bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuronal and Glial Release of [3H]GABA from the Rat Olfactory Bulb
Authors: Jaffé E. H. , Cuello A. C.
Published: October 2006

Abstract: GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [3H]‐γ‐aminobutyric acid ([3H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [3H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine‐evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine‐evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca2+‐free solution. β‐Alanine was strongly exchanged with [3H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The β‐alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine‐stimulated release of [3H]GABA was not significantly reduced after the β‐alanine heteroexchange. Stimulation of the [3H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [3H]GABA release when a high magnesium concentration was added to a calcium‐free solution. When D‐600 was used as a calcium‐flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.
Isolation and Identification of Large Overlapping Fragments of Rabbit Myelin Basic Protein Produced by Limited Peptic Hydrolysis
Authors: Martenson Russell E. , Law Mona J. , Deibler Gladys E. , Lüthy Vivian
Published: October 2006

Abstract: Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 m‐ammonium formate (pH 6.00) for 15–20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion‐exchange chromatography and gel filtration and identified by amino acid and COOH‐terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44‐Phe45, Phe87‐Phe88, Leu109‐Ser110, and Leu151‐Phe152 bonds were isolated: peptides (1–151), (1–109), (1–87), (45–168), (45–151), (45–109), (88–168), (88–151), and (110–168). Of these, peptides (1–151), (1–87), and (88–151) were recovered in the greatest yield (0.14–0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1–109) and (110–168), indicating that the Leu109‐Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe‐Phe and Leu‐Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1–44), (1–28), (45–87), (88–109), (110–151), and (152–168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above‐mentioned peptides, together with the previously isolated peptides (1–14) and (15–44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 m‐acetic acid and 8 m‐urea.
Active Uptake of Substance P Carboxy‐Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices
Authors: Nakata Yoshihiro , Kusaka Yoshiko , Yajima Haruaki , Segawa Tomio
Published: October 2006

Abstract: We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy‐terminal heptapeptide, (5–11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5–11)SP, the uptake of (5–11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5–11)SP was accumulated by means of a high‐affinity and a low‐affinity uptake system. The Km and the Vmax values for the high‐affinity system were 4.20 × 10−8m and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low‐affinity system were 1.00 × 10−6m and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 × 10−7m and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5–11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.
Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites
Authors: Mena E. Edward , Foster Alan C. , Fagg Graham E. , Cotman Carl W.
Published: October 2006

Abstract: Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X‐100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for l‐glutamate, l‐aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ‐like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for l‐glutamate (l‐GlU) and l‐aspartate (l‐Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ‐like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.
Postnatal Changes in Enzyme Activities in Synaptosomes Isolated from Hamster Optic Nerve Endings
Authors: Specht Susan C.
Published: October 2006

Abstract: Subsynaptosomal fractions isolated from optic terminal nuclei of adult and neonatal hamsters exhibited developmental changes in specific density, mitochondrial activity, and K+‐stimulated, ouabain‐inhibited p‐nitrophenylphosphatase (K‐pNPPase) activity around the time of eye opening. The specific activity of K‐pNPPase was six‐ to sevenfold higher after eye opening (14–16 days postnatal). A significant proportion of high‐specific‐activity K‐pNPPase was recovered from the lightest subsynaptosomal fraction at all ages. This fraction contained very little external membrane by galactose oxidase–NaB3H4 labeling, suggesting that it may represent an internal pool, possibly the axonally transported form of the enzyme. Synaptic mitochondrial cytochrome c oxidase activity also approximately doubled in the period between 12 and 16 days. The specific density of the external membrane increased more slowly, banding at 1.0M sucrose at 12 and 16 days, and at 1.2M in adults. These maturational events may reflect increased energetic needs of optic nerve endings following eye opening.
Evidence for Glucocorticoid Target Cells in the Rat Optic Nerve. Hormone Binding and Glycerolphosphate Dehydrogenase Induction
Authors: Meyer Jerrold S. , Leveille Paula J. , de Vellis Jean , Gerlach John L. , McEwen Bruce S.
Published: October 2006

Abstract: Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol‐3‐phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid‐inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. Correlative immunocytochemical studies found GPDH‐positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high‐affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately twofold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.
Effects of Septal Lesions on Enzymes of Acetyl‐CoA Metabolism in the Cholinergic System of the Rat Hippocampus
Authors: Szutowicz Andrzej , Srere Paul A. , Allen Charles N. , Crawford Isaac L.
Published: October 2006

Abstract: Electrolytic lesions made in the medial septum of the rat brain caused an 80% decrease in the activity of choline acetyltransferase and a 33% reduction in ATP‐citrate lyase activity in the synaptosomal fraction from the hippocampus. Decreases in the activities of the two enzymes in the cytosol (S3) fraction were 70 and 13%, respectively. The activities of pyruvate dehydrogenase, citrate synthase, acetyl‐CoA synthase, and carnitine acetyltransferase in crude hippocampal homogenates and in subcellular fractions were not affected by septal lesions. The data indicate that ATP‐citrate lyase is linked to the septal‐hippocampal pathway and that the enzyme is preferentially located in cholinergic nerve endings that terminate within the hippocampus.
Alterations of Brain Cholecystokinin Receptors in Mice Made Obese with Goldthioglucose
Authors: Saito Atsushi , Williams John A. , Waxier Samuel H. , Goldfine Ira D.
Published: October 2006

Abstract: We investigated the binding of cholecystokinin (CCK) to its receptors in brains of mice made obese by the injection of goldthioglucose. In these mice there was a significant increase in the number of binding sites in cerebral cortex, hypothalamus, and olfactory lobes in comparison with either unin‐jected mice or injected mice that did not become obese. The present study demonstrates, therefore, that obesity in mice is associated with changes in brain CCK receptors, and raises the possibility that obesity in certain animals may be related to abnormalities in the binding of CCK to its receptors.
Cyclic Nucleotides and the Release of Vasopressin from the Rat Posterior Pituitary Gland
Authors: Vale Marcus R. , Hope Derek B.
Published: October 2006

Abstract: When rat posterior pituitary glands were stimulated by a high concentration of potassium, a peak of cyclic AMP and a peak of cyclic GMP were detected after 0.5 min and 1 min, respectively, whereas the rate of release of vasopressin was maximal only after 2 min. When calcium was omitted from the medium, no significant changes in cyclic nucleotide levels were found and the vasopressin release remained at the basal rate. During cold‐stimulated (10°C) release of vasopressin, a peak of cyclic AMP was detected after 5 min simultaneously with the maximal rate of vasopressin release. The significance of the cyclic nucleotides in the release of vasopressin is discussed.
In Vitro Release of Endogenous β‐ Alanine, GABA, and Glutamate, and Electrophysiological Effect of β‐Alanine in Pigeon Optic Tectum
Authors: Toggenburger Gerhard , Felix Dominik , Cuénod Michel , Henke Hermann
Published: October 2006

Abstract: The efflux of 20 amino acids, induced by either high K+ concentration or veratrine, was determined in pigeon tectal slices. Ca2+‐dependent, K+‐induced release of β‐alanine, γ‐aminobutyric acid (GABA), and glutamate was observed. Veratrine caused release of the same amino acids plus glycine in a tetrodotoxin‐sensitive manner. β‐Alanine had a strong inhibitory effect on the activity of tectal neurons which was blocked by strychnine but not by bicuculline. The results indicated a transmitter function for β‐alanine in the optic tectum, and were consistent with the previously proposed transmitter role of GABA and glutamate in this structure.
Increased Salsolinol Levels in Rat Striatum and Limbic Forebrain Following Chronic Ethanol Treatment
Authors: Sjöquist Birgitta , Liljequist Sture , Engel Jörgen
Published: October 2006

Abstract: Endogenous levels of salsolinol and its methylated metabolite were measured by combined gas chromatography and mass spectrometry in rats chronically exposed to ethanol for 150 days. The chronic ethanol administration produced a significant increase of salsolinol concentrations in dopamine‐rich brain areas, e.g., the striatum and the limbic forebrain. A negative correlation was observed between plasma ethanol concentration and the level of salsolinol in the brain. A possible role for salsolinol in the regulation of ethanol drinking and/or in the development of ethanol dependence is discussed.
Subcellular Distribution of 3α‐Hydroxysteroid Dehydrogenase and Antiestrogen Action on Androgen‐Metabolizing Enzymes in Rat Pituitary Gland
Authors: Ghraf Rüdiger , Schneider Klaus , Kirchhoff Josef , Hiemke Christoph
Published: October 2006

Abstract: Gonadectomy of male rats led to a threefold increase of 3α‐hydroxysteroid dehydrogenase (3α‐HSDH) activity in pituitary homogenates that could be completely reversed by chronic administration of estradiol or 5α‐dihydrotestosterone (DHT). 3α‐HSDH was found to be distributed mainly between the 10,000 g and 100,000 g sediments from whole homogenates. The microsomal enzyme activity showed a substantial specificity for NADH whereas the cytosolic enzyme (100,000 g supernatant) demonstrated a slight preference for NADPH. The changes in Vmax found in homogenates following gonadectomy and gonadal steroid administration reflected changes in NADH‐ linked activity of the microsomal, but not the cytosolic enzyme. Estradiol‐induced suppression of NADH‐linked 3α‐HSDH activity in pituitary homogenates from gonadectomized rats of either sex was accompanied by a similar suppression of NADPH‐linked 5α‐reductase activity and a marked decrease of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) release. In the ovariectomized rat chronic administration of nonsteroidal antiestrogens had strong estrogenic effects on 3α‐HSDH activity and LH release, but not on 5α‐reductase activity and FSH release. In the gonadectomized male rat, which was much less sensitive to intrinsic estrogenicity of the antiestrogens tested, nafoxidine completely blocked estradiol‐induced suppression of 5α‐reductase activity and FSH release and partially antagonized suppression of LH release. The trans‐isomeric, substituted triphenylethylenes, tamoxifen, and enclomiphene, as well as nitromifene (mixture of trans and cis isomers) were able partially to counteract estradiol‐induced suppression of 5α‐reductase, but not 3α‐HSDH activity. It is concluded that estradiol action on pituitary 5α‐reductase, but not 3α‐HSDH activity, involves an estrogen receptor mechanism.
On the Phospholipid Metabolism of Glial Cell Primary Cultures: Cell Characterization and Their Utilization of 1‐Alkyl‐Glycerophosphoethanolamine
Authors: Witter Brigitte , Debuch Hildegard
Published: October 2006

Abstract: Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with l‐[3H]alkyl‐sn‐glycerophosphoethanolamine in varying concentrations for 3 h. About one‐third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one—a Km was determined with 5 nmol intracellular 1‐alkyl‐sn‐glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1‐Alkyl‐sn‐glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.
Molecular Interactions of Etazolate with Benzodiazepine and Picrotoxinin Binding Sites
Authors: Ticku Maharaj K. , Davis William C.
Published: October 2006

Abstract: Pyrazolopyridines, such as etazolate (SQ 20009), enhance [3H]diazepam binding to a Lubrol‐solubilized fraction that has specific binding sites for 3H benzodiazepines, [α‐3H]dihydropicrotoxinin (DHP) and [3H]muscimol. Etazolate enhancement of [3H]diazepam binding was inhibited by picrotoxinin. Furthermore, etazolate inhibited the [3H]DHP binding in a Lubrolsolubilized fraction with an IC50 value of 6–8 μM. These results provide evidence that etazolate, like pentobarbital, modulates benzodiazepine binding via the DHP‐sensitive site of the benzodiazepine‐GABA receptor‐ionophore complex.
Opiate Receptors from Different Tissue Sources: Solubilization and Characterization
Authors: Dornay Menashe , Simantov Rabi
Published: October 2006

Abstract: Membrane‐bound opiate receptors from neuroblastoma‐glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus‐hypothalamus and cerebellum) were labeled with the methionine‐enkephalin analogue, D‐[3H]Ala2‐Met‐enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule‐bound ligands. The solubilized receptor‐[3H]enkephalin complexes were partially purified by consecutive passages through Sephadex G‐75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol‐Sepharose or diethylaminoethanol‐Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor‐[3H]enkephalin complex from neuroblastoma‐glioma cells (apparent δ‐type receptors) binds less to the thiol‐Sepharose beads than receptor‐(3H]enkephalin prepared from the hypothalamus‐thalamus, which is rich in μ receptors. The [3H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma‐glioma receptor was monophasic, with a half‐ life of 250 min, whereas that of two brain regions was biphasic, with half‐lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross‐linked to the ligand.
Studies on α‐Latrotoxin Receptors in Rat Brain Synaptosomes: Correlation Between Toxin Binding and Stimulation of Transmitter Release
Authors: Meldolesi Jacopo
Published: October 2006

Abstract: α‐Latrotoxin (α‐LT), the major component of black widow spider venom, is a high‐molecular‐weight protein that acts presynaptically by stimulating the release of stored neurotransmitters. The purified toxin was iodinated to high specific radioactivity by the Bolton‐Hunter procedure, without appreciable loss of biological activity. By the use of the 125I‐toxin, specific receptors were revealed in synaptosome fractions isolated from various regions of the rat brain, but not in nonneural tissues. The density of α‐LT receptors [which are probably composed of, or include, membrane protein(s)] varies between 0.6 and 0.88 pmol/mg of synaptosome protein, their affinity is very high (KA of the order of 1010M−1), their association rate is fast, and their dissociation rate slow. They might belong to a single, homogeneous class. This last conclusion, however, is still uncertain, because results suggesting a possible heterogeneity were obtained by studying the dissociation of the toxin from synaptosomes incubated in high‐salt buffer. Experiments in which the binding of α‐LT and its dopamine release activity in striatal synaptosomes were investigated in parallel in a variety of experimental conditions support the hypothesis that occupation of the high‐affinity receptors is the initial step in the α‐LT activation of the presynaptic response.
A New Synaptosomal Biosynthetic Pathway of Glutamate and GABA from Ornithine and Its Negative Feedback Inhibition by GABA
Authors: Yoneda Yukio , Roberts Eugene , Dietz George W.
Published: October 2006

Abstract: In sonicates of mouse brain synaptosomes, we demonstrated that γ‐aminobutyric acid (GABA) can be formed from l‐ornithine (Orn) through l‐glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]l‐proline(Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1‐5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine δ‐amhotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30‐fold‐purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, l‐glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or β‐alanine. The inhibition by GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Om is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of l‐glutamic‐γ‐semialdehyde ⇌l‐Δ1‐pyrroline‐5‐carboxylicacid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It was confirmed that l‐canaline (δ‐aminooxy‐l‐α‐aminobutyric acid) is a potent and specific inhibitor of brain OAT, whereas much weaker inhibition was observed with two other carbonyl‐trapping agents, aminooxyacetic acid and hydrazine.
Regeneration of Cholinesterases in the Stellate and Normal and Denervated Superior Cervical Ganglia of the Cat Following Inactivation by Sarin
Authors: Koelle George B. , Ruch Gerard A. , Rickard Kathleen K. , Sanville Ursula J.
Published: October 2006

Abstract: Experiments were designed to test the hypothesis that ganglionic butyrylcholinesterase (BuChE) is derived from acetylcholinesterase (AChE). At 5 to 8 days following preganglionic denervation of the right superior cervical ganglion (SCG), cats were given sarin, 2.0 μmol/kg, i.v. At intervals of 1 h and 1, 2, 3, 6, 11, and 22 days later, they were killed, and the AChE and BuChE contents of both SCG and both stellate ganglia (StG) were assayed. The regeneration of AChE in the normal ganglia occurred in two phases: an initial rapid phase, to 25‐40% of control activity in 1 day, and a slow phase, to approximately 70% of control activity in 22 days. BuChE reached approximately 85% of control activity in normal SCG and StG at 22 days. In the denervated SCG, AChE activity reached a maximum of approximately 17% of normal at 1 day, the value prior to the administration of sarin, and did not increase appreciably above this subsequently. BuChE activity in the denervated SCG reached approximately 50% of normal ganglia at 22 days. At each interval, its activity approached 55% of that of the contralateral normal SCG, the value found in the denervated SCG prior to the administration of sarin. Hence, the regeneration of BuChE appears to be independent of the presence of AChE in the neuropil. The origin of ganglionic BuChE remains obscure.
Inactivation of Benzodiazepine Binding Sites by N‐Ethylmaleimide
Authors: Martini C. , Lucacchini A.
Published: October 2006

Abstract: The benzodiazepine receptors of bovine brain membranes have been identified by the specific binding of radiolabeled [3H]diazepam. Pretreatment of membranes with N‐ethylmdleimide causes a dose‐ and time‐dependent decrease of 45 to 60% in the number of binding sites. No decrease occurs when membranes are pretreated with N‐ethylmaleimide before administration, or in the presence, of diazepam. Binding of [3H]diazepam to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.
Effects of a Single Intravenous Glucocorticoid Dose on Biogenic Amine Levels in Cat Lumbar Spinal Cord
Authors: Hall Edward D. , McGinley Patricia A.
Published: October 2006

Abstract: The effects of a single large intravenous dose of methylprednisolone on the steady state levels of dopamine, norepinephrine, and 5‐hydroxytryptamine in cat lumbar spinal cord, as a function of dose (15, 30, or 90 mg/kg) and time (1 or 24 h) after administration, were examined by high performance liquid chromatography with electrochemical detection. Methylprednisolone produced a dose‐related increase in the levels of dopamine and 5‐hydroxytryptamine, but not norepinephrine, measured at 1 h. The effect of the single glucocorticoid dose was biphasic, however, as measurement of the three amines at 24 h showed each to be depressed below the levels found in untreated animals. The possible mechanistic basis and the significance of these glucocorticoid effects are discussed.
Ontogeny of Cerebral Oxidative Metabolism in the Chick Embryo
Authors: Gonya‐Magee Teresa , Vannucci Robert C.
Published: October 2006

Abstract: The low cerebral energy requirements of most mammals at birth reflect an immaturity of the central nervous system, and it has been suggested that energy demands in fetuses are even less well developed than in newborns. Furthermore, fetal cerebral energy requirements are presumed to be met predominantly or exclusively by anaerobic glycolysis. To clarify these issues, we investigated cerebral oxidative metabolism in 9‐, 14‐, 16‐, and 19‐day‐old chick embryos and in newly hatched peeps. Animals were decapitated and quick‐frozen in liquid Freon 0‐5 min post‐mortem. Forebrain extracts were prepared and assayed for ATP, phosphocreatine, glucose, and lactate. Alterations in these metabolites post‐decapitation were used to calculate cerebral metabolic rates (Δ∼P) and rates of maximal anaerobic glycolysis (Δ lactate). Rates of lactate accumulation during cerebral ischemia increased progressively from embryonic day 9 through hatching. Cerebral metabolic rates were not different in 9‐, 14‐, and 16‐day‐old embryos, but increased steadily thereafter. The extent to which total cerebral energy utilization could be derived from anaerobic glycolysis (Δ lactate/Δ∼ P) increased from a low at day 9 (0.29) to a maximum at day 16 (0.78). The data suggest that, despite the low cerebral metabolic activity of the chick embryo, at no time during development is anaerobic glycolysis capable of entirely supporting the energy needs of the developing brain.
Acetylcoenzyme A and Acetylcholine in Slices of Rat Caudate Nuclei Incubated with (‐)‐Hydroxycitrate, Citrate, and EGTA
Authors: R̀íčíný Jan , Tuček Stanislav
Published: October 2006

Abstract: The effects of (‐)‐hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl‐CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 mM K+, 0 or 2.5 mM Ca2+, and 0, 1, or 10 mM EGTA. OHC diminished the concentration of acetyl‐CoA in the slices under all conditions used: in experiments with 2.5 mM OHC, the concentration of acetyl‐CoA was lowered by 25‐38%. Citrate, in contrast, had no effect on the level of acetyl‐CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 mM K+ and 1 mM EGTA, they had different effects on the content of ACh during incubations in the presence of Ca2+. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca2+ and 31.2 or 6.2 mM K+, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP‐citrate lyase; with glucose as the main metabolic substrate, ATP‐citrate lyase appears to provide about one‐third of the acetyl‐CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca+2, is interpreted to be a consequence of the chelation of intracellular Ca2+; this interpretation is supported by the observation of a similar effect caused by 10 mM EGTA.
Altered Ontogenesis of Muscarinic Receptors in Agranular Cerebellar Cortex
Authors: Soreq Hermona , Gurwitz David , Eliyahu Daniel , Sokolovsky Mordechai
Published: October 2006

Abstract: The developmental pattern, the agonist binding properties and the cellular origin(s) of muscarinic binding sites were investigated in agranular cerebellum of x‐irradiated rats, of Gunn rats with hereditary hyperbilirubinemia, and of staggerer mutant mice. The density of muscarinic binding sites was found to be higher than normal in all of these cerebellar types, indicating that granular neurons do not greatly contribute to binding of acetylcholine in the rodent cerebellum. The total number of muscarinic binding sites as measured by binding of [3H]4NMPB remains unchanged in the agranular cerebellum of x‐irradiated rats. However, the number of muscarinic sites is reduced by about 30% in the agranular cerebellum of homozygous Gunn rats (jj), in which fibrous astrocytes and Purkinje cells are also damaged. In the cerebellum of staggerer mice (sg/sg), where a cascade of events leads to massive damage to mossy fibers and Golgi cells in addition to granular neurons and Purkinje cells, the content of muscarinic receptors is reduced by 50%. Thus, the number of muscarinic binding sites in the rodent cerebellum seems to depend on the integrity of the additional cell types and cellular elements, damaged in these agranular models. The ontogenetic variations in the affinity of cerebellar muscarinic sites for binding of carbamylcholine in normal and Gunn rat cerebellum were compared with those observed in x‐irradiated and staggerer cerebellum, where elimination of granular neurons induces the formation of ‘heterologous’ synapses. Muscarinic binding affinity increases 10‐fold during postnatal development in the cerebellum of normal and Gunn rats. In the immature x‐irradiated cerebellum, the affinity of muscarinic binding sites was found to be nearly as high as that detected in the adult normal cerebellum. In contrast, cerebella of 5‐month‐old staggerer mice display 5‐fold lower affinity than their normal counterpart values, as low as that determined in normal immature cerebellum. The characteristic ontogenetic pattern of muscarink binding is therefore indicated to be related to the formation of correct circuitry, but not to the presence of granular neurons, in the developing rat cerebellum.
MgATP‐Stimulated Release of Luteinizing Hormone Releasing Hormone from Isolated Hypothalamic Granules: Evidence for a Selective Requirement for Monovalent Cations
Authors: Burrows G. Howard , Barnea Ayalla
Published: October 2006

Abstract: The purpose of this study was to characterize the kinetics and monovalent ion requirements of the MgATP‐stimulated release of Iuteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. LHRH granules were obtained from homogenates of hypothalami of adult male rats by differential centrifugation and then incubated in buffered media containing KCl. Under these conditions, the pH optimum for the MgATP‐stimulated release of LHRH was between 7.5 and 8.0; the rate of release was linear for the first 5 min of incubation; and the magnitude of the release was a saturable function of KCl concentration. When the potassium in the incubation mixture was replaced with various monovalent cations (Li+, Na+, Cs+, choline, or tetraethylammonium), the magnitude of the MgATP‐stimulated release of LHRH was inversely related to the size of the cation; release in the presence of Li+ was 12%, whereas release in the presence of tetraethylammonium was 0% of the total LHRH. When the chloride in the incubation mixture was replaced with various monovalent anions (Br−, I−, or isethionate), the magnitude of the MgATP‐stimulated release of LHRH was not related to the size of the anion. Moreover, inclusion of anion transport inhibitors in the incubation mixture did not affect LHRH release, suggesting that anion transport is not required for the MgATP‐stimulated release of LHRH. In summary, we suggest that the MgATP‐stimulated release of LHRH from isolated hypothalamic granules involves an enzymatic event and that this release process does not have a selective requirement for monovalent anions, but has a selective requirement for monovalent cations.
Isolation from Cholinergic Synapses of a Protein That Binds to Membranes in a Calcium‐Dependent Manner
Authors: Walker J. H.
Published: October 2006

Abstract: A protein that binds to membranes in a calcium‐dependent manner between calcium concentrations of 10−5 and 10−6M has been isolated in large amounts (20 mg/kg tissue) from the entirely cholinergic electric organ of Torpedo marmorata. The protein bound reversibly to membrane fractions in a calcium‐specific and saturable manner. The protein also bound to lipids isolated from Torpedo electric organ and to clathrin‐coated vesicles prepared from pig brain. The protein bound to a Triton X‐100‐sensitive site. It had an apparent subunit molecular weight of 32,000 by polyacrylamide gel electrophoresis and of 35,900 by amino acid analysis; a broad isoelectric range of 4.8 to 5.5; and 27% of its amino acids after hydrolysis were observed to be aspartic and glutamic acids. Synaptosomes derived from electric organ were enriched in the protein which is probably localised within the nerve ending. It was localised in the synaptic region of the electric organ by means of immunofluorescence. In the electric lobe, discrete patches of fluorescence were seen within the cell bodies that innervate the electric organ. The protein may be involved in the recognition of membranes within the cholinergic neuronc. Proteins with similar purification properties were found in all tissues investigated so far, and polypeptides of subunit molecular weight 32,000 were identified in bovine adrenal medulla and guinea pig brain synaptosomes.
Regional Mapping of Neostriatal Neurotransmitter Systems as a Function of Aging
Authors: Strong R. , Samorajski T. , Gottesfeld Zehava
Published: October 2006

Abstract: The purpose of the present investigation was to map chemically the distribution of certain neurotransmitter systems in the neostriatum of rats aged 6, 16, and 26 months. This mapping was carried out by microdissection of discrete striatal regions coupled with radiometric assays for choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), dopamine (DA), and norepinephrine (NA). In all age groups, ChAT, DA, and NA were highest in the rostral relative to the caudal neostriatum. Additionally, ChAT was higher in the lateral than in the medial region, whereas GAD was more homogeneously distributed within the striatum. ChAT activity was decreased significantly primarily in the caudal regions in rats aged 16 and 26 months. DA levels were decreased in the caudal striatum in rats aged 26 months. NA levels were found to be significantly decreased primarily in the rostral neostriatal regions of the otdest rats. GAD activity remained unchanged in all age groups. These regional changes in selected neurotransmitter systems may underlie specific motor and cognitive deficits that often occur during aging.
Avermectin B1a Modulation of γ‐Aminobutyric Acid Receptors in Rat Brain Membranes
Authors: Pong Sheng‐Shung , Wang Ching Chung
Published: October 2006

Abstract: Avermectin B1a stimulates high‐affinity binding of [3H]‐γ‐aminobutyric acid (GABA) to receptors in washed rat brain membranes. Scatchard analysis of the data indicates that the drug does not significantly alter the apparent dissociation constant of GABA binding, but increases the detectable number of binding sites from 3.2 to 5.1 pmol/mg protein. (+)‐Bicuculline completely blocks control and avermectin B1a‐stimulated GABA binding, whereas picrotoxin antagonizes specifically the avermectin B1a‐stimulated GABA binding. The avermectin B1a‐stimulated GABA binding is also chloride ion‐dependent, whereas GABA binding in the control is not. These observations suggest that the mechanism of avermectin B1a stimulation of GABA binding may involve the chloride ion channel.
Comparison of the Effects of ATP, Mg2+, and MgATP on the Release of Luteinizing Hormone‐Releasing Hormone from Isolated Hypothalamic Granules
Authors: Burrows G. Howard , Barnea Ayalla
Published: October 2006

Abstract: The effect of ATP, Mg2+, or MgATP on the release of luteinizing hormone‐releasing hormone (LH‐RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult male hypothalami, were incubated at 37°C in a buffered (pH 7.8) medium containing 0.15 m‐KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH‐RH. In contrast, the addition of MgATP stimulated the release of LH‐RH, the release being 62% greater than control. The addition of Mg2+ to the incubated granules also stimulated the release of LH‐RH. However, the magnitude of this Mg2+‐stimulated release of LH–RH was significantly (P < 0.01) lower than that of the MgATP‐stimulated release, indicating that ATP stimulates LH‐RH release in a Mg2+‐dependent manner. As both MgATP and Mg2+ alone stimulated LH‐RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4°C in a buffered medium containing 0.15 m‐KCl or incubation at 37°C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP‐stimulated release of LH‐RH was not manifested, whereas the Mg2+‐stimulated release of LH‐RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH‐RH from isolated hypothalamic granules: one process involves ATP and Mg2+ (MgATP) and another process involves Mg2+ alone.
Presence of Two Benzodiazepine Binding Sites in the Rat Hippocampus
Authors: Volicer Ladislav , Biagioni Thomas M.
Published: October 2006

Abstract: Characteristics of receptor binding of diazepam and flunitrazepam in three brain areas were compared. It was found that in the cerebral cortex and cerebellum the number of sites was similar for both ligands and that the affinity of diazepam was four times lower than the affinity of flunitrazepam. In contrast, when binding in the hippocampus was analyzed (assuming the presence of homogenous binding sites), it was found that the number of binding sites was higher and that the affinity was 17 times lower for diazepam than for flunitrazepam. This difference is due to the presence of two diazepam binding sites in this brain area, as demonstrated by a Scatchard analysis.
Characterization of Catecholamine‐Storage Organelles in Transplantable Phaeochromocytoma and Adrenal Glands of Rats
Authors: Oberlechner E. , Westhead E. , Neuman B. , Schmidt W. , Fischer‐Colbrie R. , Weber A. , Sperk G. , Winkler H.
Published: October 2006

Abstract: The properties of the catecholamine‐storing organelles from transplantable rat phaeochromocytoma and rat adrenal glands were compared by density gradient centrifugation. It was shown that tumour granules are more heterogeneous and less dense than adrenal granules. Both granule preparations can take up catecholamines and nucleotides by a process driven by an electrochemical proton gradient. Dopamine β‐hydroxylase and glycoprotein III were analysed by immunological techniques. Glycoprotein III was shown to be a specific component of chromaffin granules. Tumour tissue (average weight 700 mg) contains amounts of these antigens comparable to those in 210 adrenals. The biosynthesis of granules in the tumour apparently occurs at a low rate, making turnover studies difficult. The transplantable rat phaeochromocytoma is very useful for studies on the uptake properties and the immunological characteristics of rat catecholamine storage granules because one tumour provides an amount of material that could otherwise be obtained only from a large number of adrenal glands.
Axonal Transport, Deposition, and Metabolic Turnover of Glycoproteins in the Rat Optic Pathway
Authors: Goodrum Jeffry F. , Morell Pierre
Published: October 2006

Abstract: Following intraocular injection of [3H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half‐life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half‐lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half‐lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.
Brain (Na+, K+)‐ATPase and Noradrenergic Activity: Effects of Hyperinnervation and Denervation on High‐Affinity Ouabain Binding
Authors: Swann Alan C. , Grant Steven J. , Maas James W.
Published: October 2006

Abstract: To examine the role of nerve‐specific (Na+, K+)‐ATPase in chronic changes in noradrenergic activity, we examined the effects of noradrenergic denervation and hyperinnervation on p‐nitrophenylphosphatase activity and on total and nerve‐specific ouabain binding. High‐affinity and erythrosin B‐sensitive binding were compared as measurements of nerve‐specific binding. Hyperinnervation and denervation was produced in cerebellum and cerebral cortex, respectively, by 6‐hydroxydopamine lesions of the dorsal noradrenergic bundle. Hyperinnervation increased, and denervation decreased, enzyme activity, high‐affinity ouabain inhibition, and erythrosin B‐sensitive ouabain binding. As (Nat+, K+)‐ATPase has a major role in the regulation of neural excitability and energy metabolism, and the ouabain binding site has been shown to have endogenous ligands, these changes in (Na+, K+)‐ATPase may be important in the long‐term regulation of neuron function by norepinephrine.
Glycoproteins of Axonal Transport: Affinity Chromatography on Fucose‐Specific Lectins
Authors: Gustavsson S. , Ohlson C. , Karlsson J. O.
Published: October 2006

Abstract: Rapidly transported fucose‐labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose‐specific lectins. A recently characterized fucose‐specific lectin from Alcuria aurantia bound reversibly approximately 60% of the applied protein‐bound radioactivity. The lectins from Lotus tetragonolohus and Ulex europaeus bound very small proportions of the labeled rapidly transported glycoproteins.
Effects of Cap Analogue or Cap Removal on the Translation of Rat Brain mRNA In Vitro
Authors: Ven Murthy M. R.
Published: October 2006

Abstract: The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5′‐terminal 7‐methylguanosine (m7G) was removed by oxidation and β‐elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7‐methylguanosine 5′‐triphosphate (pppm7G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain‐specific protein S‐100 was inhibited by β‐elimination of mRNA, by pppm7G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.
DNA Turnover in Rat Cerebral Cortex
Authors: Perrone‐Capano C. , D'Onofrio G. , Giuditta A.
Published: October 2006

Abstract: After the intracranial injection of [methyl‐3H]thymidine the specific activity of rat cortical DNA increases rapidly, reaching a maximum at about 5 h. More than half of the radioactive DNA disappears from the tissue in the following few hours. During the same period of time the concentration of radioactive DNA in liver remains essentially constant. Minor variations occur in both organs after 41 h. An apparent rapid turnover of DNA is also present in a fraction of purified neuronal perikarya prepared from the cerebral cortex.