bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ‐Opioid Receptors
Authors: Mulder Arie H. , Hogenboom Francois , Wardeh George , Schoffelmeer Anton N. M.
Published: October 2006

Abstract: Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium‐free Krebs‐Ringer‐bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7‐Dihydroxy‐N,N‐dimethyl‐2‐ami‐notetralin (TL‐99) strongly inhibited synaptosomal K+‐in‐duced [3H]NA release (EC50= 5–10 nM) by activating α2‐adrenoceptors. Release was also inhibited (maximally by 40–50%) by morphine (EC50= 5–10 nM), [Leu5]enkephalin (EC50=∼300 nM), [d‐Ala2,d‐Leu5]enkephalin (DADLE), and Tyr‐d‐Ala‐Gly‐(NMe)Phe‐Gly‐ol (DAGO) (EC50 values =∼30 nM). In contrast to the μ‐selective opioid receptoragonists morphine and DAGO, the highly δ‐selective agon ist [d‐Pen2, dPen5]enkephalin (1 μM) did not affect [3H]‐NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both δ‐and μ‐opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like α2‐adrenocep‐tors, μ‐opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.
Protein Phosphorylation in Rat Pineal Gland and Its Regulation in Supersensitive and Subsensitive States
Authors: Cimino Mauro , Benfenati Fabio , Farabegoli Costanza , Cattabeni Flaminio , Agnati Luigi F.
Published: October 2006

Abstract: The phosphorylation of specific proteins in pineal homogenate was analyzed by sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis and autoradiography. Cyclic AMP had the capacity to stimulate in a dose‐dependent manner the incorporation of 32P in protein bands of apparent molecular weights 59K, 56K, and 35K with a maximal effect at 1 μM. On the other hand, calcium alone did not induce a marked increase in 32P incorporation with the exception of a dose‐dependent phosphorylation of a 46K protein with a peak effect at 0.2 mM calcium concentration. The addition of exogenous calmodulin enhanced 32P incorporation in proteins migrating in the 62K and 52K regions, an effect that was antagonized by the calmodulin inhibitor trifluoperazine. However, also under these conditions, the stimulation of pineal protein phosphorylation was rather weak compared to that observed in other brain areas. In an attempt to investigate the functional changes of these biochemical processes during environmental lighting and ad‐renergic stimulation, it was found that the administration of (‐)‐isoproterenol (5 mg/kg, s.c), a β‐receptor agonist, induced a clear‐cut enhancement of 32P incorporation into the cyclic AMP‐sensitive 59K and 56K proteins only in animals exposed for 18 h to the light, whereas it was almost ineffective in those kept in the dark for the same period. This effect was antagonized by (—)‐propranolol pretreatment (20 mg/ kg), suggesting that the changes in cyclic AMP‐dependent protein phosphorylation observed in supersensitive pineals may represent a β‐receptor mediated process. Thus the cyclic AMP‐dependent protein kinase activity seems to be correlated with the sensitivity state of the β‐adrenergic receptor system and may take part in the development of pineal su‐persensitivity. On the other hand, the phosphorylation of the 46K calcium‐sensitive protein was completely unaffected, confirming the action specificity of the adrenergic system on cyclic AMP‐induced pineal protein phosphorylation.
Susceptibility of the Wolfgram Proteins and Stability of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase of Rat Brain Myelin to Limited Proteolytic Digestion
Authors: Mohan P. Maruthi , Sastry P. S.
Published: October 2006

Abstract: The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N‐tosyl‐l‐phenylalanine chlo‐romethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dode‐cyl sulfate‐polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydro‐lyzed in 5–10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross‐reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (>20 units/mg of myelin) resulted in the degradation of all the myelin proteins. Essentially all the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (< 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.
Phylogenetic Comparison of the Photoaffinity‐Labeled Benzodiazepine Receptor Subunits
Authors: Hebebrand Johannes , Friedl Waltraut , Breidenbach Bernd , Propping Peter
Published: October 2006

Abstract: The late evolutionary appearance of the benzodiazepine receptor (BZR) allows an experimental approach for evaluation of the qualitative development of its sub‐units. Photoaffinity labeling of brain membranes with [3H]flunitrazepam followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography offers a suitable method for tracing the qualitative evolution of the BZR. A systematic comparison of the subunit patterns in fishes, amphibians, reptiles, birds, and mammals revealed that the subunit of 53K is phylogenetically the oldest photo‐affinity labeled subunit; whereas it is the only band present in the lungfish and most amphibians, additional bands are apparent in higher tetrapods. In fishes, the evolution of the BZR subunits leads to the loss of the 53K subunit. KD values are discussed in relation to specific subunit patterns. Possible explanations for the observed variation of the subunits are discussed, with special emphasis placed on the possible evolution by gene duplication and subsequent divergence.
Synaptosomal Uptake and Release of Dopamine in Rat Striatum After Hypoxia
Authors: Odarjuk Jutta , Hetey L. , Gross J.
Published: October 2006

Abstract: Hypoxia induces alterations of central mono‐aminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long‐lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short‐term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+‐evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2–11) caused long‐lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+‐stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.
Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes
Authors: Dosemeci Ayse , Rodnight Richard
Published: October 2006

Abstract: The endogenous phosphorylation of synapsin 1 in cyclic AMP‐containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmo‐dulin‐dependent phosphorylation of the same protein. The Ca2+/calmodulin‐dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52‐kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.
Concomitant Increases in the Levels of Choline and Free Fatty Acids in Rat Brain: Evidence Supporting the Seizure‐Induced Hydrolysis of Phosphatidylcholine
Authors: Flynn Cheryl J. , Wecker Lynn
Published: October 2006

Abstract: The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 μg/kg, s.c), at a dose that produced toxic effects, increased the levels of both free fatty acids (175–250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman‐induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman‐induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant‐mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.
Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain
Authors: Takemura Motohiko , Fukui Hiroyuki , Yamamoto Yohichi , Hosomi Naohiro , Wada Hiroshi
Published: October 2006

Abstract: The nitrendipine receptor associated with the voltage‐dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3‐[(3‐cholamidopropyl)dimethylammonio] ‐ 1 ‐ propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S‐500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is ∼10 times that of the membrane‐bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 ∼ 10−7M, and they caused maximal inhibitions of 70 and 100%, respectively.
Effects of 1‐Methyl‐4‐Phenyl‐1,2,5,6‐Tetrahydropyridine and Its Metabolite 1‐Methyl‐4‐Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain
Authors: Cheeseman Andrea J. , Clark John B.
Published: October 2006

Abstract: 1‐Methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP) and its metabolite, 1‐methyl‐4‐phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro‐striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH‐CoQ reductase) of the mitochondrial respiratory chain.
Changes in β‐Adrenergic Receptor Subtypes in Alzheimer‐Type Dementia
Authors: Shimohama Shun , Taniguchi Takashi , Fujiwara Motohatsu , Kameyama Masakuni
Published: October 2006

Abstract: Using ligand binding techniques, we studied β‐adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer‐type dementia (ATD). Inhibition of [3H]dihydroalprenolol ([3H]DHA) binding by the selective β, antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of β1/β2‐adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of β‐adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. β1‐Adrener gic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas β2‐adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that β1‐and β2‐adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.
Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes
Authors: Gregerson K. A. , Selmanoff Michael
Published: October 2006

Abstract:Release of preaccumulated, tritium‐labeled dopa‐mine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1–20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+‐and the veratridine‐stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+‐induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]oand the [Ca2+]odose‐response curves, consistent with actions of Mg2+on screening surface membrane charges and blocking voltage‐dependent Ca2+channels. In all studies, steady‐state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/ or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.
Enhancement of Brain Dopamine Metabolism by Tyrosine During Immobilisation: An In Vivo Study Using Repeated Cerebrospinal Fluid Sampling in Conscious Rats
Authors: Marcou M. , Kennett G. A. , Curzon G.
Published: October 2006

Abstract: Central dopamine (DA) and 5‐hydroxytrypta‐mine (5‐HT) metabolism was monitored in conscious, freely moving rats by determination of levels of the DA metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and ho‐movanillic acid (HVA) and the 5‐HT metabolite 5‐hydroxy‐indoleacetic acid (5‐HIAA) in CSF samples withdrawn repeatedly from the cisterna magna and treated with acid to hydrolyse DOPAC and HVA conjugates. The effect of tyrosine on DA metabolism was investigated. Time courses of metabolite concentrations in individual rats in a quiet room showed that tyrosine (20, 50, or 200 mg/kg i.p.) was without significant effect; brain changes were essentially in agreement. However, the increases of CSF DOPAC and HVA levels that occurred on immobilisation for 2 h were further enhanced by tyrosine (200 mg/kg). The associated increases of 5‐HIAA level were unaffected. The corresponding increases of DA metabolite concentrations in the brains of immobilised rats given tyrosine were less marked than the CSF changes and only reached significance for “rest of brain” DOPAC. The CSF studies revealed large interindividual variation in the magnitude and duration of the effects of immobilisation on transmitter amine metabolism. These results may help toward the elucidation of possible relationships between the neurochemical and behavioural effects of stress.
Effect of Lithium on Schwann Cell Proliferation Stimulated by Axolemma‐ and Myelin‐Enriched Fractions
Authors: Yoshino Jun E. , DeVries George H.
Published: October 2006

Abstract: Cultured Schwann cells stimulated with an axolemma‐ or myelin‐enriched fraction incorporated 2.5 to three times as much [3H]thymidine when 10 mM lithium was added to the extracellular medium. The ability of lithium to enhance the mitogenic activity of either fraction was dose dependent. This result was not due to an increase in osmolarity, because addition of 10 mM NaCl had no effect on the amount of labeled thymidine accumulated by Schwann cells treated with either membrane fraction. In an earlier study, the effect of either membrane fraction could be potentiated with active phorbol esters. Lithium significantly enhanced the incorporation of [3H]thymidine into Schwann cells treated with a myelin‐enriched fraction and phorbol esters. In contrast, lithium slightly increased the amount of labeled thymidine incorporated into Schwann cells stimulated with an axolemma‐enriched fraction and phorbol esters. The mitogenic activity of either membrane fraction was impaired when the calcium channel blockers Mn2+ and nifedipine were added. Addition of lithium stimulated an increase in the amount of [3H]thymidine accumulated by Schwann cells treated with either the axolemma‐ or myelin‐enriched fraction in the presence of either Mn2+ or nifedipine.
Developmental Change in the Amount of Polysialosyl Glycopeptides Isolated from the Rat Brain
Authors: Oohira Atsuhiko , Matsui Fumiko , Matsuda Motoko , Shoji Ryujiro
Published: October 2006

Abstract: Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two‐dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue‐stained spot in the electrophoreto‐gram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at ∼30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.
Separation and Characterization of Four Forms of β‐N‐Acetylhexosaminidase from Chicken Brain
Authors: Rodríguez‐Hernández José A. , Martín‐Barrientos Josefa , Sánchez‐Bernal Carmen , Cabezas José A.
Published: October 2006

Abstract: Chicken brain β‐N‐acetylhexosaminidases from embryos (16 and 21 days old), newborns (1 and 4 days old), and adults (31/2 months and 2 years old) were separated into four different forms by ion exchange chromatography on diethylaminoethyl‐cellulose. Three of these forms were “acid” hexosaminidases (I, IIA, and IIB), and the fourth was a “neutral” form. Throughout development of the chicken, forms IIA and III maintained the same activity ratio, whereas that for form I decreased and that for form IIB showed an increase.
Prenatal Monosodium Glutamate Causes Long‐Lasting Cholinergic and Adrenergic Changes in Various Brain Regions
Authors: Frieder Brina , Grimm Veronika E.
Published: October 2006

Abstract: Prenatal monosodium glutamate (MSG) given through the mother's diet was found previously to cause behavioral changes in the offspring, including learning disabilities. In the present study, neurochemical parameters were measured in the brains of prenatally exposed rats at various ages throughout development up to adulthood. At 15 days of age, choline uptake and choline acetyltransferase (ChAT) activity in the frontal cortex were significantly reduced (by 80 and 25%, respectively) in MSG‐exposed animals, whereas the same cholinergic parameters in hippocampus were not changed. During later development, choline uptake gradually increased, until in adulthood it became significantly higher in MSG‐exposed animals than in the controls. This enhancement was found in both males and females. Our previous study showed that only the male offspring were learning disabled. Choline uptake and ChAT activity were enhanced in the hippocampus of adult male animals. Norepinephrine (NE) uptake was reduced (by 25%) in the frontal cortex of males only. There was no change in NE uptake in the hypothalamus.
Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin
Authors: Tachikawa Eiichi , Tank A. William , Weiner David H. , Mosimann Werner F. , Yanagihara Nobuyuki , Weiner Norman
Published: October 2006

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β‐phorbol‐12β‐myristate‐13α‐acetate (PMA), an activator of Ca2+/phospholipid‐dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca2+. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo‐perazine (TFP). Treatment of PC 12 cells with l‐oleoyl‐2‐acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2‐diolein and 1, 3‐diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca2+ and are not inhibited by TIT5. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca2+ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA‐treated cells is different from that phosphorylated in forskolin‐treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP‐treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP‐dependent and Ca2+/ phospholipid‐dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.
An In Vivo Model for Studying Function of Brain Tissue Temporarily Devoid of Glial Cell Metabolism: The Use of Fluorocitrate
Authors: Paulsen R. E. , Contestabile A. , Villani L. , Fonnum F.
Published: October 2006

Abstract: The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastruc‐tural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of ≥2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24–72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostria‐tum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.
Histamine Acting on H2 Receptors Stimulates Phospholipid Methylation in Synaptic Membranes of Rat Brain
Authors: Ozawa Koichiro , Nomura Yasuyuki , Segawa Tomio
Published: October 2006

Abstract: Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs‐Ringer solution containing the methyl donor S‐adenosyl‐[wethyl‐3H]methionine. The transient increase of [3H]‐methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 μM) and decreased the basal level in 60 s. Histamine‐stimulated [3HJmethyl incorporation was increased linearly in a protein concentration‐dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl‐N,N‐dimethylethanolamine, and phosphatidyl‐N‐monomethylethanolamine) accounted for 60–65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine‐induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01–10 μM) or famotidine (0.01–1.0 μM) in a concentration‐dependent manner but not by mepyramine (0.1–10 μM) or diphenhydramine (0.1–10 μM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01–10 μM) or dimaprit (1.0 μM‐1.0 mM) in a concentration‐dependent manner but not by 2‐pyridylethylamine (1.0 μM‐1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.
Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum
Authors: Olianas Maria C. , Onali Pierluigi
Published: October 2006

Abstract: The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide‐binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.
Postnatal D2‐CAM/N‐CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase
Authors: Breen Kieran C. , Kelly Paul G. , Regan Ciaran M.
Published: October 2006

Abstract: Golgi‐enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2‐CAM/N‐CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12‐day‐old animals the majority of the transferred [14C]sialic acid was found to be associated with the high‐molecular‐weight [>200 kilodaltons (kd)] form of D2‐CAM/N‐CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2‐CAM/N‐CAM polypeptides was also demonstrated in both 12‐day and adult animals and transfer was evident only in the 180‐kd and 115‐kd components and not in the 140‐kd component. In contrast, Golgi‐enriched fractions prepared from adult animals showed little capability of heavily sialylating D2‐CAM/N‐CAM to any significant extent.
Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain
Authors: Asarch Kenneth B. , Shih Jean C.
Published: October 2006

Serotonin, (5‐hydroxytryptamine1, 5‐HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1 % Nonidet P‐40 and 0.3% digitonin in a low‐salt buffer containing 0.1 % ascorbic acid. The affinity of [3H]5‐HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane‐bound binding sites (2.1 nM). [3H]8‐Hydroxy‐2‐(di‐n‐propylamino)tetraIin ([3H]DPAT), a selective 5‐HT1a, radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5‐HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5‐HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5‐HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5‐HT1a and 5‐HT1b binding sites have been cosolubilized. [3H]5‐HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone‐specific binding was not detectable in this preparation, suggesting that 5‐HT2 sites were not cosolubilized.
Subcellular Localization of Sulfated Glucuronic Acid‐Containing Glycolipids Reacting with Anti‐Myelin‐Associated Glycoprotein Antibody
Authors: Kohriyama Tatsuo , Kusunoki Susumu , Ariga Toshio , Yoshino Jun E. , DeVries George H. , Latov Norman , Yu Robert K.
Published: October 2006

Abstract: Peripheral nerve glycolipids, with which anti‐myelin‐associated glycoprotein (MAG) antibodies from patients with demyelinating neuropathy and plasma cell dyscrasia cross‐react, proved to be novel glycosphingolipids containing a sulfated glucuronyl residue. Consequently, there has been much interest in the immunological role that these sulfated glucuronyl‐glycosphingolipids (SGGLs) may play in the pathogenesis of this disorder. For the determination of the distribution of these glycolipids in various nervous tissues and, thereby, the elucidation of their pathoge‐nicity, a quantitative immunostaining‐TLC method for their detection has been devised. Using this method, we demonstrated that these glycolipids were distributed in greatly different amounts in the peripheral nerves from human, bovine, chicken, rat, and rabbit. Subcellular localization studies of bovine peripheral nerve also demonstrated that they were enriched in the axolemma‐enriched fraction and present in glial‐related membranes in lower concentrations. In addition, these glycolipids were present in bovine dura mater and transformed rat Schwann cells. These biochemical results suggest that not only myelin but also axons could be involved as targets of the anti‐MAG antibody in macroglobulinemia neuropathy, and it may also be necessary to examine anti‐SGGL activity in patients with axonal neuropathy associated with plasma cell dyscrasia.
Solubilisation of the Benzodiazepine Receptor from Rat Cerebellum by the Detergent n‐Octylpentaoxyethylene
Authors: Bristow David R. , Martin Ian L.
Published: October 2006

Abstract: The detergent n‐octylpentaoxyethylene is one in the series of tenside detergents developed for membrane solubilisation. We have used this detergent to solubilise benzodiazepine receptors from rat cerebellum. The soluble receptor has an affinity (KD) for [3H]flunitrazepam of 1.8 nM± 0.2, which is not significantly different from that observed in synaptic membranes. Under optimal conditions (0.6% wt/vol), the number of soluble flunitrazepam binding sites (Bmax) of 0.35 pmol/mg protein suggests an apparent solubilisation of 40% of sites from the membrane. However, the absence of the characteristic facilitation of [3H]flunitrazepam binding by γ‐aminobutyric acid (GABA), cartazolate, and pentobarbital in this soluble receptor preparation suggests that such a preparation is unlikely to be a useful preparation for further studies on the molecular mechanisms underlying GABAA receptor function.
Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue
Authors: Nilsson Goran E. , Tottmar Olof
Published: October 2006

Abstract: When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85–95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only ∼30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite‐aldehyde compounds were stable during storage at −20°C. Bisulfite‐free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from do pamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase‐ and aldehyde reductase‐independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.
Determination of Serotonin Turnover in the Rat Brain Using 6‐Fluorotryptophan
Authors: Miwa Soichi , Fujiwara Motokazu , Lee Ken , Fujiwara Motohatsu
Published: October 2006

Abstract: After intraperitoneal injection of rats with 6‐fluorotryptophan (6‐FT), brain 5‐hydroxytryptamine (5‐HT) levels decreased exponentially over 1 h. Depletion was dose‐dependent and maximum depletion was observed at 200 mg/kg. 6‐FT (200 mg/kg) did not significantly alter the content of 5‐hydroxyindoleacetic acid. Turnover rates of 5‐HT obtained by the 6‐FT and other methods were fairly consistent. 6‐FT had little effect on the content of noradrenaline and dopamine. These data suggest that 6‐FT completely inhibits tryptophan hydroxylase, in vivo, without affecting the release of 5‐HT from 5‐HT neurons and with little effect on the activities of tyrosine hydroxylase. Therefore, 6‐FT is a good pharmacological tool for studying the turnover rate of 5‐HT in the brain.
Hydroxyindole‐O‐Methyltransferase in Y‐79 Human Retinoblastoma Cells: Effect of Cell Attachment
Authors: Kyritsis Athanassios P. , Wiechmann Allan F. , Bok Dean , Chader Gerald J.
Published: October 2006

Abstract: Hydroxyindole‐O‐methyltransferase (HIOMT), the enzyme in the final step of melatonin synthesis, is present in the Y‐79 human retinoblastoma cell line. Using elec‐troblot immunolabellings, a single band corresponding to HIOMT was observed. Immunofluorescence, sodium dode‐cyl sulfate‐polyacrylamide gel electrophoresis, and quantification of enzyme activity all revealed dramatic increases in HIOMT in cells attached to substrate compared to cells in suspension culture.
Highly Sensitive Immunoassay for the α Subunit of the GTP‐Binding Protein Go and Its Regional Distribution in Bovine Brain
Authors: Asano Tomiko , Semba Reiji , Ogasawara Nobuaki , Kato Kanefusa
Published: October 2006

Abstract: Antisera were raised in rabbits against the α sub‐unit of a GTP‐binding protein, Go. Because the antisera cross‐reacted weakly with the α subunit of inhibitory GTP‐binding protein of adenylate cyclase (Gi), they were purified with a Goα‐coupled Sepharose column. Purified antibodies reacted only with Goα and did not cross‐react with the Giα subunit or βγ subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Goα was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Goa, and it did not cross‐react with Giα or βγ. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Goα were determined. Goα was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neu‐ropil was rich in Goα.
Modulation of Bradykinin‐Induced Inositol Trisphosphate Release in a Novel Neuroblastoma X Dorsal Root Ganglion Sensory Neuron Cell Line (F‐11)
Authors: Francel Paul C. , Miller Richard J. , Dawson Glyn
Published: October 2006

Abstract: In the mouse neuroblastoma X dorsal root ganglion hybrid cell line F‐l 1, bradykinin receptor stimulation induced the release of inositol‐1,4,5‐trisphosphate (IP3) and inositol‐1,4‐bisphosphate (IP2). Maximal stimulation of [2‐3H]IP3 and [2‐3H]IP2 release by bradykinin in the absence of LiCl occurred at 7 (or less) and 15 s, respectively, with average levels of 5.7‐ (IP3) and 3.4‐ (IP2) fold of control values. The EC50 for bradykinin was 33 ± 5 nM. IP3 and IP2 concentrations returned to basal levels ∼ 1 min after bradykinin addition. Bradykinin‐induced IP3 release was blocked by several novel bradykinin analogues. In particular, [D‐Arg0]‐Hyp3‐Thi58‐[D‐Phe7]‐bradykinin [Hyp, hydroxypro‐line; Thi, β‐(2‐thienyl)‐L‐alanine] blocked IP3 production in a dose‐dependent fashion. Several of these analogues alone showed little or no agonist activity. The bradykinin receptor may be coupled to phospholipase C via a GTP‐sensitive protein (Gi or Go), as preincubation for 18–20 h with pertussis toxin decreased IP3 concentrations by 45%. Bradykinin is also known to modulate the concentrations of other second messengers in neurons, increasing the concentrations of Ca2+, diacylglycerol (DG), and cyclic GMP and decreasing the concentration of cyclic AMP. These second messengers modulated bradykinin‐dependent IP3 release to varying degrees. A23187, a Ca2+ ionophore, produced a 37% decrease in IP3 concentration. 12‐O‐Tetradecanoylphorbol‐13‐ace‐tate, which mimics the effects of DG and activates protein kinase C, inhibited IP3 release by 80%. Dibutyryl cyclic GMP produced little or no inhibition of IP3. [D‐Ala2,D‐Leu5]Enkephalin (DADLE), an opioid peptide that decreases cyclic AMP concentrations, likewise had no effect. However, elevation of cyclic AMP concentrations by pros‐taglandins I2 or E2 or forskolin inhibited IP3 formation in a dose‐dependent fashion. This inhibition was reduced by DADLE in a naloxone‐reversible manner, a result suggesting that the inhibition is a cyclic AMP‐mediated effect. These results show that (a) bradykinin acutely stimulates IP3 release from F‐11 cells in a transient fashion, (b) bradykinin‐induced IP3 release may be subject to negative feedback control mediated through protein kinase C, (c) IP3 release is partially inhibited by pertussis toxin and by increases in cyclic AMP content, and (d) inhibition of bradykinin‐induced IP3 release can be produced by several novel bradykinin analogues.
Molecular Properties of the Growth‐Associated Protein GAP‐43 (B‐50)
Authors: Benowitz Larry I. , Perrone‐Bizzozero Nora I. , Finklestein Seth P.
Published: October 2006

Abstract: The protein that has been identified in different contexts as growth‐associated protein (GAP)‐43, GAP‐48, protein 4, B‐50, F‐1, γ5, and pp46, has been implicated in neural development, axonal regeneration, and the modulation of synaptic function. The present study investigated various properties of this protein (designated here as GAP/ B‐50), including its correct molecular weight and possible polymeric structure. GAP/B‐50 was purified to >90% homogeneity using an alkaline extraction procedure followed by a two‐stage separation on a size‐exclusion HPLC column. The equivalence of the purified protein to the B‐50 phosphoprotein was confirmed by peptide digests, comigra‐tion, immunostaining, and amino acid composition. On a series of sodium dodecyl sulfate‐polyacrylamide gels the apparent molecular weight of the protein was seen to vary inversely with the concentration of acrylamide in the gels. Using these data in the method of Ferguson, the molecular weight of GAP/B‐50 was calculated to be 32.8 kilodaltons (kD), considerably lower than the previously reported values of 43–67 kD. The low molecular weight of the protein in the presence of detergent was confirmed by density cen‐trifugation. In the absence of detergent, however, the protein was found to be part of a polymeric structure whose retention time by size‐exclusion chromatography indicated a size of 124 kD; this property was also confirmed by density centrifugation under nondetergent conditions. These data suggest the possibility that the native form of GAP/B‐50 in the presynaptic membrane may be a tetramer of four identical subunits.
Adrenocortical Steroids Modify Neurotransmitter‐Stimulated Cyclic AMP Accumulation in the Hippocampus and Limbic Brain of the Rat
Authors: Harrelson Allan L. , Rostene William , McEwen Bruce S.
Published: October 2006

Abstract: Glucocorticoid hormones are known to affect limbic system structures that have high levels of specific receptors for glucocorticoids, especially the hippocampus (HIPP). To understand how glucocorticoids may affect syn‐aptic transmission, we have tested the effects of adrenal removal and glucocorticoid replacement on neurotransmit‐ter‐stimulated cyclic AMP accumulation in brain slices from the rat limbic system. Adrenalectomy (ADX) caused an enhancement of vasoactive intestinal peptide (VIP)‐stimulated cyclic AMP accumulation in HIPP, amygdala (AMYG), and septum (SEP). In HIPP, ADX increased the cyclic AMP response to isoproterenol (ISOP) and decreased the response to histamine (HIST). In the AMYG and SEP, ADX did not affect significantly the action of ISOP, but ADX did decrease the response to HIST in AMYG. Administration of dexamethasone or corticosterone reversed the effects of ADX on cyclic AMP accumulation in the HIPP. The dexamethasone action on VIP‐stimulated cyclic AMP accumulation takes place within 48 h and is most apparent in the mid‐range of the VIP dose‐response curve. These results demonstrate that glucocorticoids regulate neurotrans‐mitter‐stimulated cyclic AMP generation in a fashion that is specific, both for the neurotransmitter involved and for the brain regions affected.
Molecular Forms of Butyrylcholinesterase in the Human Neocortex During Development and Degeneration of the Cortical Cholinergic System
Authors: Atack John R. , Perry Elaine K. , Bonham James R. , Candy John M. , Perry Robert H.
Published: October 2006

Abstract: The total levels of butyrylcholinesterase (BChE) activity and, more specifically, the distribution of BChE molecular forms were measured in the human neocortex during fetal development. Both the amount of total activity and the abundance of the different molecular forms (G1 and G4) remained relatively constant between gestational ages of 8‐22 weeks and were similar to those observed in samples of cortex from aged brain. In addition, in both Alzheimer‐type and parkinsonian dementia, the levels of total BChE activity as well as the relative abundance of the G1 and G4 molecular forms were similar to those observed in control tissue. Hence, both the levels of total activity and the distribution of molecular forms did not change significantly either during fetal development or in the neurodegenerative disorders of Alzheimer‐type and parkinsonian dementias. Because these situations are accompanied by changes in the cortical cholinergic system (including an increase and decrease in levels of the G4 form of acetylcholinesterase, respectively), it is concluded that, at least in the human neocortex, BChE is unrelated to cholinergic neurotransmission associated with subcortical cholinergic projection fibres.
Identification and Characterization of an N‐Methyl‐D‐Aspartate‐Specific L‐[3H]Glutamate Recognition Site in Synaptic Plasma Membranes
Authors: Monahan Joseph B. , Michel Jacquelyn
Published: October 2006

Abstract: Conditions have been developed for an L‐[3H]glu‐tamate binding assay in which 85‐95% of the specific binding is to a site that corresponds to the N‐methyl‐D‐aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L‐[3H]glutamate in 50 mM Tris/acetate, pH 7.4, for 2‐20 min at 2°C results in binding with pharmacological characteristics of the electrophysio‐logically defined N‐methyl‐D‐aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0‐7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mM concentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to this N‐methyl‐D‐aspartate site.