bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractDiethyldithiocarbamic 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

AbstractThe 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

AbstractThe 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

AbstractWe 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

AbstractThe 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

AbstractThe 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

Abstract: 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

Abstract: 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

Abstract: 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

Abstract: 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

Abstract: 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

Abstract: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.