bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Neurotoxic Lesions of Descending Serotonergic and Noradrenergic Pathways in the Rat
Authors: Berge Odd‐Geir , Fasmer Ole Bernt , Tveiten Lars , Hole Kjell
Published: October 2006

Abstract: The ability of neurotoxic substances to induce selective lesions of the descending monoaminergic pathways in rats was investigated. Saline, 6‐hydroxydopamine, 5,6‐dihydroxytryptamine, or 5,7‐dihydroxytryp tamine were administered into the lumbar subarachnoid space through a chronically indwelling catheter. The lesions were evaluated 2–3 weeks later by in vitro uptake of [3H]noradrenaline and [14C]5‐hydroxytryptamine into synaptosomal preparations from the frontal cortex, brain stem, cervical spinal cord, and lumbar spinal cord of each animal. There was no difference in uptake between saline‐injected and noncatheterized controls and no significant changes in cortcial uptake after any of the treatments (dose range of neurotoxins: 0.6–80 μg). In the lumbar spinal cord, 6‐hydroxydopamine (5–80 μg) reduced the [3H]noradrenaline uptake by approximately 90% with no effects on [14C]5‐hydroxytryptamine uptake, whereas 5,6‐dihydroxytryptamine reduced the uptake of [14C]5‐hydroxytryptamine by 90% (20–80 μg). [3H]Noradrenaline uptake was unaffected by lower doses of 5,6‐dihydroxy tryptamine but fell by 45–55% after 40–80 μg. 5,7‐Dihydroxytryptamine (10–80 μg) reduced [3H]noradrenaline uptake by 90–95% and [14C]5‐hydroxytryptamine uptake by approximately 80% (5–80 μg) in the lumbar cord. It is concluded that intrathecal administration of suitable doses of neurotoxins may produce extensive selective lesions of descending noradrenergic and serotonergic path ways.
Deamination of Aliphatic Amines by Monoamine Oxidase A and B Studied Using a Bioluminescence Technique
Authors: Tenne M. , Youdim M. B. H. , Ulitzur S. , Finberg J. P. M.
Published: October 2006

Abstract: Deamination of n‐octylamine and n‐decylamine has been studied in various tissues using a new bioluminescence technique. Selectivity of n‐octylamine and n‐decylamine as substrates for monoamine oxidase (MAO) A or B has been determined using both clorgyline and (–)‐deprenyl inhibition curves and kinetic parameters. Homogenates of rat brain, liver and heart containing predominantly MAO‐A or ‐B were prepared by preincubation for 60 min with (–)‐deprenyl or clorgyline (30 nM), respectively. Human placenta (MAO‐A) and platelet (MAO‐B) were used as reference tissues containing only one MAO form. In tissues (rat liver, brain) containing both MAO forms in equal proportion, inhibition curve studies showed a preference of both substrates for the B form of the enzyme; however, where MAO‐A was the major form (rat heart, human placenta), clorgyline was the more effective inhibitor. In the beef brain cortex n‐octylamine showed marked preference for MAO‐B, whereas n‐decylamine was selective toward MAO‐A. Kinetic studies in general supported the picture of greater selectivity of the aliphatic amine substrates for deamination by MAO‐B, as reflected by lower Km values for this enzyme type. However, n‐octylamine was more selective for MAO‐B than n‐decylamine in both kinetic and inhibition curve studies. The deamination of these aliphatic amine substrates cannot be explained only by reference to the binary classification of MAO into types A and B.
Polar Head Group Decarboxylation and Methylation of Phospholipids: An Alternate Route for Phosphatidylcholine Formation in Cultured Neuronal Cells
Authors: Yavin Ephraim
Published: October 2006

Abstract: Decarboxylation of phosphatidylserine (SPG) and methionine‐dependent, stepwise methylation of phosphatidylethanolamine (EPG) to form phosphatidylcholine (CPG) were examined in monolayer cultures of rat cerebral cells. Ethanolamine, monomethylaminoethanol, or dimethylaminoethanol nitrogenous bases (N‐bases) added to culture medium at millimolar level result each in synthesis of the corresponding phospholipid via a de novo pathway at initial rates of 0.18, 0.30, and 0.36 nmol/h/μg DNA, respectively. Addition of methyl‐labeled methionine to culture medium at tracer levels or at millimolar concentration enabled measurements of the rates of phospholipid methylation from EPG phosphatidylmonomethylaminoethanol (Me1EPG) and phosphatidyldimethylaminoethanol (Me2EPG) precursors. At tracer doses, the rates of methylation from the above respective phospholipids are 0.45, 1.17, and 1.70 pmol/h/μg DNA. At 1 mM methionine, synthesis of CPG proceeds from [14C]EPG or [14C]Me2EPG at initial rates of 8 and 17 pmol/h/μg DNA, respectively. Although the latter phospholipid analog can be generated from its monomethyl precursor, methylation of EPG does not result in the accumulation of Me2EPG, suggesting two segregated and metabolically distinct pathways. In the presence of N‐bases, of the total [3H]serine incorporated into cellular phospholipids 30–36.5% of labelled SPG is converted into decarboxylation products. The decarboxylation and methylation routes contribute a significant portion of choline from endogenous sources, most likely through conversion of SPG.
Purification of Somatostatin from Frog Brain: Coisolation with Retinal Somatostatin‐Like Immunoreactivity
Authors: Takami Mimi , Reeve Joseph R. , Hawke David , Shively John E. , Basinger Scott , Yamada Tadataka
Published: October 2006

Abstract: Somatostatin‐like immunoreactivity (SLI) was purified from frog brain and retina, and the structure of the brain peptide was determined. Frog brain (101 g) and retinal (45 g) tissues were extracted with 3% acetic acid, yielding 9.6 and 0.44 nmol of SLI, respectively. SLI was further purified by chromatography on a somatostatin immunoaffinity column followed by sequential application to reverse‐phase C‐18 HPLC columns. The brain and retinal peptides, purified roughly 100,000‐fold with net yields of 7.5 and 2.3%, respectively, appeared identical in the final steps of purification. The amino acid sequence of brain SLI, as determined by a gas‐phase automated Edman degradation technique, was as follows: Ala‐Gly (Cys)‐Lys‐Asn‐Phe‐Phe‐Trp‐Lys‐Thr‐Phe‐Thr‐Ser‐ (Cys). Our data indicate that despite structural variations in somatostatins of other lower vertebrates, the amino acid sequence of frog brain and, by deduction, retinal SLI is identical to that of somatostatin tetradecapeptide. These findings support the physiological relevance of studies directed at elucidating the neurotransmitter function of somatostatin using the well‐established models of frog brain and retina.
Isolation and Identification of α‐(γ‐Aminobutyryl)‐Hypusine
Authors: Sano Akira , Kotani Kazuhiko , Kakimoto Yasuo
Published: October 2006

Abstract: A new dipeptide, α‐(‐γ‐aminobutyryl)‐hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid‐soluble fraction of bovine brain with five steps of ion‐exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.
Apparent Inhibition of ATP Citrate Lyase by l‐Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase
Authors: Rider C. C.
Published: October 2006

Abstract: Preincubation in assay mixture for 30 min at 37°C of ATP citrate lyase from rat brain and liver results in 65–70% inhibition in the presence of 10 mMl‐glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate‐suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol‐precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor l‐methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elswhere, l‐glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.
Phosphate‐Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex
Authors: Svenneby Gerd , Roberg Bjørg , Hogstad Svein , Torgner Ingeborg Aa. , Kvamme Elling
Published: October 2006

Abstract The kinetics and other properties of phosphate‐activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37°. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate‐activated glutaminase‐like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate‐treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double‐inverse plots of enzyme activity against phosphate are concave‐upward, and more so in the presence of an inhibitor. The inhibition by glutamate ap pears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate‐activated glutaminase from pig brain and pig kidney.
Developmental Changes in Levels of Translatable mRNAs for Enolase Isozymes in Chicken Brain
Authors: Tanaka M. , Sugisaki K. , Nakashima K.
Published: October 2006

Abstract Using chicken brain mRNAs, α and γ enolase precursors were synthesized in the rabbit reticulocyte cell‐free translation system. The product proteins showed molecular weights almost identical to those of the mature subunits. The levels of translatable mRNAs for α and γ subunits were determined by the cell‐free translation system and immuno‐precipitation with specific antisera, during development of chicken brain. The level of α mRNA was high at any developmental stage of the brain. On the other hand, the γ mRNA level was very low at the early embryonic stage, and increased rapidly during development of the brain. These changes were closely correlated with those of the corresponding enzyme activities, indicating that the levels of enolase activities in developing brain were controlled primarily by the level of the translatable α and γ mRNAs.
Characteristics of Synthesis of Very‐Long‐Chain Saturated and Tetraenoic Fatty Acids in Swine Cerebral Microsomes
Authors: Yoshida Satoshi , Takeshita Masazumi
Published: October 2006

Abstract: The elongation of arachidoyl‐CoA (20:0‐CoA) yielded 22:0 and 24:0 concomitantly, whereas the elongation of behenoyl‐CoA (22:0‐CoA) yielded only a negligible amount of 24:0 in adult swine cerebral microsomes. The dependence on time, pH, and the substrate concentrations were examined for the synthesis of 22:0 and 24:0 from 20:0‐CoA. A microcomputer‐aided simulation study suggested that there were two parallel pathways in the elongation of 20:0‐CoA to 22:0 and 24:0. The elongation of 22:0‐CoA could not be observed in adult swine cerebral microsomes; however, it was observed clearly in newborn swine and rat brain microsomes. A dilution experiment with the addition of cold 22:0‐CoA in the reaction of elongation of 20:0‐CoA confirmed the above suggestion that no intermediate 22:0 appeared during the synthesis of 24:0 from 20:0‐CoA. The elongation of endogenous 20:4‐CoA to 22:4 and 24:4 was examined in newborn swine cerebral microsomes, and the presence of two parallel pathways in the elongation of 20:4‐CoA to 22:4 and 24:4 similar to those involved in the elongation of 20:0‐CoA to 22:0 and 24:0 was suggested.
Effect of Intrastriatal Injection of Diisopropylfluorophosphate on Acetylcholine, Dopamine, and Serotonin Metabolism
Authors: Robinson Susan E. , Rice M. Andrea , Hambrecht Kristi L.
Published: October 2006

Abstract: Diisopropylfluorophosphate (81.5 nmol) was injected directly into the striata of rats to study changes in striatal metabolism of acetylcholine (ACh), 3,4‐dihydroxyphenylethylamine (dopamine), and 5‐hydroxytryptamine (serotonin) at early time points following acute irreversible inhibition of cholinesterase. Twenty minutes following the intrastriatal injection of diisopropylfluorophosphate, levels of striatal acetylcholine were elevated by 50%, but a decrease in KACh compensated for this change. At 1 h, levels of ACh were still elevated, but not significantly different from control values. However, KACh and, hence, ACh turnover were greatly enhanced at this time. Finally, at 24 h, striatal ACh content was only slightly elevated and KACh and the turnover rate of ACh had returned to control values. Striatal cholinesterase activity remained significantly inhibited at all three times. At none of these times was ACh content or turnover affected in the parietal cortex, hippocampus, hypothalamus, or medulla/pons. Neither dopamine and its metabolites 3,4‐dihydroxyphenylacetic acid and homovanillic acid nor serotonin and its metabolite 5‐hydroxyindoleacetic acid were significantly affected at any of the three times by intrastriatal diisopropylfluorophosphate treatment. Possible mechanisms of the changes in cholinergic parameters are discussed.
A Thermodynamic Study of 5‐[3H]Hydroxytryptamine Binding to Human Cortex Membranes
Authors: Todd Richard D. , Babinski Joseph
Published: October 2006

Abstract: Kinetic and equilibrium measurements of [3H]‐serotonin (5‐hydroxytryptamine) binding to human frontal cortex membranes have been made between 4 and 30°C. The effects of spiperone and ascorbate on binding have also been determined. Under the conditions used, binding was saturable and reversible. Affinity constants derived from kinetic and equilibrium data were comparable. Serotonin binding to several sites had substantial enthalpic as well as entropic components.
Characterization of D2 Dopamine Receptors in Dopamine‐Resistant Prolactin‐Secreting Rat Pituitary Tumors 7315a and MtTW15
Authors: Bouvier C. , Lagacé G. , Lafond J. , Beauregard G. , Potier M. , Collu R.
Published: October 2006

Abstract: We have investigated the structure of D2 receptors present in two prolactin‐secreting, dopamine‐resistant, transplantable rat pituitary tumors, 7315a and MtTWIS. These receptors specifically bind with high affinity the do‐pamine antagonist [3H]spiroperidol when membrane bound or solubilized by [3‐(3‐cholamidopropyl)‐dimethyl‐ammonio]‐1‐propane sulfonate 10 mM and are pharmacologically characterized as D2 type. Target‐size analysis by radiation inactivation indicated a molecular mass of approximately 100,000 and 200,000 daltons for receptors present respectively in 7315a and MtTWIS tumors either membrane bound or solubilized. The minimal size of the D2 binding site was evaluated at 94,000 daltons by photoaffinity labeling with [125I]azido‐N‐(p‐aminophenethyl)‐spiperone followed by sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis. A guanine nucleotide had no effect on the displacing potency of the agonist N‐propyl‐norapomorphine evaluated with membrane‐bound or solubilized receptors obtained from either tumor. These results suggest the absence or inactivation of a guanine nucleotide binding protein in the receptorial complex of these tumors. Thus, our data indicate that a structural anomaly is present in the D2 receptorial complex of these prolactin‐secreting rat pituitary tumors, which may be responsible for their resistance to the inhibitory effects of dopamine.
Depolarization‐Induced Increase in Surface Binding and Internalization of 125I‐Nerve Growth Factor by PC 12 Pheochromocytoma Cells
Authors: Koike Tatsuro
Published: October 2006

The binding and internalization of 125I‐nerve growth factor (NGF) by PC 12 pheochromocytoma cells was studied as a function of extracellular potassium concentration. Both surface‐bound and internalized fractions of 125I‐NGF associated with the cells under depolarizing conditions (50 mM K+) increased to 144 ± 28% (average ± SEM, six different cell preparations) and to 176 ± 12% (n = 6), respectively, of those observed at 6.0 mM K+. Scat‐chard‐type analysis of the data indicates increased sites for the binding and internalization of iodinated NGF by the cells. Similar enhancement was observed for cells treated with NGF as well. This voltage‐dependent phenomenon was reversible, and also observed in the presence of veratri‐dine. Moreover, withdrawal of extracellular Ca2+ abolished high K+‐induced modulation of 125I‐NGF binding and internalization, indicating that this effect may be mediated by Ca2+.
Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co‐Release of 7B2 and Catecholamine as Induced by Nicotine
Authors: Iguchi H. , Natori S. , Nawata H. , Kato K. , Ibayashi H. , Chan J. S. D. , Seidah N. G. , Chrétien M.
Published: October 2006

We observed the presence of the novel pituitary protein „7B2” and its release in the bovine adrenal medulla. The 7B2 concentration (mean ± SEM) in extracts of the bovine adrenal medulla was 952 ± 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co‐release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. On reverse‐phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.
Rapid Degradation of Neurotensin by Intact Murine Neuroblastoma Cells (Clone N1E‐115)
Authors: Gilbert Judith A. , Hanson Peter D. , J. McCormick, Daniel , Richelson Elliott
Published: October 2006

Murine neuroblastoma clone N1E‐115, which possesses receptors for neurotensin mediating the formation of intracellular cyclic GMP and the stimulation of inositol phospholipid hydrolysis, exhibited only partial desensitization to neurotensin. This result led to the observation that neurotensin was very rapidly degraded by intact N1E‐115 cells. In experiments measuring the time course of [3H]neurotensin degradation, a minimum of six major tritiated products were found, with the breakdown peptides formed and the degree of proteolysis of [3H]neurotensin being dependent upon the length of incubation and the concentration of cells. Clone N1E‐115 degraded [3H]neurotensin in an apparently sequential fashion; the primary initial cleavage of intact neurotensin was at the peptide bond between residues Arg8 and Arg9. Initial degradation peptides from the active carboxyl‐terminal portion of neurotensin were more rapidly degraded, after formation, than were the peptides from the inactive amino‐terminal half of neurotensin. The final two degradation products found were tyrosine, from the carboxyl‐terminal portion of neurotensin, and an as yet unidentified peptide from the amino‐terminal half of neurotensin. [3H]Neurotensin(8–13) was more rapidly hydrolyzed under identical conditions than was [3H]neurotensin itself. A combination of the protease inhibitors 1, 10‐phenanthroline and Z‐Pro‐Prolinal was able to inhibit almost completely the degradation of neurotensin by clone N1E‐115.
Ontogeny of Glucose and Lipid Metabolism in Dorsal Root Ganglia of Chickens.: Similarities and Contrasts with Sympathetic Ganglia
Authors: Larrabee Martin G.
Published: October 2006

Dorsal root ganglia, excised from the lumbar roots of the sciatic nerve of white Leghorn chicken embryos 6–13 days of age, were incubated usually for 5 h, at 36°C in 20 μI of a bicarbonate‐buffered physiological salt solution containing 5.5 mM glucose. [U‐14C]Glucose, [1‐14C]glucose, [6‐14C]glucose, or [5‐3H]uridine was also added. Lipid synthesis and lactate output were measured by incorporation of 14C from [U‐14C]glucose and RNA synthesis by incorporation of 3H from [5‐3H]uridine. Glucose uptake and labeled lactate output declined rapidly from 6 to 8–9 days of age, more slowly thereafter. Synthesis of lipids was relatively constant throughout the ages studied, without the increased rate at intermediate ages seen previously in sympathetic ganglia of the same species. RNA synthesis declined progressively throughout the ages studied. The output of C‐6 of glucose to CO2 was about the same at all ages, whereas that of C‐1 declined rapidly from 6 to 7 days of age and then more slowly, but always remained higher than that of C‐6 and thus indicated that much glucose was metabolized via the hexosemonophosphate shunt.
Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site
Authors: Newcomb Robert W.
Published: October 2006

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide “H,” based on amino acid composition additivities and pulse‐chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, “J” and “L,” and a peptide, “K,” similar to peptides with molt inhibiting activity. Here I report the amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring “overlap” peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg‐Ser‐Ala‐Asp‐Gly‐Phe‐Gly‐Arg‐Met‐Glu‐Ser‐Leu‐Leu‐Thr‐Ser‐Leu‐Arg‐Gly‐Ser‐Ala‐Glu‐Ser‐Pro‐Ala‐Ala‐Leu‐Gly‐Glu‐Ala‐Ser‐Ala‐Ala‐His‐Pro‐Leu‐Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu‐Arg‐Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu‐Ser‐Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.
Differential Effects of Nerve Growth Factor and Ciliary Neuronotrophic Factor on Catecholamine Storage and Catecholamine Synthesizing Enzymes of Cultured Rat Chromaffin Cells
Authors: Seidl K. , Manthorpe M. , Varon S. , Unsicker K.
Published: October 2006

Abstract: : The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on Catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N‐methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8‐day‐old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4‐day culture period in a dose‐dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on Catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.
Activation of Dopamine Receptors Does Not Affect Phosphoinositide Turnover in NCB‐20 Cells
Authors: Cubitt A. B. , Brown B. L. , Dobson P. R. M.
Published: October 2006

Abstract: Dopamine inhibits and serotonin stimulates adenylate cyclase activity in a neuroblastoma X Chinese hamster brain explant cell line (NCB‐20). The inhibition of cyclic AMP accumulation by dopamine was blocked by pre‐treatment of the cells with pertussis toxin. Carbachol and bradykinin stimulated the accumulation of water‐soluble inositol phosphates whereas thyrotropin‐releasing hormone, vasopressin, neurotensin, and phenylephrine were without effect. Dopamine and serotonin had no significant effect on carbachol‐induced phosphoinositide hydrolysis or the levels of the parent lipids within the membrane. Forskolin induced a much larger stimulation of cyclic AMP than did serotonin, and caused an increase in the levels of phosphatidylinositol‐4‐phosphate and phosphatidyl inositol‐4,5‐bisphosphate in the cell membrane.
4‐Aminobutyrate Can Be Released Exocytotically from Guinea‐Pig Cerebral Cortical Synaptosomes
Authors: Sihra Talvinder S. , Nicholls David G.
Published: October 2006

Abstract: : Guinea‐pig synaptosomes possess two functional pools of 4‐aminobutyrate (GABA). One is rapidly labelled by added [I4C]GABA, is steadily released in a Ca2+‐independent manner when the Na+ electrochemical potential across the plasma membrane is collapsed, and is depleted by the GABA analogue 2,4‐diaminobutyrate (DABA), all of which is consistent with a cytosolic location. A second, noncytosolic compartment only slowly equilibrates with exogenous [14C]GABA, is not depleted by DABA, but can release 350 pmol of endogenous GABA/mg of protein (8% of the total intrasynaptosomal GABA) within 15 s of depolarization in the presence of Ca2+. Ca2+‐independent release occurs by thermodynamic reversal of the plasma membrane uptake pathway following artifactually prolonged depolarization, whereas Ca2+‐dependent release is consistent with physiological exocytosis from vesicular stores.
Solubilisation of a Glutamate Binding Protein from Rat Brain
Authors: Kuonen Donald R. , Roberts Peter J.
Published: October 2006

Abstract: : Rat brain synaptic plasma membranes were solubilised in either 1% Triton X‐100 or potassium cholate and subjected to batch affinity adsorption on L‐glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L‐glutamate in 0.1 % detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L‐[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L‐glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD= 1.0 and 1.8 μM for Triton X‐100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X‐100‐solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L‐glutamate, L‐aspartate, and sulphur‐containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists—quisqualate, N‐methyl‐D‐aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless Solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since Solubilisation with Triton X‐100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.
Calcium Dependence of Vasoactive Intestinal Peptide‐Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices
Authors: Etgen Anne M. , Browning Edward T.
Published: October 2006

Abstract: Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 μM adenosine, suggesting that calcium‐dependent, VIP‐induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP‐stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 μM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1‐methyl‐3‐isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP‐dependent calcium influx, from interactions with calmodulin, or from calcium‐inhibited phosphodiester‐ase(s). The accumulation of cyclic AMP induced by two other agonists, norepinephrine (NE) and forskolin, was also dependent on the presence of calcium ions in the bathing medium. The data are also consistent with the hypothesis that VIP, NE, and forskolin stimulate cyclic AMP accumulation in hippocampal slices by interacting with the calcium‐dependent adenylate cyclase.
Ontogeny of Glucose Metabolism in Sympathetic Ganglia of Chickens. Concurrence of Maximum Rates in the Hexosemonophosphate Shunt and in Synthesis of Lipids but Not of Ribonucleic Acid
Authors: Larrabee Martin G.
Published: October 2006

Abstract: Chains of sympathetic ganglia were excised from the lumbar region of white Leghorn chicken embryos, 8–19 days of age, and incubated, usually for 5h, at 36°C in a bicarbonate‐buffered physiological salt solution containing [U‐14C]glucose, [l‐14C]glucose, [6‐14C]glucose, or [5‐3H]uri‐dine. Lipid synthesis was measured by the incorporation of 14C into lipids, and RNA synthesis by the accumulation of 3H into macromolecules. The ratio of 14C put out in CO2 during the second hour of incubation in the presence of [1‐14C]glucose to that with [6‐14C]glucose was used as an index of activity in the hexosemonophosphate shunt (HMS). Both the rate of lipid synthesis and activity in the HMS reached well‐defined maxima at about 11 days of embryonic age. There was no evidence of a similar rise and fall of RNA synthesis during the ages studied. Estimates of the rate of NADPH production by the HMS at near‐peak lipid synthesis varied over a twofold range that included the rate needed for the observed lipid synthesis. The results thus support, quantitatively as well as qualitatively, the supposition that the HMS is accelerated during development to sustain lipid synthesis.
Arachidonic Acid Oxidation by Brain and Placenta Preparations from Normal and Placental Insufficient Fetal Rabbit
Authors: Goldin E. , Harel S , Tomer A. , Yavin E.
Published: October 2006

Abstract: Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1–14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12‐hydroxyeicosatetraenoic acid (12‐HETE) and 15‐HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12‐HETE and 15‐HETE by fetal brain is time‐dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 μmol/h/g wet weight are presumably responsible for the excessive accumulation of 12‐HETE and 15‐HETE in comparison to PGE2 (Km=0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12‐HETE and 15‐HETE there is a substantial formation of 12–l‐hydroxyheptadecatrienic acid. Formation of 12‐HETE and 15‐HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.
Acetylcholine Synthesis at the Rat Neuromuscular Junction
Authors: Harris John B.
Published: October 2006

Abstract: Acetylcholine (ACh) synthesis in homogenates of rat soleus muscles had two components. One component, specifically inhibited by bromoacetylcholine (BrACh), had a Km for choline of 0.26 mM; the other, resistant to BrACh, had a Km for choline of 45 mM. The component with a low Km was absent from denervated muscle, and was identical in kinetic terms to ACh synthesising activity in homogenates of sciatic nerve. It is therefore considered choline acetyltransferase (ChAT)‐specific. The use of BrACh as a specific inhibitor of ChAT activity allowed the calculation of ACh synthesis at individual motor end‐plates in the soleus muscle of the rat: 2.1 × 10−‐3 nmol h−1. Since the number of muscle fibres and the number of motor units are known for this muscle, ACh synthesis per motor unit could be calculated: 0.15 nmol h−1. It is concluded that BrACh can be used as a specific inhibitor of ChAT activity in homogenates of skeletal muscle and that its use will obviate the necessity of dividing biopsied muscle or small rodent muscles into neural and aneural segments.
Development of Transmitter‐Releasing Capacity in Neuron‐Enriched Tissue Cultures
Authors: Barochovsky O. , Bradford H. F.
Published: October 2006

Abstract: Dissociated cell cultures derived from whole brains of foetal rats (17 days of gestation) were maintained for periods of up to 21 days in vitro for the purpose of studying the transmitter‐releasing properties of the dopaminergic neuronal cells and glial cells. In the neuron‐enriched cultures, after 3 days in vitro, [3H]dopamine was released in response to depolarizing stimuli. Both the potassium and veratrine‐evoked release of dopamine was Ca2+ dependent. Veratrine‐evoked release was reduced in the presence of the calcium channel blocker verapamil and was tetrodotoxin sensitive. Glial cultures, after 7 days in vitro, did not respond to any depolarizing stimuli, although they displayed a significant ability to take up [3H]dopamine. Comparison between static incubations and perfused cultures showed no difference in the patterns of release resulting from veratrine stimulation. Tyrosine hydroxylase activity increased progressively in neuron‐enriched cultures but was not detectable in glial cultures. These results show that neuron‐enriched cultures respond to depolarizing stimuli in a manner similar to excised adult basal ganglia tissue, with the appearance of functional ionic channels after 3 days in vitro.
Targeting of Liposomes to Cells Bearing Nerve Growth Factor Receptors Mediated by Biotinylated Nerve Growth Factor
Authors: Rosenberg Michael B. , Breakefield Xandra O. , Hawrot Edward
Published: October 2006

Abstract: We have used biologically active derivatives of β‐nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse‐phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiola‐beled streptavidin‐liposomes to rat pheochromocytoma PC 12 cells in suspension at 4°C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin‐conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin‐liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin‐liposomes containing encapsulated fluorescein isothiocyanate‐labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37°C following targeted liposome binding at 4°C, the cell‐associated fluorescence appeared to become internalized, displaying a perinu‐clear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell‐associated fluorescence when cells were held at 4°C but did not alter the fluorescence pattern in cells following incubation at 37°C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37°C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter ly‐sosomal or prelysosomal organelles.
A Study of the Mechanism of Internalisation of Tetanus Toxin by Primary Mouse Spinal Cord Cultures
Authors: R. G. Parton  , Ockleford C. D. , Critchley D. R.
Published: October 2006

Abstract: The fate of tetanus toxin bound to neuronal cells at 0°C was followed using an anti‐toxin 125I‐protein A assay. About 50%; of surface‐bound toxin disappeared within 5 min of warming cells to 37°C. Experiments with 125I‐toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilising cells with Triton X‐100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin‐gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin‐gold particles bound to the cell body at 4°C showed that it was concentrated in coated pits. After 5 min at 37°C, toxin‐gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti‐toxins, and protein A‐gold. Toxin‐gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and in‐traaxonal uncoated vesicles, respectively.
Antisera Raised Against the Drug Imipramine
Authors: O'Callaghan Ann M. , Phillips Orla M. , Williams D. Clive
Published: October 2006

Abstract: Antisera against 2‐aminoimipramine covalently coupled to albumin have been raised in two rabbits. Both antisera bind imipramine and related tricyclic compounds as if to a single class of sites with high affinity and high litres. Displacement/inhibition assays showed that the affinities of various tricyclic compounds for the antisera showed a good correlation with the affinities of these drugs for the tricyclic antidepressant inhibitory sites on plasma‐membrane 5‐hydroxytryptamine carriers of human platelets and rat brain cortex. 5‐Hydroxytryptamine and 5‐hy‐droxytryptamine‐uptake‐selective drugs did not inhibit [3H]imipramine binding to antisera. The anti‐imipramine antibodies were purified using imipramine‐Sepharose affinity chromatography and were shown to be IgG class by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and protein A‐Sepharose precipitation.
Interstitial Ascorbate in Turtle Brain Is Modulated by Release and Extracellular Volume Change
Authors: Rice Margaret E. , Nicholson Charles
Published: October 2006

Abstract: The isolated turtle cerebellum was used as a model system to study effects of depolarizing conditions on interstitial ascorbic acid concentration. The depolarizing stimulus was Leão's spreading depression, which is characterized by transient negative extracellular potentials, high potassium levels (20–60 μM), and local depression of neuronal activity. Interstitial concentrations of ascorbate (200–400 μM) and other electroactive species were monitored voltammetrically, using graphite fiber microelectrodes. Total tissue ascorbate (1,810 nmol/g tissue wet weight) was similar to mammalian levels and was several orders of magnitude higher than catecholamine and indoleamine content. During spreading depression, a large (up to 200 μM) increase in concentration of interstitial electroactive species was monitored. Use of Nafion‐and ascorbate oxidase‐coated electrodes and uricase confirmed that ascorbate was the only substance detected. Simultaneous monitoring of ascorbate, extracellular potential, and extracellular volume (using tetramethylammonium and ion‐selective microelectrodes) indicated that (a) the ascorbate increase began with the decrease in extracellular volume during spreading depression, and (b) much of the increase was the result of extracellular volume decrease. In sucrose‐substituted medium, in which volume changes are eliminated, a 50 μM increase in interstitial ascorbate, caused by release from intracellular stores, was also seen. The ascorbate concentration increase was prolonged in sucrose medium, suggesting that an uptake process involving sodium may further regulate interstitial ascorbate concentration.
Modulation by Docosahexaenoic Acid of the Epinephrine‐Stimulated Adenylate Cyclase Activity of the Bovine Retina
Authors: Tesoriere Luisa , Bongiorno Antonino , Livrea Maria A. , Bono Antonino
Published: October 2006

Abstract: This work shows that unsaturated fatty acids enhance the epinephrine‐stimulated adenylate cyclase activity in bovine retina. The modulating effect on the epinephrine‐stimulated formation of cyclic AMP seems to be linked to the degree of unsaturation of the fatty acid. Treatment of the intact retina with docosahexaenoic acid in the concentration range 0.5 × 10‐6‐l × 10‐3M does not affect the enzyme activity measured in the absence of the hormone but markedly increases the cyclase activity when the tissue is incubated in the presence of 0.1 mM epineph‐rine. Docosahexaenoic acid enhances the maximal response to epinephrine without affecting the apparent ED50 value for this effector. Docosahexaenoic acid at 0.5 mM also increases the hormone‐stimulated adenylate cyclase activity in retinal cell‐free homogenate, whereas it has no effect on the epinephrine‐sensitive enzyme solubilized from the membrane fraction with 1% Triton X‐305. When docosahexaenoic acid‐preincubated intact retina and cell‐free Homogenate are incubated in the presence of defatted albumin, both the observed activating effect of the fatty acid on the epinephrine‐stimulated adenylate cyclase activity and the enhancement of the enzyme response to the hormone significantly diminish.
γ‐Aminobutyric Acid Release from Synaptosomes Prepared from Rats Treated with Isonicotinic Acid Hydrazide and Gabaculine
Authors: Wood J. D. , Kurylo E. , Lane R.
Published: October 2006

Abstract: The potassium‐stimulated release of γ‐aminobutyric acid (GABA) from synaptosomes was determined in preparations from control rats and from rats treated with a convulsant agent [isonicotinic acid hydrazide (INH)] and an anticonvulsant agent (gabaculine). INH treatment brought about a significant decrease in Ca2+‐dependent release of GABA with no effect on Ca2+‐independent release, whereas gabaculine caused an increase in Ca2+‐independent release with no effect on Ca2+‐dependent release of GABA. Thus, the anticonvulsant action of gabaculine was not a simple reversal of the effects of INH on GABA release. The results indicate that there are at least two pools of GABA in nerve endings and support the hypothesis that exogenous GABA is taken up first into a pool that supplies GABA for Ca2+‐independent release and then is transferred to a second pool (Ca2+‐dependent releasable), where it mixes with newly synthesized GABA.
Kinetic Modulation by GABAergic Agents of High‐ and Low‐Affinity Binding of [3H]Methyl β‐Carboline‐3‐Carboxylate
Authors: Maksay Gabor , Simonyi Miklós
Published: October 2006

Abstract: The kinetics of dissociation of [3H]methyl β‐carboline‐3‐carboxylate (β‐CCM) binding was studied in a synaptosomal membrane preparation of rat cerebral cortex. Dissociation was biphasic: a faster phase (10–30% contribution) was followed by a slower phase. Picrotoxin pre‐treatment at 22°C enhanced the equilibrium binding of [3H]β‐CCM. The half‐life of the slower phase of β‐CCM dissociation (tII1/2) was increased by 60 μM picrotoxin from 1.7 min to 3.3 min. The dissociation of [3H]β‐CCM was identical when initiated by an excess of either diazepam or β‐CCM. Quasi‐equilibrium Scatchard analysis of [3H]β‐CCM binding was performed by a kinetic separation of the rapid and slow phases of dissociation. The slow and rapid phases represented β‐CCM binding sites of high and low affinity, respectively. The dissociation of [3H]β‐CCM (control tII1/2= 2.0 min) was decelerated by the γ‐aminobutyric acid (GABA) antagonist 3–α‐hydroxy‐16‐imino‐5β‐17‐azaandrostan‐11‐one (R 5135) (tII1/2= 2.5 min) and accelerated by GABA (tII1/2= 1.6 min). GABA inhibited both high‐ and low‐affinity β‐CCM bindings.
Solubilization and Characterization of Calcitonin Gene‐Related Peptide Binding Site from Porcine Spinal Cord
Authors: Hiroshima Osamu , Sano Yoshihisa , Yuzuriha Teruaki , Yamato Chiyuki , Saito Akira , Okamura Naomichi , Uchiyama Yasuo , Kimura Sadao , Goto Katsutoshi
Published: October 2006

The binding site for calcitonin gene‐related peptide (CGRP) was solubilized with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propane sulfonate (CHAPS) in an active form from porcine spinal cord. l25I‐labeled human α‐CGRP (125I‐CGRP) binding to the solubilized protein was determined by filration using a GF/B glass filter. The maximal binding activity (˜60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I‐CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.