting Squalene Metabolism: Preferential Vulnerability to This Metabolic Block Leads to Peripheral Nervous System Demyelination
Authors: Wagner‐Recio Maria , Toews Arrel D. , Morell Pierre
Published: October 2006

Inclusion of 1.1% elemental tellurium in the diet of postweanling rats produces a peripheral neuropathy due to a highly synchronous primary demyelination of sciatic nerve; this demyelination is followed closely by remyelination. Sciatic nerves from animals fed tellurium for various times were removed and incubated ex vivo for 1 h with [14C]acetate, and radioactivity incorporated into individual lipid classes was determined. In nerves from rats exposed to tellurium, there was a profound and selective block in the conversion of radioactive acetate to cholesterol. Another radioactive precursor, [3H]water, gave similar results. We suggest that tellurium feeding inhibits squalene epoxidase activity and that the consequent lack of cholesterol destabilizes myelin, thereby causing destruction of the larger internodes. Ex vivo incubation experiments were also carried out with liver slices. As with nerve, tellurium feeding caused accumulation in squalene of label from radioactive acetate, whereas labeling of cholesterol was greatly inhibited. Unexpectedly, however, incorporation of label from [3H]water into both squalene and cholesterol was increased. Relevant is the demonstration that liver was the primary site of bulk accumulation of squalene, which accounted for 10% of liver dry weight at 5 days. Thus, accumulation of squalene (and other mechanisms, possibly including up‐regulation of cholesterol biosynthetic pathways) drives squalene epoxidase activity at normal levels in liver even in the presence of inhibitors of this enzyme. This is reflected by continuing incorporation of [3H]water into cholesterol; incorporation of this precursor takes place at many of the postsqualene biosynthetic steps for sterol formation. [14C]Acetate entering the sterol pathway before squalene in liver is greatly diluted in specific activity when it reaches the large squalene pool, and thus increased squalene epoxidase activity does not transfer significant 14C label to sterols. In contrast to the situation with liver, synthesis of sterols is markedly depressed in sciatic nerve, and squalene does not accumulate to high levels.
Axonal Transport Reversal of Acetylcholinesterase Molecular Forms in Transected Nerve
Authors: Hässig Raymonde , Tavitian Bertrand , Pappalardo Francesca , Giamberardino Luigi Di
Published: October 2006

Reversal of anterograde rapid axonal transport of four molecular forms of acetylcholinesterase (AChE) was studied in chick sciatic nerve during the 24‐h period following a nerve transection. Reversal of AChE activity started ∼1 h after nerve transection, and all the forms of the enzyme, except the monomeric ones, showed reversal of transport. The quantity of enzyme activity reversed 24 h after transection was twofold greater than that normally conveyed by retrograde transport. We observed no leakage of the enzyme at the site of the nerve transection and no reversal of AChE activity transport in the distal segment of the severed nerve, a result indicating that the material carried by retrograde axonal transport cannot be reversed by axotomy. Thus, a nerve transection induces both quantitative and qualitative changes in the retrograde axonal transport, which could serve as a signal of distal injury to the cell body. The velocity of reverse transport, measured within 6 h after transection, was found to be 213 mm/day, a value close to that of retrograde transport (200 mm/day). This suggests that the reversal taking place in severed sciatic nerve is similar to the anterograde‐to‐retrograde conversion process normally occurring at the nerve endings.