bluuebunny/crossref_metadata_2025 huggingface dataset:
1‐Piperideine as an In Vivo Precursor of the γ‐Aminobutyric Acid Homologue 5‐Aminopentanoic Acid
Authors: Callery Patrick S. , Geelhaar Linda A.
Published: October 2006

Abstract: Intraperitoneal injection of the cyclic imine I‐piperideine in mice resulted in measurable quantities of 5‐aminopentanoic acid in brain. 5‐Aminopentanoic acid is a methylene homologue of γ‐aminobutyric acid (GABA) that is a weak GABA agonist. 5‐Aminopentanoic acid formed in the periphery was ruled out as the source of brain 5‐aminopentanoic acid based on the absence of detection in brain following injection of 100 mg/kg of 5‐aminopentanoic acid. Deuterium‐labeled I‐piperideine was prepared by exchange in deuterated phosphate buffer. Injection of [3,3‐2H2] 1‐piperideine yielded [2,2‐2H2]5‐aminopentanoic acid in brain. The results are consistent with uptake of 1‐piperideine into brain and oxidation of the precursor to 5‐aminopentanoic acid. Inhibition of GABA catabolism by pretreatment with aminooxyacetic acid increased brain concentrations of 5‐aminopentanoic acid formed from 1‐piperideine, suggesting that 5‐aminopentanoic acid is an in vivo substrate of 4‐aminobutyrate:2‐oxoglutarate aminotransferase.
Facilitated Transport of Glucose from Blood into Peripheral Nerve
Authors: Rechthand Emanuel , Smith Quentin R. , Rapoport Stanley I.
Published: October 2006

Abstract: D‐Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood–nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D‐[14C]glucose, L‐[14C]glucose, and [14C]3‐O‐methyl‐D‐glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D‐glucose. The permeability‐surface area product (PA) for D‐[14C]glucose and [14C]3‐O‐methyl‐D‐glucose decreased, whereas the PA for L‐[14C]glucose remained constant, as the perfusate concentration of D‐glucose was increased. In the presence of no added unlabelled D‐glucose in the perfusate, the PA for L‐[14C]glucose equaled one‐fifth the PA for D‐[14C]glucose. These results demonstrate self‐saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D‐monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half‐saturation constant and maximal rate of transport for the saturable component of D‐glucose influx equaled 23 ± 11 μmol ml–1 and 6.6 ± 3.2 × 10–3μmol ± s–1· g–1, respectively. The constant of nonsaturable glucose influx equaled 0.5 ± 0.1 × 10–4 s–1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D‐glucose influx into nerve. In anesthetized rats unidirectional influx of D‐glucose exceeds, by a factor of about two, published rates of glucose utilization by peripheral nerve.
Impaired Development of Rat Cerebellum Induced by Neonatal Injection of the Glycoprotein Synthesis Inhibitor, Tunicamycin
Authors: Kohsaka Shinichi , Mita Koichi , Matsuyama Masayuki , Mizuno Masafumi , Tsukada Yasuzo
Published: October 2006

Abstract: The effects of the protein glycosylation inhibitor tunicamycin on the postnatal development of the rat cerebellum were examined in vivo. Tunicamycin (0.2 μg) was injected intracranially into 1‐day‐old rats. Inhibition of glycosylation of the macromolecules in the cerebellum by tunicamycin treatment was suggested by a reduced incorporation of [3H]glucosamine into the trichloroacetic acid (TCA)‐insoluble fraction. The tunicamycin treatment did not affect gain in body weight significantly. However the cerebellar weight was significantly reduced by 30–40% compared with that of the controls. Development of GABAergic and cholinergic innervations in the hypoplastic cerebellum was examined by measuring the activities of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT). The specific activity and the total activity of GAD were significantly reduced in the tunicamycin‐treated cerebellum. In contrast the specific activity of ChAT was significantly increased, whereas the total activity of ChAT per cerebellum was identical with that of the controls. These results suggest that the intracranial injection of tunicamycin affects the postnatal development of rat cerebellum, such as GABAergic and cholinergic innervations.
[3H]Threo‐(±)‐Methylphenidate Binding to 3,4‐Dihydroxyphenylethylamine Uptake Sites in Corpus Striatum: Correlation with the Stimulant Properties of Ritalinic Acid Esters
Authors: Schweri Margaret M. , Skolnick Phil , Rafferty Michael F. , Rice Kenner C. , Janowsky Aaron J. , Paul Steven M.
Published: October 2006

Abstract: Saturable and stereoselective binding sites for [3H]threo‐(±)‐methylphenidate were characterized in rat brain membranes. The highest density of [3H]threo‐(±)‐ methylphenidate binding sites was found in the synapto somal fraction of corpus striatum. Scatchard analysis revealed a single class of noninteracting binding sites with an apparent dissociation constant (KD) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high‐affinity binding of [3H]threo‐(±)‐methylphenidate to striatal synaptosomal membranes was dependent on the presence of sodium ions. A good correlation (r = 0.88; p < 0.001) was observed between the potencies of various psychotropic drugs in displacing [3H]threo‐(±)‐methylphenidate from these sites and their potencies as inhibitors of [3H]3,4‐dihydroxyphenylethyl‐ amine ([3H]dopamine) uptake into striatal synaptosomes. A good correlation (r = 0.85; p < 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting [3H]threo‐(±)‐methylphenidate binding to striatal synaptosomal membranes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for [3H]threo‐(±)‐methyl‐phenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.
In Vivo Phosphorylation of the Postsynaptic Density Glycoprotein gp180
Authors: Gurd James W. , Bissoon Nankie
Published: October 2006

Abstract: Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [γ‐32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)‐agarose demonstrated that >70–80% of the 32P present in the glycoproteins eluted from Con A‐agarose with α‐methyl‐D‐mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.
Effect of Cortico‐Striate Pathway Lesion on the Activities of Enzymes Involved in Synthesis and Metabolism of Amino Acid Neurotransmitters in the Striatum
Authors: Sandberg M. , Ward H. K. , Bradford H. F.
Published: October 2006

The activities of several enzymes involved in the metabolism of aspartate and glutamate were measured in striatal (nucleus caudatus and putamen) homogenates 2–3, 6–7, and 35–40 days following frontoparietal and frontal cortical ablation. The activity of glutamine synthetase (GS) was substantially increased (46–48%) on the operated side 6–7 days following the lesion whereas smaller changes were observed at 2–3 and 35–40 days after lesion. In contrast, decreased levels of glutaminase and malate dehydrogenase (MDH) were observed by 6–7 days while no significant change was found at either 2–3 or 35–40 days after the lesion. The activities of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD) were elevated after 35–40 days whereas no changes in the levels of either GDH or aspartate aminotransferase (ASAT) were found at 2–3 or 6–7 days after the fronto‐parietal decortication. When only the frontal cortex was removed quantitatively similar changes were observed in striatal GS and glutaminase activity. The content of glutamate and glutamine in the denervated striatum followed qualitatively the changes in glutaminase and GS. The results indicate that the degeneration of cortico‐striatal terminals causes a profound glial reaction in the striatum, and both glutaminase and MDH are present in relatively high concentrations in the corticostriatal terminals.
Modulation by Monoamines of Somatostatin‐Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures
Authors: Chneiweiss H. , Glowinski J. , Prémont J.
Published: October 2006

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin‐inhibitory effect on neuronal crude membranes was still observed in the presence of (—)‐isoproterenol, 3,4‐dihydroxyphenylethylamine (dopamine, DA), or 5‐hydroxytryptamine (5‐HT, serotonin) used at a concentration (10−5M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin‐inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5‐HT did not modify the somatostatin‐inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin‐inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (—)‐isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.
Synaptosomal Phospholipase D Potential Role in Providing Choline for Acetylcholine Synthesis
Authors: Hattori Hiroshi , Kanfer Julian N.
Published: October 2006

Abstract: The phospholipase D of the rat brain synaptic membrane possesses the highest activity of this enzyme of any mammalian tissue examined. The synaptic phospholipase D activity is latent and barely detectable in the absence of 4 mM sodium oleate. Several other fatty acids were either less effective or ineffective as stimulators of activity compared to this monounsaturated fatty acid. The activity was decreased by hemicholinium‐3, an inhibitor of choline uptake and slightly activated by neostigmine, an acetylcholinesterase inhibitor. Incubation of synaptosomes in the presence of sodium oleate and acetyl‐coenzyme A resulted in the formation of a product chromatographing with acetylcholine. Acetylcholine formation was nearly undetectable in the absence of sodium oleate or acetyl‐coenzyme A. These results implicate synaptosomal phospholipase D in releasing choline from phosphatidylcholine for acetylcholine formation.
Identification, Purification, and Characterization of Two Forms of Serotonin Binding Protein from Rat Brain
Authors: Liu Kuo‐Peing , Gershon Michael D. , Tamir Hadassah
Published: October 2006

Abstract: Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5‐Hydroxytryptamine (5‐HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4‐B, Affi‐Gel‐Blue, hydroxylapatite, and phosphocellulose. The 45K form of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate‐gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two‐dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5‐HT binding capacity; the enhancement of 5‐HT binding by Fe2+; and inhibition by –SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross‐reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants—56K form showed higher affinity for 5‐HT (KD1= 0.4 nM; KD2= 32 nM), whereas the 45K form showed lower affinity (KD1= 9.7 nM; KD2= 120 nM); (2) ratio of number of 5‐HT binding sites with low affinity to those with high affinity—56K (19:1), 45K (10:1); (3) isoelectric point—the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis. We suggest that a precursor‐product relationship may exist between 45K and 56K SBP and that the two forms may be located within different parts of serotonergic neutrons.
Characterization of Calelectrin, a Ca2+‐Binding Protein Isolated from the Electric Organ of Torpedo marmorata
Authors: Südhof Thomas C. , Walker John H. , Fritsche Ulrich
Published: October 2006

Abstract: We report a fast (< 1 day) and efficient (2–3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+‐dependent and ‐specific manner with half‐maximal binding between 3 and 7 μM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 μM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+‐binding sites that are Ca2+‐displaceable. An investigation of the protein's endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca2+. Ca2+‐dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca2+ and eluted by EGTA. Calelectrin also contains high‐affinity sites for hydrophobic fluorescence probes such as N‐phenyl‐1‐naphthylamine, 2‐CP‐toluidinylnaphthalene‐6‐sulfonic acid, and 1‐anilinonaphthalene‐8‐sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca2+. We conclude that calelectrin is a Ca2+‐binding protein whose binding to the lipid moieties of membranes is regulated by physiological changes in the Ca2+ concentration. This binding must be due to specific mechanisms other than simple exposure of hydrophobic sites.
Characterization of Myelin Fractions from Human Brain White Matter
Authors: Cruz Tony F. , Moscarello Mario A.
Published: October 2006

Abstract: Myelin and myelin‐containing (P3) fractions were prepared from human white matter by discontinuous sucrose gradient centrifugation. The myelin isolated from each of the fractions of different densities was morphologically and biochemically distinct. Light myelin fractions consisted of compact, multilamellar myelin, whereas the denser fractions consisted predominantly of loose myelin with fewer lamellae. The amounts of both basic protein and lipophilin (proteolipid protein) were reduced in the denser fractions. In contrast, the high‐molecular‐weight components were elevated in the dense fractions. The lipid composition was similar in all the fractions studied. Analysis of basic protein by gel electrophoresis at pH 10.6 revealed differences in basic protein microheterogeneity among the fractions. The light myelin fraction was enriched in the more positively charged basic protein components (components 1, 2, and 3), whereas these components were reduced in the denser fractions. Myelin in the dense fractions was enriched in the more modified forms of basic protein (components 6, 7, and 8). The pattern of microheterogeneity was different for basic protein isolated from myelins of a 2‐year‐old and an adult brain; the former showed fewer components and mainly the most cationic species. On the other hand, the pattern of microheterogeneity of basic protein isolated from the different density gradient fractions was similar for both ages.
Serotonin Inhibits Acetylcholine Release from Rat Striatum Slices: Evidence for a Presynaptic Receptor‐Mediated Effect
Authors: Gillet G. , Ammor S. , Fillion G.
Published: October 2006

Abstract: Rat brain striatum slices were incubated with [3H]choline, perfused with a physiological buffer, and stimulated by perfusion with a K+‐enriched buffer for 2 min. The tritium overflow evoked by K+ was decreased by 5‐hydroxytrytamine (serotonin, 5‐HT) (maximal inhibition 10–6M). This effect of 5‐HT was mimicked by several agonists (5‐methoxytryptamine, N,N‐dimethyltryptamine, bufotenin) and blocked by serotonergic antagonists (methiothepin, methysergide, cinanserin) but not by haloperidol; methiothepin and methysergide alone slightly increased the K+‐evoked overflow of tritium (3H). Inhibition of the tritium release by 5‐HT was not suppressed in the presence of tetrodotoxin (TTX) (10–6M) These results suggest that 5‐HT tonically inhibits acetylcholine (ACh) release from striatal cholinergic neurons by acting on a presynaptic receptor localized on cholinergic terminals.
Induced Release of γ‐Aminobutyric Acid by a Carrier‐Mediated, High‐Affinity Uptake of L‐Glutamate in Cultured Chick Retina Cells
Authors: do Nascimento Jose Luiz M. , de Mello Fernando G.
Published: October 2006

Abstract: [3H]γ‐aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse‐stimulated (2 min) with 0.1 mM L‐glutamate but not with D‐glutamate. The EC50 for L‐glutamate to evoke [3H]GABA release was approximately 15 μM. This value is close to the Km for high‐affinity uptake of L‐glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L‐glutamate‐induced release of GABA was abolished. Moreover. L‐[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L‐Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D‐Aspartate, which is taken up by the same high‐affinity uptake mechanism as L‐glutamate, induced an increase in [3H]GABA efflux comparable to L‐glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L‐glutamate in eliciting [3H]GABA release. The addition of both GABA and L‐glutamate to the incubation medium indicated that [3H]GABA efflux due to L‐glutamate and GABA was additive. L‐Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L‐aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA‐promoted GABA release‐homoexchange, (b) a Ca2+‐dependent L‐aspartate‐promoted release, and (c) a Ca2+‐independent, Na+‐dependent L‐glutamate‐evoked release. In addition, our data strongly suggest that the L‐glutamate‐promoted GABA release is due to a process of exchange of L‐glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.
Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids
Authors: Klein Chava , Levy Rivka , Simantov Rabi
Published: October 2006

Abstract: A subclone of NG108–15 neuroblastoma‐glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self‐generating Percoll‐sucrose gradients and the enzymes β‐glucuronidase, galactosyltransferase, 5′‐nucleotidase, and glucose‐6‐phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu‐enkephalin induced time‐dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]‐D‐Ala2D‐Leu5‐enkephalin in vitro was increased on treatment with Leu‐enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H‐ligand‐receptors in the cells was monitored. Leu‐enkephalin and etorphine altered the distribution of receptor‐bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid‐treated cells.
[3H]5‐Hydroxytryptamine Binding to Brain Astroglial Cells: Differences Between Intact and Homogenized Preparations and Mature and Immature Cultures
Authors: Whitaker‐Azmitia Patricia M. , Azmitia Efrain C.
Published: October 2006

Abstract: [3H]5‐hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2–12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax= 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax= 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested.
Stoichiometries of Acetylcholine Uptake, Release, and Drug Inhibition in Torpedo Synaptic Vesicles: Heterogeneity in Acetylcholine Transport and Storage
Authors: Anderson D. C. , Bahr Ben A. , Parsons Stanley M.
Published: October 2006

Abstract: Highly purified Torpedo electric organ synaptic vesicles form a 49 nM suspension at 1 mg protein/ml. Under active transport conditions hundreds of molecules of [3H]acetylcholine ([3H]ACh) can be accumulated per vesicle, which requires the ACh transporter to undergo multiple turnovers. The transport blocker trans‐2‐(4‐phenylpiperidino)cyclohexanol (AH5183) has no effect on storage of endogenous ACh by vesicles. In contrast, AH5183, other blocking drugs, and nonradioactive ACh caused a rapid release of at least 30–63 molecules of newly transported [3H]ACh per vesicle. Thus AH5183 distinguishes recently transported “new” vesicular ACh from “old” endogenous ACh. l‐AH5183 inhibits transport of ACh with a half‐inhibitory concentration of 16 ± 7 nM at 12 nM vesicles and 115 ± 34 nM at 120 nM vesicles. With the assumption that AH5183 acts on a receptor in an unamplified manner about 2.7 or fewer receptors per vesicle need to be occupied to cause inhibition of ACh transport. The apparent amplification in the number of [3H]ACh molecules per vesicle that are released by AH5183 suggests that AH5183 inhibits ACh storage by an indirect mechanism that distinguishes new from old ACh.
Regional Kinetic Constants for Blood–Brain Barrier Pyruvic Acid Transport in Conscious Rats by the Monocarboxylic Acid Carrier
Authors: Miller Leonard P. , Oldendorf William H.
Published: October 2006

Abstract: The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood–brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 μ1 containing [1‐14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi‐lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 ± 0.08 μmol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22–0.27 μmol/min/g. The Vmax for the hippocampus was intermediate at 0.37 ± 0.12 μmol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier.
Increased Acetylcholine Synthesis and Release in Brains of Cats with GM1 Gangliosidosis
Authors: Jope Richard S. , Baker Henry J. , Connor Donald J.
Published: October 2006

Abstract: Cholinergic processes were measured in motor cortex, hippocampus, and striatum of cats in the terminal stages of GM1 gangliosidosis and compared to those of control cats. The greatest difference observed was elevation in the rate of K+‐stimulated release of acetylcholine (ACh) from brain slices prepared from affected cats. The K+‐stimulated release of endogenous ACh was increased by 31–43% and of newly synthesized ACh by 19–80% in brain slices from different brain regions. All regions that were examined were affected but the greatest effects occurred in cortex. The rate of synthesis of ACh was elevated in cortical and hippocampal slices. Choline acetyltransferase activity in brain regions of cats with GM1 gangliosidosis was not significantly different from that in controls, whereas high‐affinity choline transport in cortical synaptosomes was elevated. Muscarinic receptor binding sites were reduced in the cortex, hippocampus, and striatum of GM1 mutant cats, whereas the apparent affinity was not altered. These results indicate that there are major alterations of cholinergic function in the brains of cats with GM1 gangliosidosis.
Stress‐Induced Changes in t‐[35S]Butylbicyclophosphorothionate Binding to γ‐Aminobutyric Acid‐Gated Chloride Channels Are Mimicked by In Vitro Occupation of Benzodiazepine Receptors
Authors: Trullas R. , Havoundjian H. , Skolnick P.
Published: October 2006

Abstract: The allosteric modulation of t‐[35S]butylbicyclo‐phosphorothionate binding by flunitrazepam was studied in well‐washed brain membranes prepared from control and swim‐stressed rats. Swim stress has been reported to decrease the KD, and increase the Bmax of this radioligand. Flunitrazepam increased radioligand binding with equal potency (EC50∼ 11 nM) in both groups, but the maximal enhancement (efficacy) produced by this drug was significantly greater in control than in swim‐stressed rats. Ro 15–1788 (a benzodiazepine receptor antagonist) blocked the effect of flunitrazepam on t‐[35S]butylbicyclophosphorothionate binding in both groups. This increase in t‐[35S]butylbicyclophosphorothionate binding resulted from a significant reduction in KD with no alteration in Bmax. The KD values obtained in cortical membranes of control rats after addition of flunitrazepam were not significantly different from those in the swim‐stressed group. Preincubation of cortical homogenates from control animals with flunitrazepam prior to extensive tissue washing resulted in Bmax and KD values of t‐[35S]butylbicyclophosphorothionate similar to those obtained in stressed animals. These findings suggest that stress and flunitrazepam may share a common mechanism in regulating t‐[35S]butylbicyclophosphorothionate binding and support the concept that stress‐induced modification of γ‐aminobutyric acid (GABA)‐gated chloride channels in the CNS results from the release of an endogenous modulator (with benzodiazepine‐like properties) of the benzodiazepine‐GABA receptor chloride ionophore receptor complex.
Differential Postnatal Development of Monoamine Oxidases A and B in the Blood‐Brain Barrier of the Rat
Authors: Kalaria Rajesh N. , Harik Sami I.
Published: October 2006

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [3H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several‐fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO‐A and MAO‐B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO‐B. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analyses of rat microvessels after [3H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO‐A and ‐60,000 for MAO‐B. SDS‐PAGE resuits of brain microvessels obtained from 1‐, 14‐, and 42‐day‐old rats confirm the differential postnatal development of MAO‐B in rat brain microvessels.
Glutamine Synthetase of the Human Brain: Purification and Characterization
Authors: Yamamoto Hirotaka , Konno Hidehiko , Yamamoto Teiji , Ito Kitae , Mizugaki Michinao , Iwasaki Yuzo
Published: October 2006

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate‐poly‐acrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of γ‐glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double‐immunodiffusion technique disclosed cross‐reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.
Effects of Single and Repeated Electroconvulsive Shock Administration on Inositol Phosphate Accumulation in Rat Brain Slices
Authors: Newman Michael E. , Miskin Ian , Lerer Bernard
Published: October 2006

Abstract: Accumulation of inositol‐1‐phosphate after labeling with [3H]inositol and stimulation with noradrenaline, carbachol, and serotonin was measured in rat cortical, caudate nucleus, and hippocampal slices. The response to noradrenaline was significantly increased in cortical slices from animals that had received either a single electroconvulsive shock (ECS) or a series of 10 daily ECS but was unchanged in caudate nucleus or hippocampal slices. The response to carbachol, a muscarinic cholinergic agonist, was unchanged in cortical or caudate nucleus slices but was significantly reduced in hippocampal slices from animals that had received chronic ECS. The response to serotonin in cortical slices was not affected by the treatment. The results are correlated with changes in receptor number, which have been demonstrated to occur after administration of ECS.
Inhibition of Norepinephrine Transport and Reserpine Binding by Reserpine Derivatives
Authors: Parti Rajesh , Özkan Eric D. , Harnadek Gordon J. , Njus David
Published: October 2006

Abstract: Reserpine, a competitive inhibitor of catecholamine transport into adrenal medullary chromaffin vesicles, consists of a trimethoxybenzoyl group esterified to an alkaloid ring system. Reserpine inhibits norepinephrine transport with a Ki of ∼ 1 nM and binds to chromaffin‐vesicle membranes with a KD of about the same value. Methyl reserpate and reserpinediol, derivatives that incorporate the alkaloid ring system, also competitively inhibit norepinephrine transport into chromaffin vesicles with Ki values of 38 ± 10 nM and 440 ± 240 nM, respectively. Similar concentrations inhibit [3H]reserpine binding to chromaffin‐vesicle membranes. 3,4,5‐Trimethoxybenzyl alcohol and 3,4,5‐trimethoxybenzoic acid, derivatives of the other part of the reserpine molecule, do not inhibit either norepinephrine transport or [3H]reserpine binding at concentrations up to 100 μM. Moreover, trimethoxybenzyl alcohol does not potentiate the inhibitory action of methyl reserpate. Therefore, the amine binding site of the catecholamine transporter appears to bind the alkaloid ring system of reserpine rather than the trimethoxybenzoyl moiety. The more potent inhibitors are more hydrophobic compounds, suggesting that the reserpine binding site is hydrophobic.
Dopamine D‐1 Receptor and Cyclic AMP‐Dependent Phosphorylation in Parkinson's Disease
Authors: Cash Roland , Raisman Rita , Ploska Alain , Agid Yves
Published: October 2006

Abstract: D‐1 and D‐2 receptor densities, evaluated respectively by [3H]SCH 23390 and [3H]spiperone binding, and DARPP‐32 (dopamine and adenosine 3′:5′‐monophos‐phate‐regulated phosphoprotein—32K) concentrations, were studied in the brains of control and parkinsonian subjects postmortem. D‐2 receptor density was unchanged in the putamen of parkinsonian patients. D‐1 receptor density was unchanged in the putamen and substantia nigra pars reticulata (SNR) of parkinsonian patients, but decreased by 28% in the substantia nigra pars compacta (SNC). DARPP‐32, which is localized in the same structures as D‐1 receptors of which it is thought to represent the intracellular messenger, decreased by 45% in the putamen, 66% in the SNR, and 79% in the SNC. The decrease in D‐1 receptors in the SNC may be due to degeneration of pallidonigral GABAergic neurons, but some of the D‐1 receptors may be on the nigrostriatal dopaminergic neurons themselves. The dissociation between the alteration of D‐1 receptor densities and DARPP‐32 concentrations in both the striatum and substantia nigra, which are of the same order in the two structures, may be an index of functional hypoactivity of D‐1 neurotransmission.
Solubilization of Kainic Acid Binding Sites from Rat Brain
Authors: Hampson D. R. , Huie D. , Wenthold R. J.
Published: October 2006

Kainic acid binding sites were solubilized from rat brain using a combination of Triton X‐100 and digitonin. The highest percentage of solubilized binding sites (45%) was obtained by treating brain membranes with 1 % Triton‐X‐100 and 0.2% digitonin in 0.5 M potassium phosphate containing 20% glycerol. The solubilized binding sites were stable and amenable to analysis by gel nitration and lectin affinity chromatography. Computer assisted analyses demonstrated that the solubilized sites displayed high‐and low‐affinity binding constants similar to the membrane‐bound sites. Competition experiments further supported the pharmacological similarities of the solubilized and membrane‐bound sites. Gel nitration chromatography of the solubilized binding site indicated that the detergent‐bound complex had a Stokes radius of 82.7 A. The [3H]kainic acid binding site appears to be glycosylated based on its capability to bind to lectins. The lectin, wheat‐germ agglutinin, proved to be a potentially useful tool for characterization because the solubilized binding sites were bound and eluted in relatively high yield.
Preparation and Characterization of Antibodies Against a Sulfated Glucuronic Acid‐Containing Glycosphingolipid
Authors: Kohriyama Tatsuo , Ariga Toshio , Yu Robert K.
Published: October 2006

Abstract: In some patients with demyelinating neuropathy there are immunoglobulin M paraproteins that react with carbohydrate determinants shared by myelin‐associated glycoprotein (MAG) and two peripheral nerve acidic glycolipids, termed sulfoglucuronosylglycosphingolipids (SGGLs). To study the antigenicity of these glycolipids, we immunized three New Zealand white rabbits with sulfoglucuronosylparagloboside (SGPG), a major SGGL in peripheral nerve, emulsified in Freund's complete adjuvant and keyhole limpet hemocyanin. All three rabbits inoculated with SGPG showed weight loss and mild weakness, predominantly in their hind feet, 2–5 weeks postinoculation (PI). Two of the three rabbits again showed moderate weakness 3 and 8 months PI, respectively. Electrophysiological studies demonstrated a slowed nerve conduction velocity in the sciatic nerve. Anti‐SGPG antibody titers in sera were detected at dilutions of 1:1,000 to 1:2,500 by an enzyme‐linked immunosorbent assay. Although all three rabbit sera reacted with SGGLs, two reacted with a desulfated form of SGPG and the other did not, suggesting a fine heterogeneity in antigenic specificity. As with sera from patients with demyelinative paraproteinemia, all rabbit sera reacted with MAG in human CNS and PNS myelin. They also reacted with MAG from bovine CNS myelin as well as several low‐molecular‐weight glycoproteins in bovine peripheral nerve myelin. Thus, we demonstrated that the rabbit antisera generated against SGPG have the same or similar antigenic specificity as those of the anti‐MAG M‐proteins from patients with neuropathy. The results suggest that an autoimmune response against the sulfoglucuronosyl residue may participate in the immunopathogenesis of this type of neuropathy.
Primary Structure of Neuromedin U from the Rat
Authors: Conlon J. M. , Domin J. , Thim L. , DiMarzo V. , Morris H. R. , Bloom S. R. , Thim L. , DiMarzo V. , Morris H. R.
Published: October 2006

Abstract: The primary structure of neuromedin U from the rat ileum was established as: Tyr‐Lys‐Val‐Asn‐Glu5‐Tyr‐Gln‐Gly‐Pro‐Val10‐Ala‐Pro‐Ser‐Gly‐Gly15‐Phe‐Phe‐Leu‐Phe‐Arg20‐Pro‐Arg‐Asn‐NH2. There was no evidence for microheterogeneity. This amino acid sequence contains two deletions and nine substitutions compared with the neuromedin U‐25 from the pig. In particular, the replacement of the Arg'6‐Arg17 processing site in the porcine peptide by Gly14‐Gly15 in the rat means that a peptide corresponding to neuromedin U‐8 was not found in the rat intestine.
Transmitter‐Like Release of Endogenous 3,4‐Dihydroxyphenylalanine from Rat Striatal Slices
Authors: Goshima Y. , Kubo T. , Misu Y.
Published: October 2006

Abstract: Biphasic electrical field stimulation (0.5–5 Hz, 2 ms, 25 V, 3 min) and high K+ (10–30 mM, 5 min) released endogenous 3,4‐dihydroxyphenylalanine (DOPA) from superfused rat striatal slices. Characteristics of the DOPA release were compared with those of 3,4‐dihydroxyphenylethylamine (dopamine, DA). Electrical stimulation at 2 Hz evoked DOPA and DA over similar time courses, α‐Methyl‐p‐tyrosine (0.2 mM) markedly reduced release of DOPA but not of DA. Maximal release (0.3 pmol) of DOPA was obtained at 2 Hz and at 15 mM K+. The impulse‐evoked release of DOPA and DA was completely tetrodotoxin (0.3 μM) sensitive and Ca2+ dependent and the 15 mM K+‐evoked release was also Ca2+ dependent. On l‐[3,5‐3H]tyrosine (1 μM) superfusion, high K+ (15 and 60 mM) released DOPA and DA together with concentration‐dependent decreases in tyrosine 3‐monooxygenase (EC 1.14.16.2) activity as indicated by [3H]H2O formation, followed by concentration‐dependent increases after DOPA and DA release ended. These findings suggest that striatal DOPA is released by a Ca2+‐dependent excitation‐secretion coupling process similar to that involved in transmitter release.
Characterization of Seizure‐Induced Cysteinyl‐Leukotriene Formation in Brain Tissue of Convulsion‐Prone Gerbils
Authors: Simmet Thomas , Seregi András , Hertting Georg
Published: October 2006

Abstract: Tonic‐clonic seizures elicited in convulsionprone gerbils resulted in a large increase in immunoreactive prostaglandin (PG) F2α and in a smaller increase in immunoreactive leukotriene (LT) C4‐like material in brain tissue. Brain tissue contents of both eicosanoids were found to reach a maximum at 6 min after the onset of seizures and were still elevated at 54 min after the beginning of convulsions. By reversed phase HPLC the immunoreactive LTC4‐like material was identified as LTC4 and LTD4 at 6 min after the onset of convulsions, whereas at 54 min after the onset, transformation of LTD4 to LTE4 could be detected as well. In gerbils showing only weak seizure activity a small increase in PGF2α but no increase in immunoreactive LTC4‐like material could be detected at 6 min after the onset of convulsions. Pretreatment with indomethacin abolished the formation of PGF2α but significantly enhanced the biosynthesis of immunoreactive LTC4‐like material at 18 min after the beginning of seizures. The results demonstrate formation of cysteinyl‐LT following tonicclonic convulsions in spontaneously convulsing gerbils which could be enhanced by inhibition of the cyclooxygenase pathway of arachidonic acid metabolism. Since cysteinyl‐LT have potent biological actions in various organs this finding warrants further investigations on the potential role of cysteinyl‐LT in the CNS.
Excitatory Amino Acid Agonist‐Antagonist Interactions at 2‐Amino‐4‐Phosphonobutyric Acid‐Sensitive Quisqualate Receptors Coupled to Phosphoinositide Hydrolysis in Slices of Rat Hippocampus
Authors: Schoepp Darryle D. , Johnson Bryan G.
Published: October 2006

Abstract: Studies were carried out to define the relative affinities and intrinsic activities of excitatory amino acid agonists that activate receptor sites coupled to phosphoinositide hydrolysis in brain. Slices of rat hippocampus were prelabeled with myo‐[3H]inositol, and agonist stimulation was indexed by measuring the accumulation of [3H]inositol monophosphate ([3H]IP) in the presence of Li+. It was observed that ibotenic (IBO) and quisqualic (QUIS) acids both elicit highly significant, concentration‐dependent stimulation of phosphoinositide hydrolysis. Whereas maximal stimulation by IBO (10−3M) was four‐ to fivefold over basal values, the maximal effect of QUIS (10−4M) was less (about twofold). Based on the relative concentrations required for 50% maximal stimulation, QUIS was 20 times more potent than IBO. Stimulation of phosphoinositide hydrolysis by either IBO or QUIS was additive to the effects of nonexcitatory amino acid agonists (carbachol and norepinephrine) in this tissue. However, the stimulatory effects of IBO plus QUIS were not additive. At ≥ 10−4M, QUIS significantly inhibited phosphoinositide hydrolysis by a maximal stimulatory concentration of IBO (10−3M) to a level observed with QUIS alone. Other excitatory amino acid agonists, including kainate, N‐methyl‐d‐aspartate, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), had no stimulatory effects at concentrations as high as 10−3M. The d,l or l forms of 2‐amino‐4‐phosphonobutyric acid (AP4), but not d‐AP4, significantly enhanced [3H]IP levels to ∼135% of basal values. d,l‐AP4 and l‐AP4 also inhibited stimulation by either IBO or QUIS, but relative inhibition of QUIS effects was quantitatively less than that of IBO effects. These studies indicate that IBO represents a full agonist at these “AP4‐sensitive QUIS receptors” coupled to phosphoinositide hydrolysis in brain. QUIS is a partial agonist with the highest affinity for the receptor, and l‐AP4 represents a partial agonist with relatively weak intrinsic activity. Furthermore, this receptor effect is not mediated at “QUIS” receptor sites that are activated by AMPA.
Effect of Nimodipine on the Regional Cerebral Acidosis Accompanying Thiamine Deficiency in the Rat
Authors: Vogel Steven , Hakim Antoine M.
Published: October 2006

Abstract: The effect of the calcium channel blocker nimodipine on the previously described regional cerebral acidosis accompanying thiamine deficiency was investigated. Local cerebral pH (LCpH) and blood flow (LCBF) were separately determined autoradiographically in normal and 16‐day thia‐mine‐deficient rats administered the calcium antagonist drug and compared to appropriate controls. Nimodipine did not modify LCpH in normal brain. In thiamine deficiency, nimodipine significantly raised LCpH in 5 of 17 structures evaluated, two of which, the medial dorsal nucleus of the thalamus and the mammillary body, are vulnerable to the development of histological lesions in this condition. Although the calcium blocker augmented LCBF in normal brain, it had no effect on the hyperperfusion already present by day 16 of thiamine deprivation. Thus, the pH changes we are reporting are probably not related to an effect on cerebral perfusion, but could have resulted from an improved ability of the brain to reduce its proton load in the presence of nimodipine. These results may have wider therapeutic implications than in thiamine deficiency alone.