bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Opioid Peptides Containing the Sequence of Met5‐Enkephalin or Leu5‐Enkephalin on Nicotine‐Induced Secretion from Bovine Adrenal Chromaffin Cells
Authors: Marley Philip D. , Mitchelhill Ken I. , Livett Bruce G.
Published: December 2006

Abstract: Eighteen endogenous opioid peptides, all containing the séquence of either Met5‐ or Leu5‐enkephalin, were tested for their ability to modify nicotine‐induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin‐luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 μM nicotine‐induced ATP release at 10 nM. Three peptides inhibited secretion at 5 μM dynorphin,1‐13, dynorphin1‐9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10‐phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1‐3 and dynorphin1‐9 by 20–30%. Nicotine‐induced secretion of endogenous catecholamincs from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1‐13 was 1,000‐fold more potent than Leu5‐enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1‐13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5‐enkephalin showed the opposite selectivity. The structure‐activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.
Surgical Sympathetic Denervation Increases α1Adrenoceptor‐Mediated Accumulation of myo‐Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle
Authors: Akhtar Rashid A. , Abdel‐Latif Ata A.
Published: December 2006

Abstract: Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphati‐dylinositol 4,5‐bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: (1) the effects of NE on PIP, breakdown, measured as myoinositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and (2) the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: (1) The EC50 values (μM) for NE‐induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1μM). (2) The EC50 values (μM) for NE‐induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE‐induced muscle contraction was blocked by prazosin (1μM). (3) The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. (4) Denervation increased significantly (15–18%) the basallabelling of phosphoinositides from myo‐[3H]inositol, but not from 32P or [14C]arachidonic acid. (5) Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+‐ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between α1 receptors and PIP2 breakdown into IP3 and 1,2‐diacylglycerol (DG) more efficient. The NE‐stimulated hydrolysis of PIP2 could then bring about Ca2+ mobilization, necessary for muscle contraction, either directly by causing plasma membrane depolarization or indirectly by IP3 releasing Ca2+ from sarcoplasmic reticulum and by DG activating protein kinase C, or both.
Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex
Authors: Yumoto Noboru , Watanabe Yasuyoshi , Watanabe Kikuko , Watanabe Yumiko , Hayaishi Osamu
Published: December 2006

Abstract: The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS) at 4°C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time‐dependent and the equilibrium was reached at around 60 min at 37°C. By dilution of the reaction mixture, the binding site‐[3H]PGE2 complex formed after 5‐min incubation slowly dissociated, whereas that formed after 60‐min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 > PGE, × PGE2α > PGE, methyl ester > PGA2 > 13,14‐dihydro‐15‐keto‐PGE2 > PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high‐ and low‐affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion‐exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.
Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures
Authors: Corcoran James J. , Wilson Steven P. , Kirshner Norman
Published: December 2006

Abstract: The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84–88% of the labeled adenine nucleotides, ADP 11–13%, and AMP 1–3%. The turnover of 32P‐adenine nucleotides and 3H‐nucleotides was biphasic and virtually identical; there was an initial fast phase with a t½ of 3.5–4.5 h and a slow phase with a half‐life varying from 7 to 17 days, depending upon the particular cell preparation. The t½ of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half‐life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half‐lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P‐labeled compounds and relatively three times as much catecholamine as 3H‐labeled compounds.
Chronic Use of Intracerebral Dialysis for the In Vivo Measurement of 3,4‐Dihydroxyphenylethylamine and Its Metabolite 3,4‐Dihydroxyphenylacetic Acid
Authors: Westerink B. H. C. , Tuinte M. H. J.
Published: December 2006

Abstract: The intracerebral dialysis technique was studied with a method in which the rat was directly connected to the HPLC equipment. The effect of three pharmacological treatments [perfusion of 60 mmol K+ or 5 × 10−5M (+)‐amphetamine or subcutaneous injection of 2 mg/kg (+)‐amphetamine] on the release of 3,4‐dihydroxy‐phenylethylamine (dopamine) and 3,4‐dihydroxyphenylacetic acid was followed over a period of 7 days. The marked rise of dopamine output seen after infusion of K+ had almost disappeared on day 3. Tissue reactions around the membrane presumably formed a barrier preventing K+ from reaching dopaminergic terminals. In contrast, the pronounced rise in dopamine level after amphetamine (infused as well as systemically administered) was still present (although diminished) 8 days after implantation. It is concluded that, with certain restrictions, brain dialysis of dopamine is still useful several days after implantation of the membrane.
Noninvasive In Vivo Demonstration of 2‐Fluoro‐2‐Deoxy‐d‐Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy
Authors: Nakada Tsutomu , Kwee Ingrid L. , Conboy Claire B.
Published: December 2006

Abstract: The metabolism of 2‐fluoro‐2‐deoxy‐d‐glucose (FDG) in vivo was observed noninvasively in rat brain using “F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/ kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG‐6‐phosphate and 2‐fluoro‐2‐deoxy‐8‐phosphogluconolactone and/or 2‐fluoro‐2‐deoxy‐δ‐phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F‐containing metabolites of FDG in vivo.
Development of the γ‐Aminobutyric Acid Neurotransmitter System in the Rat Cerebral Cortex During Repeated Administration of the GABA‐Transaminase Inhibitor Ethanolamine O‐Sulphate
Authors: Sykes Clare C. , Horton Roger W.
Published: December 2006

Abstract: Ethanolamine O‐sulphate (400 mg/kg, i.p.) was administered to rat pups at 9 days of age and on alternate days up to 17 days of age. At 18 days of age, β‐aminobutyric acid (GABA) concentration was increased (three‐to fourfold), glutamic acid decarboxylase (GAD) activity reduced to 55% of control, and the number of GABAA and GABAB binding sites increased in the cerebral cortex. This is the same pattern of change as seen previously with oral administration of ethanolamine O‐sulphate to the adult rat but the changes occur more rapidly in the developing rat. A lower dose of ethanolamine O‐sulphate (100 mg/kg, i.p.), administered according to the same schedule, caused a twofold increase in cortical GABA at 18 days of age whereas GAD activity and GABAA binding were not significantly altered.
Peanut Agglutinin Receptor Is a Marker of Myelin in Rat Brain. Developmental Changes in Its Distribution
Authors: Momoi Takashi , Yoshida Momoi Mariko , Kurata Takeshi
Published: December 2006

Abstract: Peanut agglutinin receptor, which is a glyco‐conjugate with terminal Gal(β1–3)GalNAc residues, was found to be a specific biochemical marker of the myelin in rat brain. The localization of peanut agglutinin receptor in rat brain was studied by use of fluorescein‐conjugated peanut agglutinin. In adult rat brain, receptors were found only on myelin. In contrast, the receptors in fetal brain were not found in the white matter, but only in the ependyma. Peanut agglutinin receptors were extracted from adult and fetal rat brains with 2% Triton X‐100, subjected to electrophoresis on 10% sodium dodecyl sulfate‐polyacrylamide gel, blotted onto nitrocellulose filters, and detected by their reaction with peroxidase‐conjugated peanut agglutinin. A major receptor of Mr 130,000 and a minor one of Mr 200,000 were found in both adult and fetal rat brains.
Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes
Authors: Daniell Laura C. , Leslie Steven W.
Published: December 2006

Abstract: Voltage‐dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were <15% of 0–1 s rates. Both resting (5 mM KCl) and depolarization‐induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage‐dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1‐ and 30‐s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization‐induced NE release was dependent on the presence of external calcium. These results show that calcium‐dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.
Abnormality of α1Adrenergic Receptors in the Frontal Cortex of Epileptic Rats
Authors: Nicoletti F. , Barbaccia M. L. , Iadarola M. J. , Pozzi O. , Laird H. E.
Published: December 2006

Abstract: We found that the binding of [3H]prazosin, a selective ligand for α1‐adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy‐prone rats (GEPRs), as compared with normal Sprague‐Dawley rats. Scatchard analysis reveals a decrease in the Bmax of [3H]prazosin binding with no change in the apparent KD, suggesting that there are fewer α1‐adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [3H]inositol monophosphate ([3H]IP1) formation in frontal cortex slices prelabeled with [3H]inositol. No significant differences in [3H]prazosin binding as well as NE‐stimulated [3H]IP1 formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the α1‐adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR.
Apparent Absence of a Translocase in the Cerebral Glucose‐6‐Phosphatase System
Authors: Fishman Robert S. , Karnovsky Manfred L.
Published: December 2006

Abstract: In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose‐6‐phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent‐disrupted brain microsomes to distinguish between glucose‐6‐phosphate and mannose‐6‐phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently‐linking reagents [e.g., 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.
Specific [3H]Piflutixol Binding to CHAPS‐Solubilised Rat Striatal Preparations Involves Dopamine D‐2 but Not D‐1 Binding Sites
Authors: Kilpatrick Gavin J. , Jenner Peter , Marsden C. David
Published: December 2006

Abstract: [3H]Piflutixol binding to rat striatal membrane preparations identifies both D‐l and D‐2 sites. We used [3H]piflutixol to characterise those binding sites present in 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS)‐solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis‐flupenthixol, to CHAPS‐solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis‐flupenthixol and (±)‐sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS‐solubilised preparations involves mainly D‐2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS‐solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS‐solubilised rat striatal D‐2 sites.
Purification and Characterization of a Bovine Cerebral Cortex Cell Surface Sialoglycopeptide that Inhibits Cell Proliferation and Metabolism
Authors: Sharifi B. G. , Johnson T. C. , Khurana V. K. , Bascom C. C. , Fleenor T. J. , Chou H.‐H.
Published: December 2006

Abstract: A Sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size‐exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of ∼18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the Sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the Sialoglycopeptide produced antibodies that, when bound to protein A‐agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.
Identification of Sodium‐Dependent, High‐Affinity Choline Uptake Sites in Rat Brain with [3H]Hemicholinium‐3
Authors: Manaker Scott , Wieczorek Caroline M. , Rainbow Thomas C.
Published: December 2006

Abstract: [3H]Hemicholinium‐3 (HC‐3) was used to label sodium‐dependent, high‐affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC‐3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate‐putamen, and olfactory tubercle. This distribution of [3H]HC‐3 binding sites was in close agreement with the amounts of choline acetyltrans‐ferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC‐3 binding by 40–50%. These data indicate that sodium‐dependent [3H]HC‐3 binding sites are related to the choline transport system present in cholinergic neurons.
GM1 Ganglioside Treatment of PC 12 Cells Stimulates Ganglioside, Glycolipid, and Lipid, but Not Glycoprotein Synthesis Independently from the Effects of Nerve Growth Factor
Authors: Katoh‐Semba Ritsuko , Skaper Stephen D. , Varon Silvio
Published: December 2006

Abstract: The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1‐induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1‐induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.
Cholinergic Receptor‐Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells
Authors: Pocotte Susan L. , Holz Ronald W. , Ueda Tetsufumi
Published: December 2006

Abstract: We have identified a 56‐kilodalton protein in cultured bovine adrenal chromaffin cells that is phos‐phorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one‐ and two‐dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis. Two‐dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+‐dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP‐dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+‐dependent protein kinase. After 2 min phosphorylation by a cyclic AMP‐dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.
Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor
Authors: Bizzozero Oscar A. , Lees Marjorie B.
Published: December 2006

Abstract: The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chlorofrm/methanol, and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl‐CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl‐CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl‐CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.
Parallel Postnatal Development of Choline Acetyltransferase Activity and Muscarinic Acetylcholine Receptors in the Rat Olfactory Bulb
Authors: Large Thomas H. , Lambert Mary P. , Gremillion Maureen A. , Klein William L.
Published: December 2006

Abstract: The development of cholinergic synapses in the rat olfactory bulb was investigated by measuring changes in the activity of choline acetyltransferase (ChAT; EC 2.3.1.6.), a presynaptic cholinergic marker, and in the concentration of muscarinic receptors, components of cholinoceptive membranes. Three biochemical properties of the muscarinic system also were examined for possible differentiation: ligand binding, molecular weight, and isoelectric point. Receptors from embryonic (day 18), neonatal (postnatal day 3), and adult rat olfactory bulbs exhibited identical complex binding (nH= 0.45) of the agonist carbachol. For each age, the relative proportions of high‐affinity (Ki= 1.0 μM) and low‐affinity (Ki= 100 μM) binding states were 60% and 40%, respectively. The antagonist pirenzepine also bound to high‐affinity (Ki= 0.15 μM, RH= 70%) and low‐affinity (Ki= 2.0 μM, RL= 30%) sites in neonatal and adult rats. Sodium dodecyl sulfate/urea‐polyacrylamide gel electrophoresis of [3H]propyibenzilylcholine mustard‐labeled receptors from neonatal and adult rats showed a single electrophoretic form with an apparent molecular weight of 65,000. In contrast, analytical isoelectric focusing indicated high pI (4.50) and low pI (4.00) receptor forms were present. Neonatal rats contained approximately equal proportions of the two receptor forms, whereas adult rats contained mainly the low pI form, indicating that molecular alteration of the receptor population had occurred during development. Comparison of postnatal changes in acetylcholine receptors and ChAT activity showed a striking correlation between the development of cholinergic terminals and muscarinic receptors. Throughout the first postnatal week, ChAT activity remained at 5% of adult levels; activity began to rise on postnatal day 6 and gradually reached adult levels (56 ± 4 μnol of [3H]acetylcholine/h/g) during the fourth week. Similarly, muscarinic receptor concentration was low (30–50 fmol/mg) throughout the first week, began to rise at postnatal day 7; and reached 90% of adult levels (317 ± 17 fmol/mg) by the fourth week. In contrast, there was little increase in the concentration of nicotinic acetylcholine receptors (30 fmol/mg) during this period. The parallel postnatal development of ChAT activity and muscarinic receptors suggests the existence of factors that couple the differentiation of presynaptic cholinergic terminals and postsynaptic cholinoceptive elements.
Regional and Subcellular Distribution of Thy‐1 in Human Brain Assayed by a Solid‐Phase Radioimmunoassay
Authors: Almqvist Per , Carlsson Sven R. , Hardy John A. , Winblad Bengt
Published: December 2006

Abstract: A solid‐phase radioimmunoassay, specific for the monomeric form of human Thy‐1, was developed and used for quantitation of the Thy‐1 antigen in human brain tissue. Determination of Thy‐1 in homogenates of 12 anatomically defined brain regions showed that Thy‐1 is present throughout the human brain. However, significant variation was found in the expression of the glycoprotein in different regions. Thy‐1 appears to be generally enriched within gray matter: caudate nucleus, cerebral cortex, and putamen were found to contain the highest Thy‐1 concentration (approximately 2.5 μg Thy‐1/mg protein). Interestingly, the cerebellar cortex contained only 25% of the Thy‐1 concentration of cerebral gray matter. Cerebral subcortical white matter contained half the amount of Thy‐1 compared to cerebral cortex. Determination of Thy‐1 in subcellular fractions prepared from human brain biopsy tissue indicated that the highest relative concentration of Thy‐1 is associated with synaptosomal membranes and myelin/axonal membrane fractions.
Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application
Authors: Yamamoto Hideko , Kato Takeshi
Published: December 2006

Abstract: A sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for Cholecystokinin octapeptide sulfate (CCK‐8S) has been developed using N‐terminal specific antibody for CCK‐8S. In this assay CCK‐8S coupled with poly‐L‐Glu (CCK‐poly‐Glu), which is adsorbed on a solid phase, competes with CCK‐8S for the binding sites of rabbit anti‐CCK antibody, and the complex of the immobilized antibody and CCK‐poly‐Glu is measured using goat anti‐rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is <24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2‐6.2% and the interassay coefficient of variation is 5.9–8.5%. This assay is sensitive enough to detect 9 pg of CCK‐8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6‐hydroxy‐dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.
Comparative Study of Guanidino Compounds in Serum and Brain of Mouse, Rat, Rabbit, and Man
Authors: Marescau B. , De Deyn P. , Wiechert P. , Van Gorp L. , Lowenthal A.
Published: December 2006

Abstract: The levels of 12 guanidino compounds were determined in the serum and brain of mouse, rat, rabbit, and man using cation‐exchange column chromatography with the fluorescence ninhydrin detection method. A comparative study of these compounds was made in the four groups studied, for serum and brain. In rabbit and man the different guanidino compounds were also determined in various brain regions. This study provides basic analytical data that could facilitate the interpretation of further biochemical and neurochemical studies dealing with guanidino compounds that are identified as being toxins in hyperargininemia and uremia.
Rapid Isolation of the 7S‐Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva
Authors: Petrides Petro E. , Shooter Eric M.
Published: December 2006

Abstract: 7S‐Nerve growth factor (NGF) and its α, β‐NGF, and γ subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed‐phase liquid chromatography on short alkyl chain supports (C4 columns), and ion‐exchange chromatography on silica‐based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material, β‐NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.
Posttranslational Protein Modification by Amino Acid Addition in Regenerating Optic Nerves of Goldfish
Authors: Chakraborty G. , Leach T. , Zanakis M. F. , Ingoglia N. A.
Published: December 2006

Abstract: Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000‐g supernatant of optic nerves was analyzed for incorporation of 3H‐amino acids into proteins. No incorporation of 3H‐amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S‐200 column (removing molecules < 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4–5 times for Lys and 2–13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5–15 times more [3H]Lys and [3H]Leu into proteins than did the shafts. When optic axons were removed from the nerve by degeneration, most of the PTPM activity was lost, indicating a primarily intraaxonal locus for the components of the reaction. SDS‐PAGE of 3H‐modified proteins showed the presence of radioactivity in proteins of ∼13,000–18,000 38,000–46,000, and 53,000 daltons. These experiments indicate that the components necessary for PTPM by amino acids are transported axonally in regenerating optic nerves of goldfish and that the greatest activity is in the most advanced tips of the growing nerve.
Monoamine Neurotransmitter Metabolism and Locomotor Activity During Chemical Hypoxia
Authors: Freeman Gary B. , Nielsen Pamela , Gibson Gary E.
Published: December 2006

Abstract: The effects of hypoxia on metabolism of 5‐hydroxytryptamine (5‐HT or serotonin) and 3,4‐dihydroxy‐phenylethylamine (DA or dopamine) were compared with those on open‐field activity in male CD‐1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5‐hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (−41%) and [3H]Trp to [3H]5‐HT (39%). Hypoxia also reduced dihydroxy‐phenylacetic acid (DOPAC) levels (−27%) and DOPAC/DA ratios (−20%). Open‐field behavior, as measured in an automated activity monitor, decreased in a dose‐dependent fashion with 75–150 mg/kg of NaNO2 (−35 to −90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine.
Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells
Authors: Yavin Ephraim , Hama Tokiko , Gil Shosh , Guroff Gordon
Published: December 2006

Abstract: PC 12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48‐h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C] glucosamine‐labeled, ethanol‐precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr > 200,000 which might correspond to the NGF‐induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides in‐ cluding asialo GMl. This increase is independent of the presence of NGF. GMl and GDlB/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 (μg/ml) enhances selectively the release of various glycoproteins between 2.6‐and 8‐fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDia is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.
Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2‐(4‐Phenylpiperidino) Cyclohexanol (AH5183)
Authors: Collier B. , Welner S. A. , Říčný J. , Araujo D. M.
Published: December 2006

Abstract: These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2‐(4‐phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183was 194 ± 10 pmol, which represented 14 ± 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4‐aminopyridine (4‐AP), and not associated with inhibition of stimulation‐induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 ± 51 pmol and in its absence it was 2,547 ± 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4‐AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization. It is concluded that increased ACh synthesis during activity can occur independent of transmitter release, and that the activation of nerve terminal ACh synthesis requires increased calcium influx.
Immunological Approach to the Detection of Taurine and Immunocytochemical Results
Authors: Campistron Ghislaine , Geffard Michel , Buijs Ruud M.
Published: December 2006

Abstract: An immunological approach to the detection of taurine resulted in antibodies specific enough to be used for immunocytochemical studies. The experimental conditions were similar to those previously described for raising antibodies against some small‐sized neurotrans‐mitter molecules: antisera were obtained from rabbits immunized with taurine conjugated to carrier proteins via glutaraldehyde and purified by adsorption on the glutar‐aldehyde‐treated protein carriers. Antibody affinity and specificity were determined in competition experiments between conjugated taurine and other conjugated amino acids or derivatives by enzyme‐linked immunosorbent assay. The resulting cross‐reactivity ratios, calculated at half‐displacement, showed conjugated taurine to be the best recognized compound. Given the molecular structure of taurine and the method used to prepare the conjugate, it seemed necessary to perform an oxidation step. However, adsorption of antisera on reoxidized or non‐reoxidized taurine conjugates suggested that reoxidation did not make a significant difference. Immunocytochemical application of the sera revealed populations of strongly immunopositive nerve cells in the cerebellum, striatum, and septum. The results confirmed that anti‐taurine antibodies can be used as specific tools for a better understanding of the role of taurine in the central nervous system.
Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat
Authors: Walker John H. , Kristjansson Gunnar Ingi , Stadler Herbert
Published: December 2006

Abstract: Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000‐150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron‐specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle‐free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergicspecific. The function of the Mr 86,000 antigen remains to be identified.
Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex
Authors: Sher Phyllis Kammerman , Neale Elaine A. , Machen Valynda L.
Published: December 2006

Abstract: Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 μM) and Ro 5‐4864 (0.1 μM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58‐68% of specific grains over background cells and Ro 5‐4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.
62K Proteins Constitute the Major Neurofilament Proteins in the Frog Optic Nerve
Authors: Quitschke Wolfgang , Schechter Nisson
Published: December 2006

Abstract: The frog optic nerve contains a major group of proteins at a molecular weight of 62K. These proteins are insoluble in nonionic detergents, reactive with a general antibody to intermediate filament proteins, and not labeled by ex vivo incubations of optic nerve. They were therefore considered neurofilament proteins. Axonal transport and enucleation studies were performed to characterize further the origin of these proteins. The results show that the 62K proteins are transported into the optic nerve at a very slow rate (0.1 mm/day). After enucleation, these proteins are substantially reduced in concentration to 20% of the control value at 13 weeks. The predominant neurofilament proteins of the frog optic nerve are 62K in molecular weight. These results are discussed in terms of the anatomy of the frog optic nerve and also contrasted to findings obtained for the goldfish optic nerve.
4‐(1‐Naphthylvinyl)pyridine Decreases Brain Acetylcholine In Vivo, but Does Not Alter the Level of Acetyl‐CoA
Authors: Budai D. , Říčný J. , Kása P. , Tuček S.
Published: December 2006

Abstract: The effects of intraperitoneally administered 4‐ (1‐naphthylvinyl)pyridine (NVP; 200 mg/kg) on the concentrations of acetylcholine (ACh), choline (Ch), and acetyl‐CoA (AcCoA) in rat striatum, cortex, hippocampus, and cerebellum were investigated. Twenty minutes after treatment, the content of ACh was significantly diminished, whereas that of Ch was increased. In response to stress (swimming for 20 min), these changes were enhanced. However, the AcCoA content did not change in any of the brain regions. It is thus very likely that the decrease of brain ACh concentration induced by NVP is due to the drug's effect on choline acetyltrans‐ ferase (ChAT) and/or the reduction of the high‐affinity Ch uptake, and not on the availability of AcCoA. Presumably, the pharmacologically diminished activity of ChAT may become the rate‐limiting factor in the maintenance of ACh levels in cholinergic neurons.
Aminergic Neurotransmitter and Water Content Changes in Rats After Transient Forebrain Ischemia
Authors: Bentué‐Ferrer D. , Reymann J. M. , Bagot H. , Van den Driessche J. , de Certaines J. , Allain H.
Published: December 2006

Abstract: We have studied changes of cerebral monoamine metabolism and water content, during recirculation following global transient ischemia (20 min) using the four‐vessel occlusion model in rats. Levels of monoamines and their metabolites were determined in cortex, striatum, hippocampus, and hypothalamus. Water content was evaluated by weight and by the analysis of Tl and T2 relaxation times in 1H‐nuclear magnetic resonance. Norepinephine levels decreased; 3,4‐dihydroxyphenylethylamine, 3,4‐dihydroxyphenylacetic acid, and 5‐hydroxytryptamine levels oscillated and levels of the end products homovanillic acid and 5‐hydroxyindole‐3‐acetic acid increased. The regional changes were qualitatively similar but quantitatively different, and were greatest in the hippocampus, illustrating the concept of neuronal selective vulnerability. The changes suggest an initial monoarhine depletion and catabolism due to massive release from stores followed by autoregulatory processes. The water content increased moderately, with a maximum at 1 h. The variations of Tl were similar, positively correlated with water content and more pronounced in the cortex than in the white matter. T2 was markedly altered over the entire 24‐h period. Those latter parameters are positively correlated with 5‐hydroxytryptamine concentration in the hypothalamus consistent with a relationship between 5‐hydroxytryptamine and cerebral edema.