bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis
Authors: Akiyama Yoshinori , Koshimura Kunio , Ohue Tetsuya , Lee Ken , Miwa Soichi , Yamagata Sen , Kikuchi Haruhiko
Published: October 2006

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4‐dihy‐droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage‐dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O2 in N2) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.
Physicochemical Characterization of Recombinant Human Nerve Growth Factor Produced in Insect Cells with a Baculovirus Vector
Authors: Barnett Jim , Chow Joan , Nguyen Binh , Eggers Daryl , Osen Eric , Jarnagin Kurt , Saldou Natalie , Straub Ken , Gu Leo , Erdos Lisa , Chaing Hi‐Shi , Fausnaugh Jodi , Townsend R. Reid , Lile Jack , Collins Frank , Chan Hardy
Published: October 2006

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion‐exchange and reversed‐phase chromatography to near homogeneity. The Nterminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (<0.08 mol of N‐acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF frominsect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.
Urinary but Not Brain Isatin Levels Are Reduced in Germ‐Free Rats
Authors: Sandier M. , Przyborowska Anna , Halket J. , Watkins P. , Glover Vivette , Coates Marie E.
Published: October 2006

Germ‐free rats excreted considerably smaller amounts of the monoamine oxidase‐inhibiting compound isatin than the substantially larger output by conventional animals of the same strain, although concentrations in brain and other tissues were similar in the two groups. Thus, isatin is likely to be elaborated both endogenously in rat tissues and “exogenously” by flora inhabiting the lumen of the alimentary tract.
Distribution of Na+, K+‐ATPase α‐Subunit Isoforms in Rat Pituitary
Authors: Rowe Paul M. , Link William T. , Osborn Charlene P. , Gainer Harold , Albers R. Wayne
Published: October 2006

The distributions of a‐subunit isoforms of the Na+, K+‐ ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only α1 and α2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only α1. but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+, K−‐ATPase activity in the neural lobe is consistent with the presence of both α2 and α3 subunits.
A Monoclonal Antibody That Reacts Immunohistochemically with Amyloid Deposits in the Brain Tissue of Alzheimer Patients Binds to an Epitope Present on Complement Factor 4
Authors: McDonald B. , Esiri M. M. , McIlhinney R. A. J.
Published: October 2006

The mouse monoclonal antibody SMP has previously been demonstrated to react immunohistochemically with neurofibrillary tangles, argyrophilic plaques, and lep‐tomeningeal vascular amyloid deposits in the brain tissue of individuals dying from pathologically diagnosed Alzheimer's disease. In preliminary studies the antibody was shown, by size exclusion chromatography, to bind to a protein with an apparent molecular mass of 260 kDa present in the CSF and serum of demented individuals. Chromatographic separation of a 40% ammonium sulphate precipitate of CSF and serum yielded immunoreactive fractions that were subjected to 9% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis followed by western blotting. Probing the nitrocellulose blot with the antibody revealed that the antibody selectively binds to a protein chain with an apparent molecular mass of 100 kDa. By using a combination of affinity chromatography and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, coupled with western blotting, the serum component with which the antibody reacts has been identified as complement factor 4. In addition, the antibody has been shown to react specifically with an epitope on the α‐chain of this protein.
Effect of Tetrahydrobiopterin on Serotonin Synthesis, Release, and Metabolism in Superfused Hippocampal Slices
Authors: Wolf William A. , Ziaja Ellen , Arthur Robert A. , Anastasiadis Panagiotis Z. , Levine Robert A. , Kuhn Donald M.
Published: October 2006

The effects of 6R‐5, 6, 7, 8‐tetrahydro‐L‐biopterin (6R‐BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R‐BH4did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KC1. Under the same incubation conditions, 6R‐BH4 altered neither the synthesis (basal or tryptophan‐stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6‐methyl‐5, 6, 7, 8‐tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R‐BH4, did not. The 6S isotner of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R‐ BH4 in stimulating serotonin release. 6R‐BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R‐BH4, like that produced by p‐chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KC1‐induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R‐BH4 increases the depolarization‐induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R‐BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.
Ca2+‐Independent, Ca2+‐Dependent, and Carbachol‐ Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane
Authors: Strosznajder Joanna , Samochocki Marek
Published: October 2006

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C] arachidonic acid ([14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly‐PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C(PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 μM CaC12 enhance AA liberation by approximately 25%, whereas 2 mM CaC12 resulted in a 50% increase in AA release relative to EGTA. The source for Ca2+‐dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca2+‐dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10–15%. This agonist‐mediated AA release occurs specifically from phosphoinositides (PI + poly‐PI). Quinacrine almost completely suppresses calcium‐ and carbachol‐mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca2+‐ independent AA release. Ca2+‐dependent and carbachol‐mediated AA liberation occurs mainly as the result of PLA2action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.
Exogenous Gangliosides GD1b and GD1b‐Lactone, Stably Associated to Rat Brain P2 Subcellular Fraction, Modulate Differently the Process of Protein Phosphorylation
Authors: Bassi Rosaria , Chigorao Vanna , Fiorilli Amelia , Sonnino Sandro , Tettamanti Guido
Published: October 2006

GD1b and GD1b‐lactone (GD1b‐L) gangliosides bind to the same extent to a P2 crude membrane preparation from rat brain. After 30 min of incubation with 10−4, 105, and 10−6 Absolutions of ganglioside, 1,800, 450, and 100 pmol of ganglioside/mg of protein, respectively, were found to be stably associated to the P2 fraction. This association modifies the phosphorylation process of the P2 membrane proteins in a dose‐dependent manner, the maximal effect being reached at a ganglioside association of 1.85 nmol/mg of protein and in large part at 450 pmol/mg of protein. The effects of GD1b and GD1b‐L on the phosphorylation of five proteins, showing apparent molecular masses of 17, 20, 36, 41, and 44 kDa, were different after 0.5 min of phosphorylation reaction as well as after 15 min. After 0.5 min of reaction, in the presence of stably associated GD1b, the phosphorylation of the 36‐, 41‐, and 44‐kDa proteins was increased with reference to the control, whereas the phosphorylation of the 17‐ and 20‐kDa proteins was decreased. GD1b‐L exerted qualitatively similar effects only on the 44‐, 41‐, and 36‐kDa proteins and to a strongly reduced degree. After 15 min of reaction, only the phosphorylation of the 36‐kDa protein was stimulated by GD1b; GD1b‐L exerted a similar effect, but to a low degree.
Astroglial‐Induced In Vitro Angiogenesis: Requirements for RNA and Protein Synthesis
Authors: Laterra John , Goldstein Gary W.
Published: October 2006

Astrocytes are believed to affect microvascular endothelial cell differentiation in brain and retina. Bovine retinal microvessel endothelial cells formed capillary‐like structures when cocultured with C6 astroglial cells or in the absence of C6 cells in response to the reconstituted basement membrane protein Matrigel. Using quantitative computer‐assisted image analysis, the requirements for RNA and protein synthesis in these two complementary models of in vitro microvessel morphogenesis were examined. Astroglial‐dependent capillary‐like structure formation was inhibited by up to 87% in a dose‐dependent fashion by cycloheximide (0.01–0.1 μg/ml), puromycin (0.1–0.25 μg/ml), and actinomycin D (0.01– 0.025 μg/ml). In contrast, the astroglial‐independent process in response to Matrigel was not affected by these metabolic inhibitors. These findings suggest that capillary‐like structures form in response to astroglial cells in two distinct sequential stages. The first consists of inductive astroglial‐endothelial interactions requiring both RNA and protein synthesis. This initiates endogenous endothelial morphogenic events that do not appear to require RNA or protein synthesis, consistent with posttranslational regulatory mechanisms. The first astroglial‐dependent step is relevant to the regulation of microvessel formation in brain and retina, whereas the second may represent a morphogenic pathway common to micro‐vessel formation in many tissues.
Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells
Authors: Manev Hari , Favaron Marco , Siman Robert , Guidotti Alessandro , Costa Erminio
Published: October 2006

Glutamate‐induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate‐induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose‐dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK‐801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N‐acetyl‐Leu‐Leu‐ norleucinal) prevented glutamate‐induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate‐induced neurotoxicity is independent of calpain I activation.
Characterization and Localization of a Long‐Chain Isoprenyltransferase Activity in Porcine Brain: Proposed Role in the Biosynthesis of Dolichyl Phosphate
Authors: Crick Dean C. , Rush Jeffrey S. , Waechter Charles J.
Published: October 2006

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1‐14C] isopentenyl pyrophosphate ([1‐MC] I‐P‐P) into long‐chain polyisoprenyl pyrophosphates (Poly‐P‐P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly‐P‐P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 μM I‐P‐P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (S) was stimulated by dithiothreitol. The major products were identified as C90and Q95 allyhic Poly‐P‐P on the basis of the following chemical and chromatographic properties: (1) the intact product cochromatographed with authentic Poly‐P‐P on silica‐gel‐impregnated paper, (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly‐P) by alkaline hydrolysis; (3) treatment of the labeled Poly‐P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly‐P from lipophilic Sephadex LH‐20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (Q95) isoprene units when analyzed by reverse‐phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long‐chain Poly‐P‐P. The enzymatic reactions by which the Poly‐P‐P intermediate is converted to dolichyl phosphate remain to be elucidated.
Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum
Authors: Farooqui Shakeel M. , Brock Jeffery W. , Hamdi Anwar , Prasad Chandan
Published: October 2006

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG‐KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24–34 and 176–185 from the NH2 terminus, respectively, were conjugated to keyhole limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross‐reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90‐kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I‐AP‐azidospiperone (125I‐NAPS). Photoaffinity labeling of the 90‐kDa protein by 125I‐NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90‐kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H] YM‐09151‐2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration‐dependent manner, a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H] YM‐09151‐2 binding to striatal membranes were examined in the presence of antibody and showed a 25–30% decrease in Bmax (203.5 ± 11.0 and 164.6 ±3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD. These results suggest that polyclonal antisera raised against peptide I exhibited specific antibodies for the dopamine D2 receptor protein. The primary epitope for this antibody is at or near the ligand binding site which can be recognized in both denatured and native receptor protein in striatal membranes.
Quantitative and Qualitative Alterations of Neuronal and Glial Intermediate Filaments in Rat Nervous System After Exposure to 2, 5‐Hexanedione
Authors: Karlsson J.‐E. , Rosengren L. E. , Haglid K. G.
Published: October 2006

The precise mechanism for the neurotoxicity of 2, 5‐hexanedione is not known, but cross‐linking of neurofilament proteins has been suggested as one possibility. In this study the effects of long‐term exposure to 2, 5‐hexanedione were studied in the rat nervous system with special reference to regional changes in the quantities of neuronal and glial intermediate filaments. Using enzyme‐linked immunosorbent assays the concentrations of 68‐ and 200‐kDa neurofilament polypeptides were shown to be reduced in all brain regions studied. Similar results were obtained in the sciatic nerve. The concentration of glial fibrillary acidic protein was decreased in the cerebellar vermis and the dorsal cerebral cortex, whereas it was increased in the spinal cord, a result suggesting a regional variation in glial sensitivity. The intermediate filaments of the exposed animals were also immunoblotted using polyclonal antisera against the various neurofilament polypeptides and glial fibrillary acidic protein. In all tissues studied, several aggregates with molecular weights higher than those of the monomeric polypeptides were demonstrated. Contrary to clinical observations, these data indicate pronounced effects in both CNS and PNS and call for further studies on CNS effects in humans.
D1 Dopamine Receptors Can Interact with Both Stimulatory and Inhibitory Guanine Nucleotide Binding Proteins
Authors: Sidhu Anita , Sullivan Molly , Kohout Trudy , Balen Paul , Fishman Peter H.
Published: October 2006

Pretreatment of striatal membranes with N‐ethylmaleimide in the presence of a D1specific agonist inactivated endogenous guanine nucleotide binding proteins (G proteins), but not D, dopamine receptors, resulting in a loss of high‐affinity agonist binding sites. Such D1 receptors were solubilized, mixed with exogenous G proteins from cells not containing D1receptors, and reconstituted into phospholipid vesicles. These reconstituted receptors were able to couple to the exogenous G proteins, and the proportion of agonist high‐affinity sites of the receptor (40–57%) was similar to levels obtained with naive receptors coupling to endogenous G proteins (40%) upon solubilization and reconstitution. These hybrid high‐affinity sites were fully modulated by guanine nucleotides. Pretreatment of cells with pertussis toxin prior to extraction of G proteins resulted in a 50% decrease in the proportion of high‐affinity sites; these sites remained sensitive to guanine nucleotides. When D, receptors were reconstituted with extracts of cyc− cells, which lack stimulatory G proteins, the proportion of high‐affinity sites was reduced to 31% of the total. Pertussis toxin treatment of the cyc− cells completely abolished the formation of high‐affinity sites. These results demonstrate that D1‐dopaminergic receptors are able to couple to not only stimulatory G proteins (Gs), but also to inhibitory G proteins (Gi).
B‐50 (GAP‐43): Biochemistry and Functional Neurochemistry of a Neuron‐Specific Phosphoprotein
Authors: Coggins Philip J. , Zwiers Henk
Published: October 2006

Abstract: The biochemistry and functional neurochemistry of the synaptosomal plasma membrane phosphoprotein B‐50 (GAP‐43) are reviewed. The protein is putatively involved in seemingly diverse functions within the nervous system, including neuronal development and regeneration, synaptic plasticity, and formation of memory and other higher cognitive behaviors. There is a considerable amount of information concerning the spatial and temporal localization of B‐50 (GAP‐43) in adult, fetal, and regenerating nervous tissue but far less is known about the physical chemistry and biochemistry of the protein. Still less information is available about posttranslational modifications of B‐50 (GAP‐43) that may be the basis of neurochemical mechanisms that could subsequently permit a variety of physiological functions. Hence, consideration is given to several plausible roles for B‐50 (GAP‐43) in vivo, which are discussed in the context of the cellular localization of the protein, significant posttranslational enzymes, and regulatory proteins, including protein kinases, phosphoinositides, calmodulin, and proteases.
Induction of 5‐Deiodinase Activity in Astroglial Cells by 12‐O‐Tetradecanoylphorbol 13‐Acetate and Fibroblast Growth Factors
Authors: Courtin F. , Liva P. , Gavaret J. M. , Toru‐Delbauffe D. , Pierre M.
Published: October 2006

Abstract: In the brain, 5′‐deiodinase (5′‐D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3′‐triiodothyronine (T3) and 5‐deiodinase (5‐D) deiodinates T4 and T3 into inactive metabolites. This study examines the effects of factors known to induce astroglial 5′‐D activity on the 5‐D activity in cultured rat astroglial cells. The potencies of these factors were compared after 8 h of incubation, when stimulations by these factors near their maximal effects. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) at 10−7M was a potent inducer of 5‐D activity, producing a 30‐ to 80‐fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5‐D activity was not dependent on glucocorticoids, unlike 5′‐D activity. In comparison with TPA, 8‐bromo‐cyclic AMP (10−3M) was a poor inducer of 5‐D activity whereas it is an excellent inducer of 5′‐D activity. It produced a 2‐ to 20‐fold increase in 5‐D activity after 8 h. Natural acidic fibroblast growth factor (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic fibroblast growth factor was observed after about 16 h (until a 120‐fold increase). Recombinant acidic fibroblast growth factor also induced 5‐D activity. Basic fibroblast growth factor was less potent than acidic fibroblast growth factor for increasing 5‐D activity (maximal increase by 40‐ to 50‐fold after 8 h). Platelet‐derived growth factor (20 ng/ml) and epidermal growth factor (100 ng/ml) were poor inducers of 5‐D activity (8‐ to 12‐fold increase at 8 h); insulin (10−6M) was without effect. The 5‐D activity induced by TPA and acidic fibroblast growth factor manifested the characteristics of type III 5‐D (Km for T3 0.5–0.8 nM, thiol‐dependent, 6‐n‐propyl‐2‐thiouracil‐insensitive). The results demonstrate that, like 5′‐D activity, 5‐D activity is induced by multiple pathways. The relative potencies of TPA, fibroblast growth factors, and cyclic AMP on 5‐D and 5′‐D activities were different, as were the time courses of their actions. These data indicate that 5‐ and 5′‐D are distinct enzymes and support the view that T3 availability may be controlled not only by regulating T3 production, but also by regulating T3 and T4 degradation.
A Method to Measure Simultaneously Cyclic AMP and Inositol Phosphate Accumulation in Rat Brain Slices
Authors: Morin D. , Zini R. , Querol‐Ferrer V. , Sapena R. , Tillement J. P.
Published: October 2006

Abstract: The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion‐exchange chromatography and their concentrations were determined by means of a double‐labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70–80% for inositol phosphates and 40–50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with α‐ and β‐adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H‐inositol phosphates and [14C]cyclic AMP in a concentration‐dependent process. EC50 values were similar for the two, 8.5 ± 3.9 μM for 3H‐inositol phosphates and 20.2 ± 6.3 μM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H‐inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor‐coupled effectors.
Autoradiographic Imaging of [3H]Phorbol 12,13‐Dibutyrate Binding to Protein Kinase C in Alzheimer's Disease
Authors: Horsburgh Karen , Dewar Deborah , Graham David I. , McCulloch James
Published: October 2006

Abstract: Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13 dibutyrate ([3HIPDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being >60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CAI of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex, e.g., layers 1–11 control, 491 ± 46 versus AD, 537 ± 39 pmol/g of tissue; middle temporal cortex, e.g., layers 1–11 control, 565 ± 68 versus AD, 465 ± 72 pmol/g of tissue; and hippocampal formation, e.g., CAI control, 51 ± 28 versus AD, 498 ± 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well‐defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical correlates of AD.
Output, Tissue Levels, and Synthesis of Acetylcholine During and After Transient Forebrain Ischemia in the Rat
Authors: Kumagae Yoshihiro , Matsui Yoshiki
Published: October 2006

Abstract: Biochemical changes in the rat brain cholinergic system during and after 60 min of ischemia were studied using a four‐vessel occlusion model. Extracellular acetylcholine (ACh) concentrations in the unanesthetized rat hippocampus markedly increased during ischemia and reached a peak (about 13.5 times baseline levels) at 5–10 min after the onset of ischemia. At 2–5 h after reperfusion, extracellular ACh concentrations were reduced to 64–72% of the levels of controls. ACh levels in the hippocampus, striatum, and cortex decreased significantly during ischemia and exceeded their control values just after reperfusion. A significant increase in hippocampal ACh level after 2 days of reperfusion and a decrease in [14C]ACh synthesis from [14C]glucose in hippocampal slices excised at 2 days after reperfusion were observed. The extracellular concentrations and tissue levels of choline markedly increased after ischemia. These results show that ACh is markedly released into the extracellular space in the hippocampus during ischemia, and they suggest that ACh synthesis is activated just after reperfusion and that cholinergic activity is reduced after 2–48 h of reperfusion in the hippocampus.
Decreased Release of D‐Aspartate in the Guinea Pig Spinal Cord After Lesions of the Red Nucleus
Authors: Benson Christina G. , Chase Michael C. , Potashner Steven J.
Published: October 2006

Abstract: This study attempts to determine if fibers that project from the guinea pig red nucleus to the spinal cord use L‐glutamate and/or L‐aspartate as transmitters. Unilateral injections of kainic acid were placed stereotaxically in the red nucleus to destroy the cells of origin of the rubrospinal tract. Six days after the injection, Nissl‐stained sections through the lesion site showed that the majority of neurons in the red nucleus ipsilateral to the kainic acid injection were destroyed. In addition, the lesioned area included parts of the surrounding midbrain reticular formation. Silver‐impregnated, transverse sections of the cervical spinal cord revealed the presence of degenerating fibers contralaterally in laminae IV–VII of the gray matter. Ipsilaterally, very sparse degeneration was evident in laminae VII and VIII of the gray matter. Two to six days after surgery, the electrically evoked, Ca2+‐dependent release of both D‐[3H]aspartate, a marker for glutamatergic/aspartatergic neurons, and γ‐amino[14C]‐butyric acid ([14C]GABA) was measured in dissected quadrants of the spinal cervical enlargement. Lesions centered on the red nucleus depressed the release of D‐[3H]aspartate by 25–45% in dorsal and ventral quadrants of the cervical enlargement contralaterally. The release of [14C]GABA was depressed by 27% in contralateral ventral quadrants. To assess the contribution of rubro‐ versus reticulospinal fibers to the deficits in amino acid release, unilateral injections of kainic acid were placed stereotaxically in the midbrain reticular formation lateral to the red nucleus. Nissl‐stained sections through the midbrain revealed the presence of extensive neuronal loss in the midbrain and rostral pontine reticular formation, whereas neurons in the red nucleus remained undamaged. In the spinal cord, degenerating axons were present ipsilaterally in laminae VII and VIII of the gray matter. Some fiber degeneration was also evident contralaterally in laminae V and VI of the gray matter. This lesion did not affect the release of either D‐[3H]aspartate or [14C]GABA in the spinal cord. The substantial decrements in D‐[3H]aspartate release following red nucleus lesions suggests that the synaptic endings of rubrospinal fibers mediate the release of D‐[3H]aspartate in the spinal cord. Therefore, these fibers may be glutamatergic and/or aspartatergic. Because other evidence suggests that rubrospinal neurons are probably not GABAergic, the depression of [14C]GABA release probably reflects changes in the activity of spinal interneurons following the loss of rubrospinal input.
Interaction of 1‐Methyl‐4‐Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase
Authors: Ramsay Rona R. , Krueger Matthew J. , Youngster Stephen K. , Gluck Martin R. , Casida John E. , Singer Thomas P.
Published: October 2006

Abstract: Nigrostriatal cell death in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1‐methyl‐4‐phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4′‐analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4′‐heptyl‐MPP+, being 6 μM. The 4′‐analogs compete with rotenone for the binding site in a concentration‐dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4′‐propyl‐MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.
Positive and Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Cortical Prisms Incubated Under “Resting”Conditions
Authors: Doležal Vladimír , Tuček Stanislav
Published: October 2006

Abstract: The effects of tacrine (1,2,3,4‐tetrahydro‐9‐aminoacridine) and 7‐methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low‐potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under “resting”conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium‐3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium‐3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis. Most experiments were performed with tacrine and methoxytacrine at concentrations of 100 μmol/L, but a positive action of tacrine on the synthesis of acetylcholine was apparent at a concentration as low as 10 μmol/L. The mechanism by which tacrine and methoxytacrine increase the utilization of intracellular choline or its esters for the synthesis of acetylcholine has not been identified.
Coexistence of γ‐Aminobutyric Acid Type A and Type B Receptors in Testicular Interstitial Cells
Authors: Ritta Mónica N. , Campos María B. , Calandra Ricardo S.
Published: October 2006

Abstract: The existence of specific γ‐aminobutyric acid (GABA)ergic receptors in testicular interstitial cells was investigated in the present study. Specific binding of [3H]GABA to interstitial cell membranes was found to be time‐ and temperature‐dependent and varied according to Ca2+ concentration present in the incubation medium. We analyzed the ability of different GABAergic agonists and antagonists to displace the bound radioactivity. In the absence of Ca2+ (1 mM EDTA), GABA and the GABAergic agonist isoguvacine displaced the bound radioactivity. When the radioligand assay was performed in the presence of 2.5 mM CaCl2, the [3H]GABA specifically bound increased twofold. Under such conditions, the specific GABAergic agonist baclofen, as well as GABA and isoguvacine, displaced the [3H]GABA bound. Saturation analysis revealed the presence of a population of GABAA binding sites with a KD value of 45.2 nM and a maximal number of binding sites of 57.4 fmol/mg of protein. The maximal binding increased on addition of 2.5 mM CaCl2 to 102 fmol/mg of protein, indicating the existence of a second population of GABAergic receptors, i.e., type B, with essentially the same affinity. In addition, the incubation of testicular interstitial cells with GABA and baclofen resulted in an increase in androgen production. These results support a functional role of GABA in the neuroendocrine control of the male gonad.
Differences in the Pharmacokinetics of Cocaine in Naive and Cocaine‐Experienced Rats
Authors: Pan Hwai‐Tzong , Menacherry Stanley , Justice J. B.
Published: October 2006

Abstract: Enhanced cocaine concentrations in brain and blood observed after an intraperitoneal challenge dose in rats exposed to cocaine for 10 days by subcutaneous administration are traced to a change in the absorption process from the site of an intraperitoneal injection to general circulation. This conclusion is reached by three sets of corroborating results: (a) Adipose tissue of rats treated for 10 days with repeat subcutaneous injections of cocaine did not reveal a buildup of cocaine in sufficient concentrations to account for the twofold increase in brain and blood concentrations seen during intraperitoneal administration; (b) administration of the drug by an intravenous route after 10‐day cocaine treatment did not show a significant difference between treatment and control groups; (c) nonlinear regression on the intravenous and intraperitoneal data sets using a two‐compartment open model indicated a difference in the absorption process but not in the metabolic and blood‐brain transfer processes.
Postnatal Development and Isolation of Peroxisomes from Brain
Authors: Lazo Oscar , Singh Avtar K. , Singh Inderjit
Published: October 2006

Abstract: We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl‐CoA oxidase and β‐oxidation of [1‐14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10‐day‐old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self‐generating gradient) banded at a density of 1.036 ± 0.012 g/ml and in Nycodenz continuous gradient at 1.125 ± 0.014 g/ml. Acyl‐CoA oxidase, D‐amino acid oxidase, L‐pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.
Neurofilament Protein Phosphorylation in Spinal Cord of Experimentally Diabetic Rats
Authors: Pekiner Can , McLean W. Graham
Published: October 2006

Abstract: This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate‐dependent (RT97) and ‐independent (RMd09) antibodies against high‐molecular‐mass NFPs on Western blots. Neurofilament‐associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein‐bound radioactivity in NFPs from diabetic animals was reduced to 86.7 ± 3.4 and 54.3 ± 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p < 0.001 and p < 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.
Dopaminergic Regulation of Enkephalin Release
Authors: Llorens‐Cortes C. , Zini S. , Gros C. , Schwartz J.‐C.
Published: October 2006

Abstract: The effects of dopamine receptor stimulation on enkephalin release were evaluated in vitro and in vivo by measuring the changes in the levels of [Met5]enkephalin (YGGFM) and Tyr‐Gly‐Gly (YGG), a characteristic extracellular enkephalin metabolite produced under the action of enkephalinase. In rat striatal slices, D1‐receptor agonists or antagonists did not modify enkephalin release. By contrast, D2‐receptor agonists enhanced the potassium‐induced release of YGGFM and YGG without affecting spontaneous release from nondepolarized slices. This response was prevented by the D2‐receptor antagonists haloperidol and RIV 2093, the latter compound being more potent, which suggested the involvement of a putative D2‐receptor subtype. Acute administration of apomorphine or selective D2‐receptor agonists, but not that of a D1‐receptor agonist, enhanced the steady‐state level of YGG without affecting the YGGFM level in rat striatum. The effect was blocked selectively by D2‐receptor antagonists which, administered alone, had no effect. These observations indicate that D2‐receptor stimulation in vitro or in vivo facilitates enkephalin release from striatal neurons, but that endogenous dopamine does not exert any tonic influence upon the opioid peptide neuron activity under basal conditions. However, chronic administration of haloperidol resulted in increases in striatal YGGFM and YGG, an effect presumably reflecting a long‐term adaptive process.
Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2‐Adrenergic Agonists
Authors: Ong Mei‐Leng , Ball Stephen G. , Vaughan Peter F. T.
Published: October 2006

Abstract:Noradrenaline (NA) and the α2‐adrenergic agonists clonidine, BHT‐920, and UK 14304‐18 inhibit potassiumevoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10−5M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304‐18, clonidine, and BHT‐920, respectively, all at 10−5M.Potassium‐evoked release was enhanced by both forskolin (10−5M) and 1 mMdibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mMdibutyryl cyclic AMP in the presence of 3‐isobutyl‐1‐methylxanthine partially reversed the α2‐adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10−5Mforskolin. The effects of clonidine, BHT‐920, UK‐14308–18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these α2‐adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to α2‐adrenergic receptors. These results are discussed in relation to recent findings that several α2‐adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that α2‐adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.
Effect of Cholecystokinin Octapeptide on Endogenous Amino Acid Release from the Rat Ventromedial Nucleus of the Hypothalamus and Striatum
Authors: Barnes Susan , Whistler Harold L. , Hughes John , Woodruff G. N. , Hunter John C.
Published: October 2006

Abstract: The sulphated octapeptide of cholecystokinin (CCK‐8S) was found to cause a dose‐dependent increase in the basal release of aspartate, glycine, and γ‐aminobutyric acid from the striatum and the ventromedial nucleus of the hypothalamus (VMH). No effect on amino acid release was observed after electrical (VMH) or potassium (striatum) stimulation. Experiments performed using the CCKB‐selective antagonist L‐365,260 and the CCKA‐selective antagonist L‐364,718 suggested that this action of CCK‐8S was mediated via the CCKB receptor. The ability of CCK‐8S to evoke amino acid release was not dependent on the presence of extracellular calcium, though the effect was abolished by tetrodotoxin. Inhibition of protein kinase activity by staurosporine prevented the excitatory effects of CCK‐8S on amino acid release.
Carassin: A Tachykinin That Is Structurally Related to Neuropeptide‐γ from the Brain of the Goldfish
Authors: Conlon J. Michael , O'Harte Finbarr , Peter Richard E. , Kah Olivier
Published: October 2006

Abstract: A 21‐amino‐acid residue tachykinin‐related peptide, carassin, was isolated in pure form from an extract of the brain of the goldfish, Carrassius auratus, by reversedphase HPLC. The primary structure of the peptide was established as the following: Ser‐Pro‐Ala‐Asn‐Ala‐Gln‐IIe‐Thr‐Arg ‐ Lys ‐ Arg ‐ His ‐ Lys ‐ Hle ‐ Asn ‐ Ser ‐ Phe ‐ Val ‐ Gly ‐ Leu‐Met · NH2. This amino acid sequence is the same length as and shows structural similarity (57% homology) to the mammalian tachykinin, neuropeptide‐γ, which is a product of the posttranslational processing of γ‐preprotachykinin. The mammalian tachykinins, substance P and neurokinin B, were not detected in the extract by using specific antisera directed against the NH2‐termini of the peptides, but an antiserum directed against the COOH‐terminal region of substance P did detect a low concentration of immunoreactive material.