bluuebunny/crossref_metadata_2025 huggingface dataset:
A Unique Neurofilament from Torpedo Electric Lobe: Sequence, Expression, and Localization Analysis
Authors: Linial Michal , Scheller Richard H.
Published: October 2006

Abstract: A set of cDNA clones encoding a protein highly homologous to the mammalian middle‐size class of neurofilaments (NF‐M) was characterized. The amino acid similarity between the Torpedo and rat NF‐M approaches 90% in the amino‐terminal “rod‐like”domain and is significantly lower in the carboxy‐terminal tail. The Torpedo protein contains 13 tandem repeats of a unique six amino acid core, containing a Lys‐Ser‐Lys putative phosphorylation site. Surprisingly, the 3’untranslated region contains stretches of 80‐90% nucleic acid homology with the mammalian, but not with the chicken sequences. This homology is greater than much of the coding region, suggesting that the 3’untranslated region of the message has an important functional role, perhaps governing RNA stability or localization. This Torpedo NF‐M mRNA is expressed specifically in the electric lobe and was not detected in other tissues, including brain and spinal cord. A polyclonal antibody generated against a fusion protein synthesized in E. coli detects a 150‐kDa protein in the electric lobe and organ, as well as a small amount of material in the brain. Cytochemical studies reveal immunoreactivity in electromotor neuron axons and terminals. Specific expression of neurofilament genes in subsets of central neurons may be important in determining the morphology and functional characteristics of specific neuronal subtypes.
Relationship Between Serotoninergic Measures in Blood and Cerebrospinal Fluid Simultaneously Obtained in Humans
Authors: Sarrias Maria J. , Cabré Pere , Martínez Emili , Artigas Francesc
Published: October 2006

Abstract: The relationships between the concentration of serotonin (5‐HT) and related metabolites in human blood and CSF have been studies. Plasma tryptophan (TP), 5‐HT, 5‐hydroxyindoleacetic acid (5‐HIAA), and indoleacetic acid (IAA), whole‐blood 5‐HT, and CSF TP, 5‐HT, 5‐HIAA, IAA, homovanillic acid, and 3‐methoxy‐4‐hydroxyphenylethylene glycol were determined in 35 unmedicated outpatients who underwent minor surgical operations and had no history of psychiatric or neurological illnesses. Significant correlations were found between the serotoninergic parameters analyzed in blood and CSF. Plasma free 5‐HT correlated significantly with CSF 5‐HT (r= 0.411, p < 0.02), and plasma 5‐HIAA correlated with the CSF 5‐HIAA/5‐HT ratio (r= 0.508, p < 0.004). The concentration of 5‐HIAA in CSF correlated with the plasma 5‐HIAA/5‐HT ratio (r= 0.405, p < 0.026) (which can be taken as an index of monoamine oxidase type A activity in peripheral tissues) and with the platelet 5‐HT/plasma 5‐HT ratio (r= 0.375, p < 0.05). The concentrations of IAA in CSF and plasma were strongly correlated (r= 0.899, p < 0.001). The significance of these results and their relationship to the use of “in vivo”measures of 5‐HT and related metabolites in plasma and platelets as an index of serotoninergic function in affective disorders are discussed.
Aromatic L‐Amino Acid Decarboxylase Is Modulated by D1 Dopamine Receptors in Rat Retina
Authors: Rossetti Zvani L. , Silvia Christopher P. , Krajnc Dimitrij , Neff Norton H. , Hadjiconstantinou Maria
Published: October 2006

Abstract: Aromatic L‐amino acid decarboxylase (AAAD) activity of rat retina increases when animals are placed in a lighted environment from the dark. The increase of activity can be inhibited by administering the selective dopamine D1 receptor agonist SKF 38393, but not the selective D2 agonist quinpirole, or apomorphine. Conversely, in the dark, enzyme activity can be enhanced by administering the selective D1 antagonist SCH 23390 or haloperidol, but not the selective D2 antagonist (–)‐sulpiride. Furthermore, in animals exposed to room light for 3 h, the D1 agonist SKF 38393 reduced retinal AAAD activity, and this effect was prevented by prior administration of SCH 23390. In contrast, quinpirole had little or no effect when administered to animals in the light. Kinetic analysis indicated that the apparent Vmax for the enzyme increases with little change in the apparent Km for the substrate 3,4‐dihydroxyphenylalanine or the cofactor pyridoxal‐5′‐phosphate. We suggest that dopamine released in the dark tonically occupies D1 receptors and suppresses AAAD activity. When the room light is turned on, D1 receptors are vacated and selective D1 agonists can either prevent the rise of AAAD or reverse light‐enhanced AAAD activity.
Effects of Transient Forebrain Ischemia and Pargyline on Extracellular Concentrations of Dopamine, Serotonin, and Their Metabolites in the Rat Striatum as Determined by In Vivo Microdialysis
Authors: Damsma G. , Boisvert D. P. , Mudrick L. A. , Wenkstern D. , Fibiger H. C.
Published: October 2006

Abstract: Striatal microdialysis was performed in rats subjected to 20 min of transient forebrain ischemia produced by occlusion of the carotid arteries during hemorrhagic hypotension. Extracellular changes of dopamine, serotonin, and their metabolites were monitored before, during, and after the ischemic insult at 10‐min intervals by on‐line HPLC analysis. During ischemia, extracellular dopamine increased dramatically (156 times baseline), as did 3‐methoxytyramine (3‐MT), whereas 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) decreased (15–25% of baseline). Upon reperfusion, dopamine was cleared from the extracellular fluid within 40 min and reached a stable level (70% of baseline). DOPAC and HVA increased (250–330%) transiently and reached their maximum 1 h following reperfusion, whereas 3‐MT decreased to undetectable levels within 20 min. Although baseline levels of serotonin were not detectable, serotonin and 5‐hydroxyindoleacetic acid showed a qualitatively similar temporal pattern to dopamine and its acid metabolites. Killing rats by cervical dislocation produced changes in extracellular dopamine, serotonin, and their metabolites that were almost identical to those seen during ischemia. Pargyline pretreatment 2 h before ischemia had marginal effects on the postischemic clearing of dopamine. The pargyline pretreatment, however, did increase the survival rate of rats subjected to ischemia, and this protective effect might be due to the pargyline‐induced blockade of the postischemic monoamine oxidase‐mediated increase in dopamine metabolism and the concurrent production of the potentially neurotoxic molecule, hydrogen peroxide.
Solubilisation of the 5‐Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes
Authors: McKernan Ruth M. , Quirk K. , Jackson R. G. , Ragan C. Ian
Published: October 2006

Abstract: 5‐Hydroxytryptamine3 (5‐HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205–930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested { 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X‐100, and 1% octyl glucoside}, deoxycholate (0.5%) yielded the best solubilisation (54.6 ± 6% of receptor, 70.5 ± 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205–930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G‐25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205–930 to these soluble sites was saturable and specific (Bmax, = 46.1 ± 6 fmol/mg of protein; KD= 0.33 ± 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205–930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4°C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists competed for the soluble receptors with a rank order of potency typical for binding at a 5‐HT3 receptor site: zacopride (Ki= 0.26 nM) > quipazine (0.37 nM) = Q ICS 205–930 (0.33 nM) > ICS 205–930 (0.93 nM) > GR 38032F (2.2 nM) > BRL 24924 (4.1 nM) > MDL 72222 (23.4 nM) > ketanserin (6,000 nM). The agonists 5‐HT and 2‐methyl‐5‐HT also competed for [3H]Q ICS 205–930 binding with high affinity (39.6 and 55.6 nM respectively). Therefore, we conclude that the 5‐HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.
Different Patterns of Agonist‐Stimulated Increases of 3H‐Inositol Phosphate Isomers and Cytosolic Ca2+ in Bovine Adrenal Chromaffin Cells: Comparison of the Effects of Histamine and Angiotensin II
Authors: Stauderman Kenneth A. , Pruss Rebecca M.
Published: October 2006

Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine‐ and angiotensin II‐induced changes of inositol mono‐, bis‐, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four‐ to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II‐ and histamine‐stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5‐tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2+‐mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+], changes in BCC. Different patterns of second‐messenger formation are likely to be important in signal recognition and may encode agonist‐specific information.
Release of Calcitonin Gene‐Related Peptide‐Like Immunoreactive Substance from Neuromuscular Junction by Nerve Excitation and Its Action on Striated Muscle
Authors: Uchida Shuji , Yamamoto Hiromasa , Iio Sadaharu , Matsumoto Naoko , Wang Xiao‐Bing , Yonehara Norifumi , Imai Yasuo , Inoki Reizo , Yoshida Hiroshi
Published: October 2006

Abstract: In a rat phrenic nerve‐hemidiaphragm preparation, calcitonin gene‐related peptide (CGRP) increased the twitch contraction induced by nerve or transmural stimulation dose dependently. Either electrical or high K+ stimulation of the phrenic nerve caused release of a CGRP‐like immunoreactive substance (CGRP‐LIS) in a Ca2+‐dependent manner. Electrical stimulation of the phrenic nerve also increased the cyclic AMP content in diaphragm. This increase was not observed in Ca2+‐free medium and was blocked by antiserum against CGRP. These results indicate that excitation of the motor nerve causes release of CGRP‐LIS at nerve terminals and that the released CGRP‐LIS increases the cyclic AMP content of skeletal muscles and potentiates twitch contraction.
Cholinotoxic Effects of Aluminum in Rat Brain
Authors: Gulya K. , Rakonczay Z. , Kása P.
Published: October 2006

Abstract: The in vivo and in vitro effects of A1 on the cholinergic system of rat brain were studied. The amount of A1 accumulated after the chronic, intraperitoneal administration of aluminum gluconate (Al‐G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al‐G and AlCl3 administrations resulted in a general decrease (to 40–70% of the control values) in the specific l‐[3H]nicotine binding, involving all brain areas studied. The specific (–)‐[3H]quinuclidinyl benzilate binding was reduced (to 40–60% of the control values) only after 25 days of Al treatment. Al‐G and AlCl3 were equivalent in eliciting these reductions. In vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al‐G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10–8–10–6M), the activity was slightly increased, whereas at higher concentrations (10–6–10–4M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.
Brain and Plasma Quinolinic Acid in Profound Insulin‐Induced Hypoglycemia
Authors: Heyes Melvyn P. , Papagapiou Marios , Leonard Catherine , Markey Sanford P. , Auer Roland N.
Published: October 2006

Abstract: Profound insulin‐induced hypoglycemia is associated with early‐onset neuronal damage that resembles excitotoxic lesions and is attenuated in severity by antagonists of N‐methyl‐D‐aspartate receptors. Hypoglycemia increases L‐tryptophan concentrations in brain and could increase the concentration of the L‐tryptophan metabolite quinolinic acid (QUIN), an agonist of N‐methyl‐D‐aspartate receptors and an excitotoxin in brain. Therefore, we investigated the effects of 40 min of profound hypoglycemia (isoelectric EEG) and 1–2 h of normoglycemic recovery on the concentrations of QUIN in brain tissue, brain extracellular fluid, and plasma in male Wistar rats. Plasma QUIN increased 6.5‐fold by the time of isoelectricity (2 h after insulin administration). Regional brain QUIN concentrations increased two‐ to threefold during hypoglycemia and increased a further two‐ to threefold during recovery. However, no change in extracellular fluid QUIN concentrations in hippocampus occurred during hypoglycemia or recovery as measured using in vivo microdialysis. Therefore, the increases in brain tissue QUIN concentrations may reflect elevations of QUIN in the intracellular space or be secondary to the increases in QUIN in the vascular compartment in brain per se. L‐Tryptophan concentrations increased more than twofold during recovery only. Serotonin decreased >50% throughout the brain during hypoglycemia, while 5‐hydroxyindoleacetic acid concentrations increased more than twofold during hypoglycemia and recovery. In striatum, dopamine was decreased 75% during hypoglycemia but returned to control values during recovery, while striatal 3,4‐dihydroxyphenylacetic acid and homovanillic acid were increased more than twofold during both hypoglycemia and recovery. The present results do not support a role for increased QUIN in extracellular fluid space during the earlyphase neuronal damage of profound insulin‐induced hypoglycemia.
Identification and Role of Serotonin 5‐HT1A and 5‐HT1B Receptors in Primary Cultures of Rat Embryonic Rostral Raphe Nucleus Neurons
Authors: Héry F. , Boulenguez P. , Sémont A. , Héry M. , Pesce G. , Becquet D. , Faudon M. , Deprez P. , Fache M.‐P.
Published: October 2006

Abstract: Autoregulatory mechanisms affecting serotonin [5‐hydroxytryptamine (5‐HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5‐HT1A and 5‐HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5‐HT1A and 5‐HT1B receptors in the control of the synthesis and release of [3H]5‐HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5‐HT synthesis and release was observed in the presence of 5‐HT (10−8M), the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), the 5HT1B/1A agonist 5‐methoxy‐3‐(1,2,5,6‐tetrahydro‐4‐pyridinyl)‐1H‐indole (RU 24969), the 5‐HT1B agonist 3‐(1,2,5,6‐tetrahydropyrid‐4‐yl)pyrrolo[3,2‐b]pyrid‐5‐one (CP‐93, 129), and the 5‐HT1D/1B agonist sumatriptan. Inhibition of 5‐HT synthesis and release induced by 8‐OH‐DPAT was blocked by chiral N‐tert‐butyl‐3‐[1‐[1‐(2‐methoxy)phenyl]piperazinyl]‐1‐phenylpropionamide dihydrochloride quaternary‐hydrate (WAY 100135) (10−7M) or methyl 4‐[4‐[4‐(1,1,3‐trioxo‐2H‐1,2‐benzoisothiazol‐2‐yl)butyl]‐1‐piperazinyl]‐1H‐indole‐2‐carboxylate (SDZ 216–525) (10−7M), and that of CP‐93, 129 was blocked by methiothepin (10−7M). Paradoxically, extracellular levels of [3H]5‐HT increased in the presence of 8‐OH‐DPAT and RU 24969 at 10−6M. 5‐HT uptake experiments showed that these two agonists interacted with the 5‐HT transporter. 5‐HT1 binding sites (620 fmol/mg of protein) and 5‐HT1A (482 fmol/mg of protein) and 5‐HT1B (127 fmol/mg of protein) receptors were detected in 12‐day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5‐HT1A receptors are located on nerve cell bodies, whereas 5‐HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5‐HT1A and 5‐HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.
Localization of Endo‐Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue
Authors: Oliveira Elisabeth S. , Leite Paulo E. P. , Spillantini Maria G. , Camargo Antonio C. M. , Hunt Stephen P.
Published: October 2006

Abstract: The subcellular and regional distribution of endooligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin‐containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane‐associated form of endo‐oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Sub‐cellular fractionation showed that ∼17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3‐([3‐cholamidopropyl]dimethylammonio)‐1‐propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three‐ to 10‐fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo‐oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo‐oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo‐oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide‐containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo‐oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.
Immunohistochemical Detection of Phenol Sulfotransferase‐Containing Neurons in Human Brain
Authors: Zou Jianyan , Pentney Roberta , Roth Jerome A.
Published: October 2006

Abstract: Using antibodies raised against human platelet phenol sulfotransferase (PST), immunohistochemical studies were performed to determine the cellular localization of PST in several areas of human brain. In the hippocampus PST immunoreactivity was localized in both the pyramidal and nonpyramidal neurons and was in greatest abundance in the CA2 and CA3 areas. In the striatum the immunoreactivity was most predominant in the large neurons of the globus pallidus and in the medulla the staining was scattered throughout the neurons of the raphe nucleus and the reticular formation. The selective presence of PST in the neurons of the CNS raises the issue as to the role of this enzyme in sulfating neurotransmitters because PST has been shown to be capable of conjugating a variety of neurotransmitters including the catecholamines as well as the tyrosine moiety of a number of small peptides such as enkephalin and cholecystokinin.
Effects of Dietary n‐3 Fatty Acids on the Phospholipid Molecular Species of Monkey Brain
Authors: Lin Don S. , Connor William E. , Anderson Gregory J. , Neuringer Martha
Published: October 2006

Abstract: We examined the changes in the molecular species of brain ethanolamine glycerophospholipids of monkeys fed diets containing widely ranging amounts of n‐3 fatty acids. Two groups of rhesus monkeys were fed pre‐ and postnatally either a control diet (soy oil; containing 8% of fatty acids as 18:3n‐3) or a deficient diet (safflower oil; containing <0.3% 18:3n‐3). The brains of these animals were analyzed at 22 months of age. A third group of monkeys was fed the safflower oil diet to 22 months of age and then switched to a fish oil diet (28% long‐chain n‐3 fatty acids) for 1–2 years before autopsy. The molecular species of the diacyl, alkylacyl, and alkenylacyl ethanolamine glycerophospholipids from frontal cortex were separated by HPLC. A total of 24 molecular species were identified. Fatty acids in the sn‐2 position differed markedly among the diet groups, but the sn‐1 position always contained only 16:0, 18:0, or 18:1. In the diacyl subclass of the control brain, the n‐3 molecular species represented 41% of total and the n‐6 species 45%, whereas in the deficient brain the n‐3 molecular species decreased to 9% and n‐6 molecular species increased to 77%. The fatty acid 22:5n‐6 did not replace 22:6n‐3 in a symmetrical fashion in the molecular species of the deficient brain. In the brains of the fish oil‐fed monkeys, the n‐3 molecular species amounted to 61% and n‐6 molecular species were reduced to 25%. The species 18:1–22:6, 16:0–22:6, and 18:0–22:6 generally changed proportionally in response to diet. However, 18:1–20:4, 16:0–20: 4, and 18:0–20:4 responded differently. The fish oil diet led to an increase in the proportion of 18:1–20:4 in the alkenylacyl subclass, whereas 16:0–20:4 and 18:0–20:4 decreased. Thus, total species containing sn‐1 18:1 increased at the expense of sn‐1 16:0 in the fish oil animals. Regardless of diet, each subclass of ethanolamine glycerophospholipid showed a strikingly different ratio of sn‐1 16:0 to 18:0 to 18:1 for a given sn‐2 fatty acid. In conclusion, the different diets had profound qualitative and quantitative effects on the molecular species of brain phospholipids, and these changes have implications for possible functional changes.
Permeability of the Blood‐Brain Barrier to the Immunosuppressive Cyclic Peptide Cyclosporin A
Authors: Begley David J. , Squires Linda K. , Zloković Berislav V. , Mitrović Dusan M. , Hughes Christopher C. W. , Revest Patricia A. , Greenwood John
Published: October 2006

Abstract: Uptake of the immunosuppressive lipophilic peptide cyclosporin A has been measured by a number of techniques. The brain uptake index (BUI) technique in the rat yields only a small BUI value that is not significantly different from that of sucrose and mannitol and is comparable to other published BUI values for this compound. Brain perfusion studies in the guinea pig produce a unidirectional cerebrovascular permeability constant (Kin) of 1.2 ± 0.28 μg−1 min−1 for the hippocampus. Intravenous bolus injection techniques also in the guinea pig characteristically produce a larger Kin value of 2.53 ± 0.38 μg−1 min−1 for the same brain region, even after a correction for the inulin space of the tissue has been made. Apparent penetration of cyclosporin A into the cerebrospinal fluid (CSF) determined with the intravenous bolus injection technique is small with a Kin of 0.79 ± 0.07 μ g−1 min−1. However it is suggested that the radioactivity present in CSF is largely tritiated water. Studies with cultured cerebral endothelial cells from the rat have also been carried out and show that the cultured cells take up and accumulate cyclosporin A in vitro, achieving a tissue‐to‐medium ratio of 20 after 25 min of incubation. It is suggested that cyclosporin A is primarily taken up from lipoprotein at the blood‐brain interface but, because of tight junctions at the blood‐brain and blood‐CSF barriers, becomes effectively trapped in the cerebral endothelial cells and the choroid plexus.
Determination of Gangliosides in Six Human Primary Medulloblastomas
Authors: Gottfries Johan , Fredman Pam , Mansson Jan‐Eric , Collins V. Peter , von Hoist Hans , Armstrong Dawna D. , Percy Alan K. , Wikstrand Carol J. , Bigner Darell D. , Svennerholm Lars
Published: October 2006

Abstract: The ganglioside composition of six human medulloblastomas was analyzed. The characterization was performed by thin‐layer chromatography, sialidase hydrolysis, and immunological staining with a panel of characterized antiganglioside monoclonal antibodies. The total ganglioside content ranged from 60 to 1,130 nmol of ganglioside sialic acid/g wet weight. Neuronal gangliosides (gangliotetraose series) were found in varying amounts in all medulloblastomas. Gangliosides of the neolactotetraose series (3′‐LM 1 and LD1) were present in all specimens, and the lactotetraose series ganglioside 3′‐isoLM1 was found in all cases showing astro‐cytic differentiation. This supports our previous findings that 3′‐isoLM1 is a marker for proliferating astroglial cells.
Phospholipid Transverse Diffusion in Synaptosomes: Evidence for the Involvement of the Aminophospholipid Translocase
Authors: Zachowski Alain , Gaudry‐Talarmain Yvette Morot
Published: October 2006

Abstract: We have studied in Torpedo marmorata electric organ synaptosomes the equilibration kinetics of spin‐labeled phospholipid analogues initially incorporated into the outer plasma membrane monolayer. As assayed by evoked releases of both ATP and acetylcholine, the nerve endings were closed vesicles containing an energy source. The aminophospholipids (phosphatidylethanolamine and phosphatidylserine) were translocated toward the inner membrane leaflet faster and to a higher extent than their choline‐containing counterparts (phosphatidylcholine and sphingomyelin). This difference was abolished by incubation of synaptosomal membranes with N‐ethylmaleimide, suggesting that the accumulation of aminophospholipids in the inner layer was driven by a protein. This phenomenon is comparable with what was described in plasma membranes of other eucaryotic cells (erythrocyte, lymphocyte, platelet, fibroblast), and thus we would suggest that an aminophospholipid translocase, capable of moving the aminophospholipids from the outer to the inner layer at the expense of ATP, is also present in the synaptosomal plasma membrane.
Evidence for the Importance of a Carboxyl Group in the Binding of Ligands to the D2 Dopamine Receptor
Authors: Williamson Richard A. , Strange Philip G.
Published: October 2006

Abstract: A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N‐Acetylimidazole, 5,5′‐dithiobis(2‐nitrobenzoic acid), 1,2‐cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor.N,N′‐Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH‐binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted ben‐zamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but overlapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.
Modification by Cholecystokinin Octapeptide of the Binding ofμ‐, δ‐, and K‐Opioid Receptors
Authors: Wang Xiao‐Jing , Han Ji‐Sheng
Published: October 2006

Abstract: Previous study has shown that cholecystokinin (CCK) octapeptide (CCK‐8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium‐labeled agonists for the μ‐ {[tyrosyl‐3,5‐3H][d‐Ala2,MePhe4,Gly‐ol5]enkephalin ([3H]DAGO)}, δ‐ {[tyrosyl‐3,5‐3H][d‐Pen2,5]enkephalin ([3H]DPDPE)}, and k‐ ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK‐8. In the competition experiments, CCK‐8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 μmol/L. In the saturation experiments. CCK‐8 at concentrations of 0.1 nmol/L to 1 μmol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK‐8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK‐8 inhibits the binding of μ‐ and K‐opioid receptors via the activation of CCK receptors.
Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells
Authors: Yu Victor C. , Eiger Steven , Duan Dah‐Shuhn , Lameh Jelveh , Sadée Wolfgang
Published: October 2006

Abstract: The human neuroblastoma clonal cell line SH‐SY5Y expresses both μ‐ and δ‐opioid receptors (ratio ∼4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by μ‐opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE,), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (∼65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE, and VIP cAMP response from ∼50 to ∼80%. The use of highly μ‐ and δ‐selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly μ, and not δ, receptor mediated in SH‐SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE,‐stimulated, RA‐differentiated SHSY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over 12h, a fourfold shift of the PGE,‐morphine dose‐response curve was observed, whether or not IBMX was added. However, μ‐opioid receptor number and affinity to the μ‐selective [D‐Ala2, N‐Me‐Phe4, Gly5‐ol]enkephalin were largely unaffected, and Na+‐ and guanyl nucleotide‐induced shifts of morphine‐[3H]naloxone competition curves were unchanged. Further, morphine pretreatment elicited an AC rebound effect, causing higher cAMP accumulation after the drug was removed from the medium or acutely antagonized by naloxone. These results document biochemical correlates of μ‐opiate tolerance and dependence in SH‐SY5Y cells.
Agonists and Antagonists Recognize Different but Overlapping Populations of A1 Adenosine Receptors: Modulation of Receptor Number by MgCl2, Solubilization, and Guanine Nucleotides
Authors: Olah Mark , Stiles Gary L.
Published: October 2006

Abstract: A1 selective agonist and antagonist radioligands bind to the same A1 adenosine receptor binding subunit, as documented by photoaffinity labelling and partial peptide maps. In this study we document that although these radioligands recognize the same A, adenosine receptor (A1AR), they recognize different numbers of A1ARs in bovine brain membranes, with agonist number being greater than antagonist number. Neither addition of guanine nucleotides nor removal of Mg2+ ions enhanced antagonist binding in membranes. On solubilization, agonists still recognized a greater number of A1ARs but addition of guanine nucleotides or removal of Mg2+ substantially increased the number of receptors detected with antagonist radioligands. The effects of Mg2+ and guanine nucleotides were not additive, suggesting that formation of a “low agonist‐receptor‐G protein state” by either modulating agent was sufficient to alter the receptor conformation such that it could be recognized by antagonist. These studies suggest that a proportion of the “precoupled A1AR‐G protein complex” in membranes are in a conformation that cannot be recognized by antagonists and that membrane constraints are such that ions or guanine nucleotides cannot sufficiently modulate the conformation to allow it to recognize antagonists. On removal of membrane structure by solubilization, these constraints are removed.
Effects of N‐Methyl‐d‐Aspartate on [Ca2+]i and the Energy State in the Brain by 19F‐ and 31P‐Nuclear Magnetic Resonance Spectroscopy
Authors: Ben‐Yoseph O. , Bachelard H. S. , Badar‐Goffer R. S. , Dolin S. J. , Morris P. G.
Published: October 2006

Abstract: The effects of N‐methyl‐d‐aspartate (NMDA) on the free intracellular Ca2+ concentration ([Ca2+]i) and the energy state in superfused cerebral cortical slices have teen studied using 19F‐ and 31P‐nuclear magnetic resonance spectroscopy. [Ca2+]i was measured using the calcium indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N.N.N′.N′‐tetraacetic acid (5FBAPTA). NMDA (10 μM) in the absence of extracellular Mg2+ caused the expected rise in [Ca2+]i but produced an impairment of the energy state: the phosphocreatine (PCr) content was decreased by 42%, and the Pi/PCr ratio was increased by 55%. There was no detectable change in ATP or free intracellular Mg2+ concentration. Increasing the NMDA concentration in the superfusing medium to 100 or 400 μM caused no further increase in [Ca2+]i or further decrease in PCr content, but the Pi/PCr ratio continued to rise. The impairment of the energy state preceded the effect on [Ca2+]i, and these changes were irreversible on return to control conditions. Repeating the experiments in the presence of 1.2 mM extracellular Mg2+ resulted in similar changes in the energy state, with no change in [Ca2+]i. The possibilities that the effects were due to membrane depolarisation or to the presence of 5FBAPTA within the tissues were eliminated. The results suggest that low concentrations (10 μM) of NMDA produce an impaired energy state independent of the presence of extracellular Mg2+ and that the decreased energy state is not due to the changes in [Ca2+]i, which are seen only in the absence of extracellular Mg2+.
Selective Loss of N‐Methyl‐d‐Aspartate‐Sensitive l‐[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis
Authors: Peterson Christine , Giguere Jean‐Francois , Cotman Carl W. , Butterworth Roger F.
Published: October 2006

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N‐methyl‐d‐aspartate (NMDA) subclasses of l‐glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end‐to‐side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of l‐glutamate to the NMDA receptor when compared to sham‐operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down‐regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.
Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ
Authors: Guitart Xavier , Marsal Jordi , Solsona Carles
Published: October 2006

The effect of phorbol esters and so the involvement of Ca2+/phospholipid‐dependent protein kinase (protein kinase C; PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration‐dependent manner and increased the potassium‐evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA‐induced ACh release was reversed by H‐7, an inhibitor of PKC activity. This drug showed no effect on potassium‐evoked ACh release. Botulinum toxin, a strong blocker of potassium‐induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA‐induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.
Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line
Authors: Gordon Eric A , Kohout Trudy A. , Fishman Peter H.
Published: October 2006

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK‐N‐MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide‐binding protein of adenylate cyclase, Gi Intact SK‐N‐MC cells bound radiolabeled NPY with a KD of 2 nM and contained ˜83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK‐N‐MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol‐stimulated cyclic AMP production in a dose‐dependent manner, with half‐maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose‐response curves. NPY also inhibited basal and isoproterenol‐stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP‐ribosylation of a 41‐kDa protein in SK‐N‐MC cells that corresponds to Gi. The receptors on SK‐N‐MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as α‐adrenergic, dopaminergic. muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK‐N‐MC cells may be a useful model for investigating NPY receptors and NPY‐mediated signal trans‐duction.
Aluminum Uptake by Neuroblastoma Cells
Authors: Shi Biao , Haug Alfred
Published: October 2006

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 × 106 cells/ml, pH 6.0, and 25 μM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60–70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the post‐mitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum‐fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.
1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Resistant, Flat‐Cell PC12 Variants Having a Partial Loss of Transformed Phenotype
Authors: Andersen Julie K. , Zhang Mao‐Bin , Zhong Xiao‐Hui , Rozenberg Yanina Y. , Howard Bruce D.
Published: October 2006

We have cloned and characterized two variants of PC12 cells. MPT1 cells were selected by their resistance to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and variant 2068 was isolated nonselectively as a large, flat‐cell variant commonly occurring in PC12 cultures. Variant 2068 cells also exhibit resistance to MPTP. Karyotype analysis revealed that these variants are true derivatives of wild‐type PC12 cells; however, each variant is tetraploid, whereas the wild‐type parent is diploid. The two variants contain an altered level and composition of lactate dehydrogenase isoenzymes, which could account for a previously described difference in lactate metabolism. Both variants exhibit a partial loss of transformed phenotype in culture in that they are nonrefractile, grow in monolayers, and fail to multiply in soft agar. We suggest that this alteration in transformed phenotype may result in altered mitochondria and lactate dehydrogenase and thus account for their resistance to MPTP. Compared with wild‐type PC12 cells, MPT1 cells have a decreased level of fos mRNA and an increased level of myc mRNA; the latter results from an increased level of transcription of exon 1 of the myc gene. Studies with hybrid cells obtained by fusing MPT1 cells with wild‐type‐like cells show that most, but not all, of the parameters of the MPT1 phenotype predominate.
Conformational Correlates of the Epitopes of Human Myelin Basic Protein Peptide 80–89
Authors: Whitaker John N. , Moscarello Mario A. , Herman Paula K. , Epand Richard M. , Surewicz Witold K.
Published: October 2006

Different epitopes residing within the decapeptide of residues 80–89 of human myelin basic protein (MBP) exist in the MBP‐like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80–89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80–89 to form peptides 81–89, 82–89, 83–89, and 84–89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier‐transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to β‐structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by α‐helix and β‐structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.
Sulfated Glucuronyl Paragloboside in Rat Brain Microvessels
Authors: Miyatani Nobuyuki , Kohriyama Tatsuo , Maeda Yasuhiro , Yu Robert K.
Published: October 2006

In patients with neuropathy associated with para‐proteinemia, there are monoclonal immunoglobulin M antibodies reacting with myelin‐associated glycoprotein and sulfated glucuronyl glycolipids. There are indications that the monoclonal antibodies may be responsible for these neuropathies. However, the mechanism by which the antibodies gain access to the nervous tissue, which is separated by the blood‐brain barrier or blood‐nerve barrier, is still unknown. In this study, we examined the presence of the sulfated glucuronyl glycolipid antigens on brain endothelial cells. Micro‐vessels were isolated from adult Lewis rat brain cortex. Sulfated glucuronyl paragloboside (SGPG) was detected in the acidic lipid fraction by a TLC immunostaining method. Immunofluorescence studies showed positive staining on the surface of microvessels. In addition, SGPG could be detected in the cultured endothelial cells of human umbilical vein. These findings suggest that the endothelial cells contain an‐tigenic sites for interaction with the autoantibodies. This type of interaction may result in damages to the endothelial cell function and may be responsible for changes in the blood‐brain barrier permeability and the ensuing penetration of large molecules, such as immunoglobulins, into the endo‐neurial space.