bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, Structure, and Properties of the β‐Carboline Formed from 5‐Hydroxytryptamine by the Superoxide Anion‐Generating System
Authors: Uemura Tomihiko , Kanashiro Masaru , Yamano Toshio , Hirai Kumiko , Miyazaki Noriyuki
Published: October 2006

Abstract: A yellow substance was isolated by Sephadex LH‐20 gel chromatography, silica gel TLC, and reversed‐phase HPLC after incubation of 5‐hydroxytryptamine (5‐HT) with the superoxide anion (O‐2)‐generating system, i.e., the xanthine‐xanthine oxidase system, in the presence of the Fe‐EDTA complex and glycine in alkaline medium. The product gave a blue color with Ehrlich's reagent very slowly but no color with xanthydrol and Gibbs’reagent. Its reduced form, however, gave an immediate blue‐violet color with all three reagents. No color was developed with ninhydrin, but the reduced form was orange‐red. The chemical structure of the yellow substance was identified by‘H‐nuclear magnetic resonance and field desorption‐mass spectrometry as 4,9‐dihydro‐3H‐pyrido[3,4‐b]indol‐6‐ol (6‐hydroxy‐3,4‐dihydro‐β‐carboline, 5‐hydroxy‐2,3‐dihydrotryptoline). The one carbon unit inserted into 5‐HT came from glycine, with its 14C‐2 being incorporated into C‐1 of the yellow substance. The mechanism for the formation of the yellow substance from 5‐HT is discussed. This compound inhibited 5‐HT uptake into rat brain cortical synaptosomes with an IC50 of 1.5 × 10‐4M and a Ki value of 1.2 × 10‐5M.
Two Groups of Amino Acids Interact with GABA‐A Receptors Coupled to t‐[35S]Butylbicyclophosphorothionate Binding Sites: Possible Involvement with Seizures Associated with Hereditary Amino Acidemias
Authors: Squires Richard F. , Saederup Else , Lajtha Abel
Published: October 2006

Abstract: Seven L‐amino acids (Trp, Arg, Lys, Met, lie, Val, and Phe) partially (28–81%) reversed the inhibitory action of 1 μMγ‐aminobutyric acid (GABA) on t‐[35S]‐butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes, with EC50 values ranging from 5 to 120 mM. d‐Trp, d‐Arg, d‐Lys, d‐Met, d‐Val, and d‐Phe were approximately equipotent with their L‐isomers. Tyramine, phenethylamine, and tryptamine, the decarboxylation products of the aromatic amino acids (Tyr, Phe, and Trp, respectively), reversed the inhibitory action of 1 μM GABA on [35S]TBPS binding more potently than the parent amino acids (EC50 values = 1.5–3.0 mM). Human hereditary amino acidemias involving Arg, Lys, lle, Val, and Phe are associated with seizures, and these amino acids and/or their metabolites may block GABA‐A receptors. Five other L‐aminc acids (ornithine, His, Glu, Pro, and Ala) as well as Gly and β‐Ala inhibited [35S]TBPS binding with IC50 values ranging from 0.1 to 37 mM, and these inhibitions were reversed by the GABA‐A receptor blocker R 5135 in all cases. The inhibitory effects of l‐ornithine, l‐Ala, l‐Glu, and l‐Pro were stereospecific, because the corresponding d‐isomers were considerably less inhibitory. l‐His, d‐His, and l‐Glu gave incomplete (plateau) inhibitions. Human hereditary amino acidemias involving l‐ornithine, His, Pro, Gly, and β‐Ala are also associated with seizures, and we speculate that these GABA‐mimetic amino acids may desensitize GABA‐A receptors. l‐Asn, l‐Ser, l‐Thr, l‐Cys, l‐Asp, l‐Leu, and l‐Gln at 100 mM and l‐Tyr at 10 mM had no significant inhibitory effect on [35S]TBPS binding, nor did they reverse the inhibitory effect of 1 μM GABA. Amino acidemias involving the latter eight amino acids do not appear to be associated with seizures.
Effect of Local Injection of Cysteamine and Cystamine on Somatostatin and Neuropeptide Y Levels in the Rat Striatum
Authors: Widmann Rudolf , Maas Dagmar , Sperk Günther
Published: October 2006

Abstract: Cysteamine and its dimeric form cystamine have been applied to the rat striatum by local injection. Both compounds resulted in a dose‐dependent decrease of somatostatin levels. Maximal reduction of somatostatin (by about 50%) was obtained at a dose of 50 μg of cysteamine or cystamine after about 6 h. All three molecular weight forms of somatostatin—somatostatin‐14, somatostatin‐28, and the 13,000 molecular weight form of somatostatin—were reduced, as shown by size exclusion HPLC. Injection of radiolabeled cystamine revealed a fast conversion of the compound to cysteamine, suggesting it is active in the monomeric form. The levels of neuropeptide Y, which is colocalized with somatostatin in striatal neurons, failed to be changed by local or intraperitoneal injection of cysteamine, suggesting that this treatment does not affect vesicles of somatostatin/neuropeptide Y neurons.
Agonist‐Induced Stimulation of Inositol Phosphates in Primary Rabbit Retinal Cultures
Authors: Ghazi Hani , Osborne Neville N.
Published: October 2006

Abstract: Carbachol, noradrenaline, and serotonin stimulated the accumulation of inositol phosphates in a dose‐dependent manner and maximally by 172.4%, 71.2%, and 51.6%, respectively, in 3‐day‐old rabbit retinal cultures. In contrast, dopamine, nicotine, isoproterenol, clonidine, 8‐OH‐dipropylaminotetralin, and γ‐aminobutyric acid were ineffective. In older cultures identified as containing primarily Müller cells, only carbachol and noradrenaline were able to induce a significant stimulatory response. The carbachol‐, noradrenaline‐, and serotonin‐induced responses were pharmacologically characterized and shown to be mediated by muscarinic, α1‐‐adrenergic, and 5‐hydroxytryptamine2 receptors, respectively. The results of the present study show that primary retinal cultures of 3 days of age give results comparable with those of the intact retina. Furthermore, it is demonstrated for the first time that putative Müller cells in cultures possess functional muscarinic and α1‐‐adrenergic receptors.
1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Metabolism and l‐Methyl‐4‐Phenylpyridinium Uptake in Dissociated Cell Cultures from the Embryonic Mesencephalon
Authors: Schinelli Sergio , Zuddas Alessandro , Kopin Irwin J. , Barker Jeffery L. , di Porzio Umberto
Published: October 2006

Abstract: 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1‐methyl‐4‐phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum‐free conditions). However, mesencephalic neurons in culture revealed a high‐affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 μM, respectively), whereas the number of uptake sites for MPP+is double (Vmax= 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented <10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures. These data support the view that MPTP is transformed into MPP+ by astrocytes and concentrated in DA neurons by a relatively specific uptake system, similar to that for DA.
Induction of N‐Glycosylation Activity in Cultured Embryonic Rat Brain Cells
Authors: Bhat Narayan R. , Waechter C. J.
Published: October 2006

Developmental changes in protein N‐glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4–6 days in culture), followed by a period of cellular differentiation. N‐Glycosylation activity has been measured by assaying the incorporation of [2‐3H]‐mannose into dolichol‐linked oligosaccharides and glyco‐protein over a period of 1–25 days in culture. This study revealed a marked induction of N‐glycosylation activity beginning at approximately 1 week of culture. [2‐3H]‐Mannose incorporation into the oligosaccharide‐lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol‐linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N‐glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N‐glycosylation pathway and suggest a possible relationship between N‐linked glycoprotein assembly and the growth and differentiation of glial cells.
Differential Release of Enkephalin and Enkephalin‐Containing Peptides from Perfused Cat Adrenal Glands
Authors: Vindrola Osvaldo , Ase Ariel , Finkielman Samuel , Nahmod Victor E.
Published: October 2006

We have compared the enkephalin‐like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10‐4M) and nicotine (5 X 10–6M). In addition, two doses of acetylcholine (10‐5 and 10‐4M) and 50 mM K+ were tested. Free Met‐enkephalin immunoreactivity and total Met‐enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin‐containing fragments, were coreleased with catecholamines. Free Met‐enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10‐5M acetylcholine, 22% for 10‐4M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met‐enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met‐enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low‐molecular‐weight peptide fractions revealed that Met‐enkephalin, Met‐enkephalin‐Arg‐Gly‐Leu, and Met‐enkephalin‐Arg‐Phe represented 69% of total Met‐enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low‐molecular‐weight material, whereas nicotine application also yielded high‐molecular‐weight peptides. Furthermore, increasing the acetylcholine concentration from 10‐5 to 10‐4M and using 50 mM K+ increased proportionally the high‐molecular‐weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules, either in the same cell or in different cells, containing proenkephalin‐derived peptides. It appears that, under physiological conditions, different enkephalin‐containing peptides might be secreted from the cat adrenal gland. The response might also be modified when nonphysiologic stimuli are applied.
Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution
Authors: Reader Tomás A. , Brière Richard , Gottberg Estela , Diop Laurent , Grondin Louise
Published: October 2006

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding, probably due to high‐ and low‐affinity states. Endogenous dopamine, 4‐hydroxy‐3‐methoxyphenylacetic acid, 3,4‐dihydroxyphenylacetic acid, and 3‐methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate. primary somatosensory, primary visual, and piriform‐entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r > 0.98) with the endogenous levels of dopamine and its major metabolites.
Effects of 5,6‐Dihydroxytryptamine on the Release, Synthesis, and Storage of Serotonin: Studies Using Rat Brain Synaptosomes
Authors: Wolf William A. , Bobik Alex
Published: October 2006

5,6‐Dihydroxytryptamine is a neurotoxic analogue of serotonin which can have profound cardiovascular effects within minutes of administration in vivo (Korner and Head, 1981). These effects have been attributed to 5,6‐dihydroxytryptamine‐induced serotonin release, although there has been no biochemical assessment of the extent to which this occurs. The present study utilized an in vitro synaptosomal assay to determine the short‐term effects of 5,6‐dihydroxytryptamine on endogenous serotonin release, synthesis, storage, and metabolism. 5,6‐Dihydroxytryptamine produced a rapid depletion of serotonin. At lower concentrations of 5,6‐dihydroxytryptamine (0.1–1 μM), this depletion was associated primarily with an increase in the levels of 5‐hydroxyindoleacetic acid, the deaminated metabolite of serotonin, with small increases in the amount of serotonin release. At higher concentrations (10–100 μM), a greater proportion of the depicted serotonin was released with less metabolism occurring. When metabolism was prevented by inhibiting monoamine oxidase, the amount of serotonin which was released equalled the amount of serotonin depletion. Thus monoamine oxidase activity was important in controlling the amount of serotonin which could be released by 5,6‐dihydroxytryptamine. Further studies demonstrated that an impairment in serotonin synthesis and vesicular storage could account for the rapid depletion produced by 5,6‐dihydroxytryptamine. Taken together, the results indicate that 5,6‐dihydroxytryptamine acts to displace serotonin from vesicular stores into the cytoplasm where it can either be deaminated by monoamine oxidase or be released. Moreover, it is hypothesized that the intraneuronal concentration of 5,6‐dihydroxytryptamine is important in determining the extent of serotonin release, because it can inhibit the deamination of serotonin by monoamine oxidase.
Glutamate Dehydrogenase Activity Is Markedly Higher in a “Golgi‐Bergmann”‐Like Glial Clone than in Other Astroglial Cell Lines
Authors: Cambier Danièle , Pessac Bernard
Published: October 2006

Glutamate appears to be the neurotransmitter of granule cells, the major neuronal population of the cerebellar cortex. To determine the role of astroglial cells in the synthesis of glutamate, we have measured the specific activity of glutamate dehydrogenase (GDH) in clonal cell lines that might be the in vitro equivalents of the different cerebellum astroglial cell types. In conditions where GDH operates in the direction of glutamate synthesis, the specific activity of GDH measured in the “Golgi‐Bergmann”‐like clone was 4–6 times higher than in the “velate protoplasmic”‐ or “fibrous‐like” astrocytic clones. These data correlate well with the intense immunoreactivity to GDH in Golgi‐Bergmann astrocytes in vivo that has been recently reported.
γ‐Aminobutyric Acid and Pentobarbital Enhance 2‐[3H]Oxoquazepam Binding to Type I Benzodiazepine Recognition Sites in Rat and Human Brain
Authors: Corda M. G. , Giorgi O. , Longoni B. , Ongini E. , Barnett A. , Montaldo S. , Biggio G.
Published: October 2006

2‐Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of γ‐aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ([3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]‐2oxoquaz and [3H]FNT binding in a concentration‐dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.
Induction of Glutamine Synthetase in Rat Astrocytes by Co‐Cultivation with Embryonic Chick Neurons
Authors: Wu Doris K. , Scully Sheila , Vellis Jean de
Published: October 2006

Co‐cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co‐cultivation and the number of neurons present in the co‐culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.
Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte‐Enriched Cultures from Rat Brain
Authors: Warringa Ruud A. J. , van Berlo Mario F. , Klein Wil , Lopes‐Cardozo Matthijs
Published: October 2006

Abstract: Glial cells were isolated from 1‐week‐old rat brain and cultured in a serum‐free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside‐positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement‐mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol3‐phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double‐label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.
Calmodulin‐Binding Proteins in Human Y‐79 Retinoblastoma and HTB‐14 Glioma Cell Lines
Authors: Pennypacker Keith R. , Kyritsis Athanassios , Chader Gerald J. , Billingsley Melvin L.
Published: October 2006

Abstract: The Y‐79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial‐like (glial fibrillary acidic protein positive) or neuron‐like (neuron‐specific enolase‐positive) cells. To determine whether Y‐79 retinoblastoma cells express neuronotypic calmodulin‐binding proteins, Y‐79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum‐containing medium or were maintained in serum‐free media. Using a biotinylated calmodulin blot overlay technique, we found that Y‐79 cells treated with dbcAMP or butyrate expressed low levels of membrane‐bound calmodulin‐binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate‐treated cells also expressed a calmodulin‐binding peptide of 135 kDa. Since butyrate treatment of Y‐79 cells induces the expression and the secretion of interphotoreceptor retinoid‐binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin‐binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135‐kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial‐like characteristics in Y‐79 cells, we compared the calmodulin‐binding protein pattern in these cells with that seen in human HTB‐14 glioma cells. The HTB‐14 line did not express calmodulin‐binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y‐79 cells express membrane‐bound calmodulin‐binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB‐14 glioma cells.
An Approach to Searching Protein Sequences for Superfamily Relationships or Chance Similarities Relevant to the Molecular Mimicry Hypothesis: Application to the Basic Proteins of Myelin
Authors: Weise Michael J. , Carnegie Patrick R.
Published: October 2006

Abstract: A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid‐binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus‐induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain‐Barre syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.
Cyclic GMP‐Dependent Protein Kinase and Phosphorylation of the Endogenous Substrate Proteins in the Rabbit Superior Cervical Ganglion
Authors: Takahashi Susumu Y. , Mochida Sumiko , Kobayashi Haruo
Published: October 2006

Abstract: When the homogenate of rabbit superior cervical ganglia (SCG) was incubated in the presence of [γ‐32P]ATP and Mg2+, two specific proteins were strongly labeled. Their apparent molecular weights were 90,000 and 54,000, respectively. The phosphorylation of the latter was significantly stimulated by 10–50 nM cyclic GMP but to a lesser extent by cyclic AMP, whereas that of the former was not stimulated significantly by either of the cyclic nucleotides. The purified protein kinase inhibitor from rabbit skeletal muscle did not inhibit the phosphorylation. These results indicated that the observed phosphorylation of 54K protein was dependent on cyclic GMP but not on cyclic AMP. When intact SCG was incubated in the presence of 32Pi, phosphorylation of 90K protein was stimulated by cyclic GMP, dibutyryl cyclic GMP, and 8‐bromo‐cyclic GMP (10 μM), whereas phosphorylation of 54K protein was not significantly stimulated by any of these substances. The present demonstration of endogenous cyclic GMP‐dependent protein kinase activity and its endogenous substrate proteins raises a possibility that the physiological actions of cyclic GMP in SCG are mediated by the phosphorylation of these proteins. Key Words: Superior cervical ganglion—Slow synaptic transmission—Cyclic GMP—Protein phosphorylation—G‐kinase—Muscarinic actions. Takahashi S. Y. et al. Cyclic GMP‐dependent protein kinase and phosphorylation of the endogenous substrate proteins in the rabbit superior cervical ganglion.
Changes of Phospholipid‐Metabolizing and Lysosomal Enzymes in Hypoglossal Nucleus and Ventral Horn Motoneurons During Regeneration of Craniospinal Nerves
Authors: Alberghina M. , Stella A. M. Giuffrida
Published: October 2006

In order to study the biochemical changes associated with the cell body response to axonal crush injury, two systems, hypoglossal nucleus and spinal cord ventral horn, were used. The time intervals chosen were7, 14, and 28 days after unilateral crushing of the right hypoglossal nerve and cervicothoracic nerves of the rabbit. Non‐crushed, contralateral nerves were used as controls. Three groups of enzyme activities were tested: (a) phospholipase A2, acyl CoA:2‐acyl‐sn‐glycero‐3‐phosphocholine acyl‐transferase, and choline phosphotransferase, as indicators of phospholipid degradation and biosynthesis; (b) seven hydrolases, namely, β‐D‐glucuronidase, β‐iV‐acetyl‐d‐hex‐osaminidase, arylsulfatase A, galactosylceramidase, GM1‐ganglioside β‐galactosidase, and acid RNase, as indicators of lysosomal activity; and (c) free and inhibitor‐bound alkaline RNase, as an index of RNA metabolism. Changes could be grouped into three distinct patterns. Compared to contralateral control, choline phosphotransferase showed a slight increase, whereas phospholipase A2 and most lyso somal hydrolases showed a significant increase of activity, especially evident in the ventral spinal cord neurons 14–28 days after crushing. These changes correlate with known increases of membrane and organelle numbers, including lysosomes, in motor and sensory neurons during peripheral regeneration. In contrast, free and acid alkaline RNase activity significantly decreased in the injured sides compared to the controls. This change can probably be correlated with a stabilization of RNAs needed for increased protein synthesis. No changes in total alkaline RNase and acyltrans‐ferase activities in either regeneration model were observed.
Regional Changes in the Cellular Level of Adenine Nucleotides in Ischemic Rat Brain Subjected to Single Embolization
Authors: Yamada Kenji , Uozumi Tohru , Kawasaki Takashi , Yamada Kazuo , Sogabe Takashi , Ohta Keiji
Published: October 2006

Regional changes in adenine nucleotides in the rat brain were studied after 1 h of ischemia produced by the embolization method. The animals were divided into three groups according to neurological symptoms: sham‐operation group, group A (hemiparesis only), and group B (he‐miparesis with unconsciousness). Marked ATP depletion was detected in the hippocampus on the embolized side and extended to the other regions on the same side in group B. The results suggest that this damage in regional energy metabolism arises from regional reduction in blood flow and/or tissue vulnerability. ATP levels in the hypothalamus, hippocampus, and striatum on the opposite side of embolization decreased markedly in group B, and may be caused by extension of brain edema or diaschisis.
Calcium Homeostasis in Digitonin‐Permeabilized Bovine Chromaffin Cells
Authors: Kao Lung‐Sen
Published: October 2006

The regulation of cytosolic calcium was studied in digitonin‐permeabilized chromaffin cells. Accumulation of 45Ca2+ by permeabilized cells was measured at various Ca2+ concentrations in the incubation solutions. In the absence of ATP, there was a small (10–15% of total uptake) but significant increase in accumulation of Ca2+ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca2+ into carbonyl cyanide m‐chlorophenyl hydrazone (CCCP)‐sensitive and ‐insensitive pools. The CCCP‐sensitive pool—mainly mitochondria—was active when the calcium concentration was > 1 μM and was not saturated at 25 μM. The Ca2+ sequestered by the CCCP‐insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP‐insensitive pool had a high affinity for calcium, with an EC50 of ∼1 μM. When the Ca2+ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 μM), the presumed endoplasmic reticulum pool was responsible for ∼90% of the ATP‐stimulated calcium uptake. At a calcium level similar to the acetylcholine‐stimulated level in intact cells (5–10 μM), most of the Ca2+ (>95%) went into the CCCP‐sensitive pool.
Characterization of N‐[3H]Methylcarbamylcholine Binding Sites and Effect of N‐Methylcarbamylcholine on Acetylcholine Release in Rat Brain
Authors: Araujo D. M. , Lapchak P. A. , Collier B. , Quirion R.
Published: October 2006

The present experiments show that N‐[3H]‐methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro‐β‐erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro‐β‐erythroidine and d‐tubocurarine, but not by α‐bungarotoxin or by the muscarinic antagonist atropine. The MCC‐induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.
Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine
Authors: Baker Stephen P. , Standifer Kelly M. , Kalberg Christopher J. , Pitha Josef , Sumners Colin
Published: October 2006

Abstract: The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium‐dependent, high‐affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose‐ and time‐dependent manner (IC50. 6.5 μM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN‐induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19‐fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmas or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible β‐antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synapto somes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium‐dependent uptake of [3H]NE (IC50, 5.6 μM) with no effect on sodium‐independent uptake. After pretreatment of cultures with 30 μM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24‐h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.
Heterogeneity of γ‐Aminobutyric Acid/Benzodiazepine/β‐Carboline Receptor Complex in Rat Spinal Cord
Authors: Santi M. R. , Cox D. H. , Guidotti A.
Published: October 2006

Abstract: The properties of muscimol, β‐carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high‐affinity [3H]muscimol and [3H]6,7‐dimethoxy‐4‐ethyl‐β‐carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or[3H]flumazenil (Ro 15–1788; ethyl‐8‐fluoro‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5–α][1–4]benzodiazepine‐3‐carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is ∼20–25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]‐flumazenil bound to those sites, with low affinity and a Hill slope significantly <1; the potency of the different BCs in displacing [3H]flumazenil is 25–50‐fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5–4864 (4′‐chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of < 1 (nH= 0.4) by CL 218,872 (3‐methyl‐6–(3‐trifluoromethylphenyl)‐1,2,4‐triazolol[4,3‐b]pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (∼80%) are of the central‐type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominately of the central‐type. BZD1 subclass. In both cerebellar and spinal cord membranes, micromolar γ‐aminobutyric acid (GABA) enhanced the binding of [3H]FLU; however, this effect is less efficacious and less potent in the spinal cord, observations indicating two possibilities: (a) that in spinal cord some of the BZD2 binding sites are not coupled to the GABAA binding sites, or (b) that they are coupled in a GABAA/BZD2 receptor complex containing a large proportion of BZD2 binding sites associated with a relatively small number of GABAA binding sites.
Synthesis and Nuclear Magnetic Resonance Spectroscopic, Mass Spectroscopic, and Plasma Amine Oxidase Inhibitory Properties of Analogues of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine
Authors: Bhatti A. R. , Burdon J. , Williams A. C. , Pall H. S. , Ramsden D. B.
Published: October 2006

Abstract: Seventeen analogues of l‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine were synthesized using three reaction pathways: condensation of phenols with 1‐methyl‐4‐piperidone, reaction of Grignard reagents with 1‐methyl‐4‐piperidone followed by dehydration of the product, and aminomethylation of olefins. The identity of the products of synthesis was established by nuclear magnetic resonance spectroscopy, mass spectroscopy. and elemental analysis. Thirteen analogues were shown to inhibit the oxidation of benzylamine by bovine plasma amine oxidase. Increasing the length of the aliphatic chain of N‐substituted analogues resulted in increased inhibition. In 4‐phenyl‐substituted analogues, both the position and electronic character of the substituent group affected the degree of inhibition.
Go, a GTP‐Binding Protein: Immunochemical and Immunohistochemical Localization in the Rat
Authors: Asano Tomiko , Semba Reiji , Kamiya Nobuko , Ogasawara Nobuaki , Kato Kanefusa
Published: October 2006

Abstract: The tissue and cellular distribution of a GTP‐binding protein, Go, was investigated in the rat by immunochemical and immunohistochemical methods. Because the specific antibody for the α subunit of bovine Go (Goα) cross‐reacted with rat Goα, an enzyme immunoassay method developed for bovine Goα was applied for measuring the tissue concentration of Goα in the rat. Goα was detected in all tissues examined except blood cells. The concentration of Goα was highest in the CNS (∼7.7 and 4.4 nmol/g in the cerebrum and cerebellum, respectively), followed by the pituitary gland and sciatic nerve. Among the other peripheral tissues, relatively high concentrations of Goα were observed in the urinary bladder, stomach, and intestines; however, these values were <2% of the concentration in the cerebrum. Goα in the intestine was located mostly in the muscle layer. Immunohistochemical study showed that Goα was associated mostly with the neural elements but not with cells particular to each peripheral organ. Goα was also present in the membranes of neuroen‐docrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, islets cells in the pancreas, and parafollicular cells in the thyroid. These results indicate that Go is localized exclusively in the nervous tissues and neuroendocrine cells.
Activation of Glucocorticoid‐Type II Receptor Complexes in Brain Cytosol Leads to an Increase in Surface Hydrophobicity as Determined by Hydrophobic Interaction Chromatography
Authors: Densmore Charles L. , Chou Yun‐Chia , Luttge William G.
Published: October 2006

Abstract: Hydrophobic interaction chromatography has been used to demonstrate an increase in the surface hydrophobicity of [3H]triamcinolone acetonide ([3H]TA)‐labeled type II receptors in mouse brain cytosol following transformation of these receptor complexes to the activated DNA‐binding form. After removing unbound [3H]TA and molybdate (which prevents activation) by gel nitration, [3H]TA‐type II receptors were activated by incubation at 22°C for 20 min. Gel filtration was then used to remove newly dissociated steroid and to readjust the molybdate and/or KCl concentration. Unactivated and activated receptors were then added to propyl, butyl, pentyl, hexyl, octyl, decyl, and dodecyl alkyl agarose, phenyl agarose, or unmodified agarose columns equilibrated and eluted with buffers of various molybdate and KCl concentrations and/ or other additions, including glycerol, ethylene glycol, and urea. Under high‐salt conditions, activated receptors were retained longer than unactivated receptors run on butyl, pentyl, hexyl, and phenyl agaroses. With the longer alkyl chain columns, essentially none of the [3H]TA was eluted in association with receptor macromolecules. Removal of the remaining steroid required receptor denaturation with urea. Under low‐salt conditions, both receptor forms were retained more avidly on all alkyl agarose columns; however, on phenyl agarose only activated receptors displayed this increased retention. Further studies revealed that optimal separation and subsequent recovery of unactivated and activated [3H]TA‐type II receptor complexes were achieved on pentyl agarose columns equilibrated and eluted with buffers containing 50 mM molybdate and 600–1,200 mM KCl. The activation‐associated increase in surface hydrophobicity revealed in this study may reflect mechanisms underlying receptor subunit dissociation, increased permeation through the nuclear membrane, and/or increased affinity for DNA and other nuclear acceptors.
Specific Binding of Glycosylated β‐Galactosidase to Rat Clonal Pheochromocytoma PC12h Cells: Effects of Nerve Growth Factor and Gangliosides
Authors: Naoi Makoto , Shibahara Keiichi , Suzuki Hiroko , Nagatsu Toshiharu
Published: October 2006

Abstract: Rat clonal pheochromocytoma PC12h cells were found to bind β‐galactosidase modified with specific glycosides. The enzyme modified with p‐aminophenyl β‐d‐glucoside was most effectively bound to the cells, followed by α‐d‐mannoside and α‐d‐glucoside. The binding was dependent on the number of PC12h cells, the incubation interval, and the pH; the maximal binding at 4°C was obtained by incubation with 75 μg of cell protein for 15 min at pH 4.0. The binding proved to be a saturable and receptor‐mediated process, and the apparent Km value and the maximal binding capacity of the cells with β‐d‐glucosylated β‐galactosidase were 1.03 ± 0.06 μM and 333 ± 24 pmol/ min/mg of protein, respectively. When the cells were cultured in the presence of nerve growth factor (NGF), GM1, GM2, and a ganglioside mixture, marked morphological differentiation was observed in the presence of NGF, and the specificity of the binding was also affected. By supplementation of NGF in the culture medium, the cells lost the selectivity of the glycoside binding, whereas cells cultured with GM1 supplement showed increased binding of the specific glycosides.
Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis
Authors: Metcalf R. H. , Boegman R. J.
Published: October 2006

Abstract We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10‐ to 12‐fold increase in the release of endogenous ACh above spontaneous release. Potassium‐evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 ± 13.0% decrease in spontaneous ACh release and a 60.4 ± 8.2% decrease in potassium‐evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4‐diaminopyridine (3,4‐DAP) whereas 10 mM 3,4‐DAP caused a sevenfold increase. The increase in ACh release caused by 3,4‐DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high‐affinity choline uptake and this could be inhibited by hemi‐cholinium‐3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium‐dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.
Neurofilament Phosphorylation in Cultured Bovine Adrenal Chromaffin Cells Is Stimulated by Phorbol Ester
Authors: Georges Elias , Lindenbaum Michael H. , Sacher Michael G. , Trifaró José‐María , Mushynski Walter E.
Published: October 2006

Abstract Primary cultures of bovine adrenal chromaffin cells contain neurofilament proteins that are hypophosphorylated. When the cells were grown in medium containing 32Pi and 0.1 μM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), 32P‐labelling of the three neurofilament subunits was increased 6‐ to 20‐fold relative to controls, the highest level of stimulation occurring for the mid‐sized subunit. Addition of the protease inhibitor leupeptin to the growth medium had no effect on TPA‐stimulated phosphorylation. The increased 32P incorporation was accompanied by a marked reduction in the gel electrophoretic mobilities of the two largest subunits. The augmented phosphorylation was observed 10 min after addition of TPA to a concentration of 0.1 μM or after 1 h of incubation in the presence of 0.01 μM TPA. One‐dimensional peptide mapping and phosphoamino acid analysis indicated that TPA stimulated the phosphorylation of seryl residues at new sites in the mid‐sized subunit. All of the latter subunit contained in the cytoskeletal fraction of chromaffin cells was converted to a more highly phosphorylated state after the cells were grown in the presence of TPA for 1 h.
n‐[3H]Butyl‐β‐Carboline‐3‐Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors
Authors: Medina Jorge H. , de Stein Miguelina Levi , De Robertis Eduardo
Published: October 2006

Abstract: Synthetic n‐butyl β‐carboline‐3‐carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium‐labeled from the butenyl ester. Binding of this [3H]β‐carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high‐affinity site (KD= 2.7 ± 0.1 nM) with a Bmax of 1.16 ± 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax= 2.36 ± 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10−11‐10−5M butyl β‐carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie‐Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KDi= 0.6–1.5 nM and KD2= 12–18 nM). The differentiation of a high‐ and low‐affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl β‐carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1. γ‐Aminobutyric acid (GABA) stimulated the binding of [3H]flunitrazepam, whereas the effect on the binding of [3H]butyl β‐carboline was negative. This finding, together with some pharmacological experiments in mice, suggests that n‐butyl β‐carboline‐3‐carboxylate acts as an inverse agonist on the central benzodiazepine receptor, inhibiting GABAergic neurotransmission.
Clonidine Prevents Transient Loss of Noradrenaline in Response to Cholinergic Hypofunction Induced by Ethylcholine Aziridinium (AF64A)
Authors: Hörtnagl Heide , Potter Pamela E. , Singer Ernst A. , Kindel Gisela , Hanin Israel
Published: October 2006

Abstract: Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 ± 0.84 to 10.04 ± 0.59 pmol/mg of tissue; p < 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 ± 0.08 to 1.41 ± 0.06 pmol/mg of tissue; p < 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose‐dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/ NA molar ratio (from 0.84 ± 0.06 to 1.62 ± 0.17; p < 0.002). Chronic treatment of AF64A‐injected rats with clonidine (0.02–0.2 mg/kg, i.p., every 8–12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A‐treated rats, in contrast to sedation in vehicle‐injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.