bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Phosphoinositide Hydrolysis by NaF and Aluminum in Rat Cortical Slices
Authors: Jope Richard S.
Published: October 2006

Abstract: NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 μM NaF. Addition of 10 or 100 μM A1C13 or aluminum maltol did not alter the effect of NaF, whereas at 500 μM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 μM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 μM phorbol 12–myristate 13–acetate, a phorbol ester, inhibited carbachol‐induced, but not NaF‐induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.
Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts
Authors: Barbarese Elisa , Barry Christopher , Chou Chi‐Hsin Jen , Goldstein David J. , Nakos Grace A. , Hyde‐DeRuyscher Robin , Scheld Kathy , Carson John H.
Published: October 2006

Abstract: Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos‐1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady‐state levels of MBP‐specific RNA and of MBP poly‐peptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild‐type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions. Purified MBP added prior to subcellular fractionation to ho‐mozygous shiverer oligodendrocytes (which express no MBP endogenously) is recovered predominantly in the plasma membrane fraction, whereas purified MBP added to mock‐transfected fibroblasts exhibits a dispersed distribution among subcellular fractions. On the basis of these results, we propose a model whereby localization of MBP to the plasma membrane in oligodendrocytes involves interaction between the newly synthesized MBP polypeptide and specific MBP receptors which are present in oligodendrocyte plasma membrane, but not in fibroblast plasma membrane.
Homocysteic Acid as a Putative Excitatory Amino Acid Neurotransmitter: I. Postsynaptic Characteristics at N‐Methyl‐D‐Aspartate‐Type Receptors on Striatal Cholinergic Interneurons
Authors: Lehmann John , Tsai Cindy , Wood Paul L.
Published: October 2006

Abstract: The actions of the stereoisomers of homocysteic acid (HCA) were characterized at N‐methyl‐D‐aspartate (NMDA)‐type receptors which mediate excitatory amino acid‐evoked [3H]acetylcholine ([3H]ACh) release from striatal cholinergic interneurons. Like NMDA, l‐HCA and d‐HCA evoked the release of [3H]ACh formed from [3H]choline in striatal slices. The concentration‐response curve for l‐HCA was virtually superimposable on that for NMDA, yielding an equal EC50 value (56.1 μM) and maximal response. However, d‐HCA was weaker, with an EC50 value of 81.1 μM, and an apparently smaller maximal response. l‐HCA‐evoked [3H]ACh release was inhibited by the same categories of compounds which inhibit NMDA‐evoked [3H]ACh release: the divalent ion Mg2+ (IC50= 25.8 μM); competitive NMDA antagonists 2‐amino‐7‐phosphonoheptanoate (IC50= 51.2 μM) and 3‐(2‐carboxypiperazin‐4‐yi)propyl‐1 ‐phosphonic acid (IC50= 20.1 μM); and the dissociative anesthetics tile‐tamine (IC50= 0.59 μM) and MK‐801 (IC50= 0.087 μM). Like NMDA, l‐HCA produced a tachyphylaxis in this system. Tachyphylaxis to NMDA resulted in a decreased response to l‐HCA, and conversely, tachyphylaxis to l‐HCA resulted in a decreased response to NMDA. The results suggest that l‐HCA is an agonist at the NMDA‐type receptor and may represent an endogenous ligand for this excitatory amino acid receptor.
Conservation of Amino Acid Sequences Between Human and Porcine Choline Acetyltransferase
Authors: Hersh Louis B. , Takane Karen , Gylys Karen , Moomaw Carolyn , Slaughter Clive
Published: October 2006

Abstract: The amino acid sequence of 11 peptides generated from human placental choline acetyltransferase was compared to the corresponding amino acid sequences predicted from the nucleotide sequence of a recently cloned porcine choline acetyltransferase cDNA. These peptides, which were generated by cyanogen bromide cleavage or tryptic digestion, accounted for 23% of the amino acids in the enzyme. Of the 145 amino acids sequenced eight differed between the two species, yielding an identity of 94% over the regions sampled.

Of the eight amino acids that differed six could represent single base changes in the DNA sequence. These findings demonstrate strong sequence similarity between porcine and human choline acetyltransferase and indicate that they are closely related evolutionarily.
Amphiphilic and Nonamphiphilic Forms of Torpedo Cholinesterases: I. Solubility and Aggregation Properties
Authors: Bon Suzanne , Toutant Jean‐Pierre , Méflah Khaled , Massoulié Jean
Published: October 2006

Abstract: We report an analysis of the solubility and hy‐drophobic properties of the globular forms of acetylcholin‐esterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X‐100 is ˜2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE‐depleted Triton X‐100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythro‐cyte membranes correspond to this type, which is also detectable in detergent‐soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low‐salt‐soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).
Detection of N‐Terminally Extended Substance P but Not of Substance P in Human Cerebrospinal Fluid: Quantitation with HPLC‐Radioimmunoassay
Authors: Toresson Gudrun , Brodin Ernst , Wahlström Agneta , Bertilsson Leif
Published: October 2006

Abstract: A reversed‐phase HPLC system was used to concentrate and separate components of substance P‐like immunoreactivity (SP‐LI) from human CSF. When CSF was injected and fractions collected, no SP‐LI could be detected by radioimmunoassay (RIA) at the retention time of SP or SP‐sulfoxide. Instead, SP‐LI was detected in later eluting fractions. This SP‐LI reacted with two different antisera raised against the C‐terminal part of SP, but not with an antiserum against the N‐terminal part. A compound with similar properties was also found to be present in neutral extracts of rat dorsal spinal cord. When the late‐eluting compound from human CSF was treated with trypsin and rechromatographed on HPLC, an immunoreactive component eluting at the position of SP could be detected with both the C‐ and N‐terminally directed SP antisera. These results suggest that an N‐terminally extended form of SP is present in human CSF. Trypsinization also gave two other compounds with affinity for the N‐ but not the C‐terminally directed antisera. This may indicate that N‐terminal fragments of SP extended at the N‐terminus or SP molecules extended at both the N‐ and the C‐terminus (i.e., preprotachykinins) also are present in human CSF. In 32 CSF samples from depressed patients, SP‐LI was determined with a C‐terminally directed antiserum with and without prior HPLC separation. SP itself could not be detected, but the late‐eluting form of SP‐LI could be quanti‐tated in all samples by combined HPLC‐RIA. In most samples, there was a relatively good agreement between the SP‐LI levels measured with and without HPLC.
Differential Alteration of Various Cholinergic Markers in Cortical and Subcortical Regions of Human Brain in Alzheimer's Disease
Authors: Araujo D. M. , Lapchak P. A. , Robitaille Y. , Gauthier S. , Quirion R.
Published: October 2006

Abstract: The main objective of the present study was to determine whether cholinergic markers (choline acetyl‐transferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N‐[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2‐like muscarinic sites were studied using [3H]‐acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2‐like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.
Adenosine Inhibits Choline Kinase Activity and Decreases the Phosphorylation of Choline in Striatal Synaptosomes
Authors: Wecker Lynn , Reinhardt Rick R.
Published: October 2006

Abstract: The main objective of these studies was to determine whether adenosine inhibits choline kinase in rat striata, leading to a decreased incorporation of choline into phosphorylcholine, a mechanism that may mediate seizure‐induced increases in the levels of free choline in brain. Incubation of paniculate and soluble fractions of striatal synaptosomes with adenosine or its metabolically stable analogues significantly inhibited enzyme activity. The inhibition was noncompetitive versus choline and competitive versus MgATP. Inhibitor constants for adenosine, 2‐chloroadenosine, and 2′,5′‐dideoxyadenosine at the MgATP site were 94, 49, and 207 μM, respectively; these values were less than the Michaelis constant for MgATP (340 μM). To determine whether adenosine altered the phosphorylation of choline in an intact preparation, synaptosomes were in cubated with [3H]choline in the presence or absence of adenosine or its analogues and the amount of [3H]‐phosphorylcholine formed from the [3H]choline taken up was measured. All compounds tested significantly reduced the synthesis of [3H]phosphorylcholine. Results suggest that following seizures or hypoxia, when levels of adenosine increase and the concentration of ATP decreases, inhibition of choline phosphorylation may be manifest, resulting in increased levels of free choline in brain.
Characterization of a Cytosolic Protein (P36) Isolated from Pig Brain by Benzodiazepine‐Affinity Chromatography
Authors: Kirkness Ewen F. , Turner Anthony J.
Published: October 2006

Benzodiazepine‐affinity chromatography, on a column of 1012S‐Sepharose, resulted in the detection and purification of a binding protein (P36) from the cytosolic fraction of pig cerebral cortex. Purified P36 was enriched over 3,500‐fold in a single step and was recovered with an efficiency of 50–60%. Analysis of the purified preparation by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis demonstrated a single polypeptide of Mr 36,000. TheStokes radius (3.44 nm) and sedimentation coefficient (4.43S) indicated that purified P36 is a dimeric protein. Analysis of the amino acid composition of P36 revealed a relatively high content of the hydrophobic amino acids, valine and leucine. Immunoblotting of several pig tissue preparations with an antiserum raised against purified P36 demonstrated approximately equal enrichment of P36 in cerebral cortex, cerebellum, and adrenal glands. Lesser enrichment was observed in kidney and liver, whereas a number of other tissues displayed no immunoreactivity. The γ‐aminobutyrate/benzodiazepine receptor complex and P36 showed no immunological cross‐reactivity. High‐affinity binding activity for [3H]Ro 15–4513, [3H]flunitrazepam, or [3H]PK11195 was not detected in preparations of purified P36. However, the ability of the γ‐aminobutyrate/benzodiazepine receptor inverse agonists, methyl‐ and ethyl‐#bT‐carboline‐3‐carboxylate, to inhibit the binding of P36 to 1012S‐Sepharose at relatively low concentrations indicates that P36 exhibits a degree of binding specificity.
Chemical and Surgical Lesions of Rat Olfactory Bulb: Changes in Thyrotropin‐Releasing Hormone and Other Systems
Authors: Sharif N. A.
Published: October 2006

Stereotaxic injection of kainic acid (15 μg) into rat olfactory bulbs was accompanied by a 53% (n = 4; p < 0.02) depletion of endogenous thyrotropin‐releasing hormone (TRH) as compared to sham‐operated controls 2 weeks postlesion. TRH levels remained unaltered in three other caudal regions. Bulbar kainate lesions produced a 58% (n = 5; p < 0.001) decrease in TRH receptor binding capacity without affecting the receptor affinity. Kainate lesions also reduced bulbar muscarinic and benzodiazepine receptors by 60% and 48%, respectively. Again, no changes in TRH receptors were apparent in six other brain areas after bulbar kainate treatment. Injection of the dopaminergic neurotoxin, 6‐hydroxydopamine (8 μg), into rat bulbs decreased TRH receptors by 35% (n = 4; p < 0.05) 1 week postlesion. One month after surgical bulbectomy, TRH and TRH receptor levels in a number of brain areas were unaltered compared to those of control animals. These studies suggest that TRH in the olfactory bulb originates intrinsically and may be produced predominantly for local use. Secondly, TRH receptors in the bulb appear to be postsyn‐aptically localized on intrinsic neurons, although a small proportion are also associated with presynaptic elements of dopaminergic noradrenergic neurons. Bulbar TRH receptors exhibited nanomolar affinity and a pharmacological selectivity akin to that of the pituitary gland and other brain regions.
Assay of 2‐Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography‐Mass Spectrometry
Authors: Noto Tadashi , Hasegawa Takeshi , Hashimoto Hiromichi , Nakajima Teruo
Published: October 2006

2‐Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography‐mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2‐hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and mid‐brain. The level of 2‐hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.
A 140‐Kilodalton Protein Is Released from Cultured Astrocytes by Phosphatidylinositol Phospholipase C
Authors: Amblard F. , He J.‐T. , Barbet J. , Goridis C. , Prochiantz A.
Published: October 2006

Astrocytes in culture synthesize a 140‐kilodalton (140‐kD) protein (protein 140) that is released into the medium on incubation with phosphatidylinositol phospholipase C. This molecule therefore belongs to the class of proteins anchored to the external side of the cell membrane through a glycolipid moiety. Protein 140 is present in astrocyte cultures derived from two different regions of the brain and is not expressed by neurons in vitro. It differs from neuronal cell adhesion molecule 120 or 140 and is probably identical to a protein of 140 kD present in C6 glioma cells.
[3H]Spiroxatrine: A 5‐HT1A Radioligand with Agonist Binding Properties
Authors: Herrick‐Davis Katharine , Titeler Milt
Published: October 2006

Spiroxatrine has been reported to be a 5‐HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5‐HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5‐HT1A antagonist radioligand. [3H]8‐Hydroxydipropylaminotetralin ([3H]8‐OH‐DPAT), a well‐characterized 5‐HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8‐OH‐DPAT binding produced KD values of 0.9 nAf and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r= 0.98; p < 0.001) exists between K1 values obtained for a series of drugs in competing for [3H]‐spiroxatrine and [3H]8‐OH‐DPAT binding. Of special interest was the observation that 5‐HT1A agonists such as serotonin, 8‐OH‐DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8‐OH‐DPAT‐labelled 5‐HT1A receptors. [3H]Spiroxatrine and [3H]8‐OH‐DPAT binding to 5‐HT1A receptors was inhibited by guanosine 5′‐(β,γ‐imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration‐dependent manner whereas adenosine 5′‐(β,γ‐imido)triphosphate (a non‐hydrolyzable analog of ATP) had no effect. The similarities in the 5‐HT1A receptor radiolabelling properties of [3H]‐spiroxatrine and [3H]8‐OH‐DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has “agonist‐like” binding properties in its interaction with the 5‐HT1A receptor.
Effects of Dorsal Bilateral Rhizotomy Treatment on Transmitter Systems in the Spinal Cord of Normal and Spastic Dogs
Authors: McBride W. J. , Shapiro S. , Chernet E. , Sartorius C. , Hall P. V.
Published: October 2006

The high‐affinity uptakes of [3H]serotonin, [3H]‐glutamate, and γ‐[3H]aminobutyric acid were studied using a myelin‐free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham‐operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of γ‐[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and γ‐[3H]aminobutyric acid. Comparison of sham‐operated spastic dogs and rhizotomy‐treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy‐treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.
Transient and Persistent Depolarization‐Induced Changes of Protein Phosphorylation in a Molluscan Nervous System
Authors: Naito Shigetaka , Bank Barry , Alkon Daniel L.
Published: October 2006

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea‐water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ ([Ca2+]o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10‐fold by high K+, irrespective of the level of [Ca2+]o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF and EDTA‐sensitive protein phosphatase capable of de‐phosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on [Ca2+]o even in normal low K+ (10 mM) medium. Removal of [Ca2+]o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long‐lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed. On the other hand, changes of Mr 56,000 and Mr 20,000 phosphorylation did not persist after depolarization had been terminated. A23187, a Ca2+ ionophore, partially reduced Mr 25,000 phosphorylation. These results suggested that depolarization‐induced modification of protein phosphatase and/or protein kinase activity which regulates Mr 25,000 phosphorylation may be involved in biochemical pathways important for memory storage.
Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats
Authors: Aono Sachiko , Sato Hiroshi , Semba Reiji , Kashiwamata Shigeo , Kato Kanefusa , Eng Lawrence F.
Published: October 2006

The behavior of marker proteins of glial cells [α‐enolase, β‐S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of eerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, γ‐enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of β‐S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in α‐ and γ‐enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of β‐S100 and α‐enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of γ‐enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that β‐S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of eerebellar damage, respectively, but that a‐enolase is less available.
pH Dependence of Histidine Affinity for Blood‐Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids
Authors: Oldendorf William H. , Crane Paul D. , Braun Leon D. , Gosschalk Eddy A. , Diamond Jared M.
Published: October 2006

The effects of pH (3.5–7.5) on the brain uptake of histidine by the blood‐brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single‐pass carotid injection technique. Cationic amino acid ([14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ([14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.
Effect of Maternal Alcohol Consumption on the Lipid Composition of CNS in the Offspring
Authors: Lalitha T. , Kumar Krishna , Ramakrishnan C. V. , Telang S. D.
Published: October 2006

Abstract: Maternal alcohol consumption at a level that does not affect calorie intake increases cholesterol concentration and content as well as incorporation of labeled glucose into cholesterol in the brain and spinal cord of newborn rat pups. Continued consumption of alcohol during lactation also affects the galactolipid concentration in the brain and spinal cord of pups at 21 days of age, and this increase seems mainly to be due to an increase in content of myelin lipids. Analysis of myelin shows that the concentration of phospholipids also increases in this fraction. The increase in incorporation of labeled glucose into these membrane lipids suggests an increase in the synthesis of these lipids, which prevents fluidization of the membrane by alcohol. That in the brainstem the increase in levels of cholesterol and galactolipids is higher than in other regions and that there is also an increase in content of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine suggest that the brainstem needs better protection against fluidization.
A Retinal Calmodulin‐Dependent Kinase: Calcium/ Calmodulin‐Stimulated and ‐Inhibited States
Authors: Bronstein Jeff M. , Wasterlain Claude G. , Farber Debora B.
Published: October 2006

Abstract: A calcium/calmodulin‐dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50‐kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50‐kD and 60‐kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto‐phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin‐dependent to a calcium/calmodulin‐inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin‐independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.
An In Vitro Micromethod for the Quantitative Assessment of Central Demyelination
Authors: Watson C. M. , Najbauer J. , Owen S. J. , Davison A. N.
Published: October 2006

Abstract: We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2′:3′‐cyclic nucleotide 3′‐phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 μg of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein‐primed SJL/J mice, after selective expansion with antigen and in‐terleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.
Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells
Authors: Shah Shantilal N. , Johnson Ronald C.
Published: October 2006

Abstract: Cells dissociated from brains of 1‐day‐old rats were cultured in medium containing either lipoprotein‐deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin‐precipitable sterols, and from the activity of the enzyme 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low‐density lipoproteins (LDL), high‐density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).
Identification and Characterization of the Major Proteins of Mammalian Brain Synaptic Vesicles
Authors: Floor E. , Leeman S. E.
Published: October 2006

Abstract: Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of ∼74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle‐specific monoclonal antibody. The 40,000‐Mr protein appeared to be identical to the 38,000‐Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000‐Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000‐Mr protein is ∼5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these proteins.
Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin
Authors: Volonte Cinzia , Parries Gregory S. , Racker Efraim
Published: October 2006

Abstract: The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC 12 cells cultured for 20 h in the presence [PC12 (+)] or in the absence [PC12 (−)] of nerve growth factor (NGF). BK (1 nM) induced a small stimulation of the incorporation of m′o‐[2–3H] inositol into the lipids of PC12 (−) ceils and a three‐ to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and >10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels ninefold in PC12 (−) and 30‐fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4°C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC 12 (+) cells. In these cells, LiCl in the presence of BK also increased several‐fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.
Synapsin Ia, Synapsin Ib, Protein IIIa, and Protein IIIb, Four Related Synaptic Vesicle‐Associated Phosphoproteins, Share Regional and Cellular Localization in Rat Brain
Authors: Walaas S. Ivar , Browning Michael D. , Greengard Paul
Published: October 2006

Abstract: The regional and cellular distribution of four synaptic vesicle‐associated proteins, synapsins Ia and Ib (Mr 86,000 and 80,000, collectively referred to as synapsin I) and proteins IIIa and IIIb (Mr 74,000 and 55,000, collectively referred to as protein III), has been compared in selected rat brain regions, using both radioimmunoassays and back‐phosphorylation assays. Lesions of several neuronal populations in the basal ganglia (corticostriatal fibers, intrinsic striatal neurons, striatonigral fibers, nigrostriatal fibers) induced decreases in the levels of these various proteins that were highly correlated (r= 0.96–0.97). Moreover, the synaptic vesicle‐associated phosphoproteins displayed a similar and widespread distribution throughout the CNS. This evidence for colocalization indicates that the majority of, and possibly all, CNS neurons and nerve terminals may contain both forms of synapsin I and both forms of protein III.
In Vivo Microdialysis as a Technique to Monitor Drug Transport: Correlation of Extracellular Cocaine Levels and Dopamine Overflow in the Rat Brain
Authors: Hurd Yasmin L. , Kehr Jan , Ungerstedt Urban
Published: October 2006

Abstract: A sensitive and rapid HPLC‐UV method for in vivo determinations of cocaine levels in extracellular fluid of specific brain regions and plasma is described. Free drug levels resulting from intravenous administration of cocaine were sampled using in vivo microdialysis probes simultaneously located in the jugular vein, nucleus accumbens. and anteromedial caudate‐putamen of halothane‐anes‐thetized rats. In a separate group of animals, the influence of cocaine on extracellular dopamine concentrations in the anteromedial caudate‐putamen was also assessed. The time dependences of changes in cocaine concentration in each of the above regions were congruent, and peak concentrations were reached 10 min after the drug was administered. The half‐lives of cocaine in the blood, nucleus accumbens, and anteromedial caudate‐putamen were estimated to be 31.5, 29.1, and 21.4 min, respectively. A repeated injection of cocaine, given 90 min later, produced a maximal cocaine level and pharmacokinetic profile that were indistinguishable from those of the initial infusion. Cocaine was concentrated to a greater extent in brain than in blood, a feature consistent with the action of a lipophilic drug. In addition, extracellular dopamine levels measured in the anteromedial caudate‐putamen following cocaine infusions closely mirrored those of cocaine itself. The ability to measure the free concentration of drugs by microdialysis should be applicable to a wide range of in vivo pharmacological studies.
Tissue Content of Opioid Peptides in the Myenteric Plexus‐Longitudinal Muscle of Guinea‐Pig Small Intestine
Authors: Corbett Alistair D. , McKnight Alexander T. , Kosterlitz Hans W.
Published: October 2006

We have developed a method that is based on two HPLC systems and permits the separation of endogenous opioid peptides in tissue extracts. The individual peptides are bioassayed on the mouse isolated vas deferens; nalox‐one (100 nM) ensures opioid specificity. In the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine, the tissue content of prodynorphin‐derived peptides is lower than those of proenkephalin‐derived peptides. No β‐endorphin was detected. Of the prodynorphin fragments, α‐neoendorphin, β‐neoendorphin, dynorphin A(1–8), and dynorphin B are present in equimolar concentrations (12–15 pmol/g) whereas the tissue content of dynorphin A is only 0.8 pmol/g. Processing of proenkephalin leads to at least six opioid peptides. The tissue contents of [Leu5)enkephalin, [Met5]enkephalyl‐Arg‐Gly‐Leu, and [Met5]enkephalyl‐Arg‐Phe are 90–100 pmol/g and the content of [Met5]enkephalin is 405 pmol/g. BAM‐18 and [Met5]enkephalyl‐Arg‐Arg‐Val‐NH2 are present in much lower concentrations, 24 and 5 pmol/g, respectively. Although present in low amounts, BAM‐18 and [Met5]‐enkephalyl‐Arg‐Arg‐Val‐NH2 have high affinity for the μ‐opioid binding site and to a lesser extent for the k‐site; this binding profile differs from that of the other proenkephalin fragments all of which have high affinities for the μ‐ and δ‐sites.
Identification of a Trypsin‐Like Site Associated with Acetylcholinesterase by Affinity Labelling with [3H]Diisopropyl Fluorophosphate
Authors: Small David H. , Chubb Ian W.
Published: October 2006

In addition to its ability to hydrolyze acetylcho‐line, purified eel acetylcholinesterase possesses a trypsin‐like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor Nα‐p‐tosyl‐l‐lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion‐exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate‐polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide‐containing cells, acetylcholinesterase could act as a neuro‐peptide processing enzyme in these cells.
Evidence that Alterations in γ‐Aminobutyric Acid and Acetylcholine in Rat Striata and Cerebella Are Not Related to Soman‐Induced Convulsions
Authors: Liu Duan Duan , Ueno Eiko , Ho Ing K. , Hoskins Beth
Published: October 2006

Many reports have suggested that γ‐aminobutyric acid (GABA) may play a role in organophosphate‐in‐duced convulsions. The balance between GABA and acetylcholine (ACh) in the brain also has been suggested by some investigators to be related to brain excitability. We examined these questions by studying the levels of GABA and ACh and the ratios of GABA to ACh in rat striata and cerebella (two major motor control areas in the CNS) after the administration of soman, an organophosphate acetylcholinesterase inhibitor also known as nerve gas. Male Sprague‐Dawley rats weighing 250–300 g were injected subcutaneously with three different doses of soman: a subconvulsive dose of 40 μg/kg (approximately 30% of the ED50 for convulsions in rats), a convulsive dose of 120 μg/kg (approximately one ED50 for convulsions), and a higher convulsive dose of 150 μg/kg (approximately 120% of the ED50 for convulsions). The incidence and severity of convulsions were monitored in individual rats until they were sacrificed by focused microwave irradiation of the head at the following time points after soman administration: 4 min, a time prior to the onset of convulsions; 10 min, the time of onset of convulsions; 1 h, the time of peak convulsive activity; and 6 h, a time at which rats were recovering from convulsions. Results showed that in rat striata and cerebella, neither changes in levels of GABA and ACh nor changes in ratios of GABA to ACh were related to soman‐induced convulsions, i.e., none of the changes in either levels or ratios of these two neurotransmitters were related to the initiation of, maintenance of, or recovery from soman‐induced convulsions.
Bovine Chromaffin Cells Have Insulin‐Like Growth Factor‐I (IGF‐I) Receptors: IGF‐I Enhances Catecholamine Secretion
Authors: Dahmer Mary K. , Perlman Robert L.
Published: October 2006

The binding of 125I‐insulin‐like growth factor‐I (125I‐IGF‐I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 ± 40,000 IGF‐I binding sites/cell. These sites bound IGF‐I with a KD of 1.1 ± 0.3 nM and had a much lower affinity for insulin. Cross‐linking studies showed that 125I‐IGF‐I bound to a protein that had an Mr of ∼ 125,000, similar to the Mr of the α subunit of the IGF‐I receptor in other tissues. Cells cultured with IGF‐I (10 nM) for 4 days exhibited an almost twofold increase in high K+‐evoked catecholamine secretion. Insulin was much less potent than IGF‐I in enhancing catecholamine secretion. These data indicate that binding of IGF‐I to its receptors on chromaffin cells can modulate the function of these cells.
Muscarinic Modulation of Purine Release from Electrically Stimulated Rat Cortical Slices
Authors: Pedata Felicita , Magnani Maurizio , Pepeu Giancarlo
Published: October 2006

Abstract: The release of 3H‐labeled purines at rest and during electrical stimulation was investigated in slices of rat cortex prelabeled with [3H]adenine and perfused with Krebs solution. A linear relationship was found between radioactivity efflux and stimulation frequency from 2.5 to 20 Hz. At frequencies of <2.5 Hz, no increase in radioactivity efflux was detected. The amount of tritium released per pulse increased with stimulation frequency up to 10 Hz and declined at 20 Hz. The tritium efflux from the slices at rest and at a stimulation frequency of 10 Hz, analyzed by HPLC with ultraviolet absorbance detection at 254 nm, consisted mostly of adenosine, inosine, and hypoxanthine. The 3H‐labeled purine release evoked by 10‐Hz stimulation increased with current intensity from 15 to 100 mA/cm2. At 20 mA/cm2, addition of 0.5 μM tetrodotoxin to the superfusing Krebs solution brought about a 98% decrease of 3H‐labcled purine release. At higher current strength, the percentage of tetrodotoxin‐sensitive‐evoked tritium efflux was smaller. At 30 mA/cm2. 86% of the evoked release was tetrodotoxin sensitive. Under these stimulation conditions, tritium efflux showed a 69% decrease when the slices were superfused with calcium‐free Krebs solution containing 0.5 mM EGTA. The muscarinic agonist oxotremorine (30 μM) significantly enhanced the 10‐Hz‐stimulated 3H‐labeled purine release. The effect of oxotremorine was partially prevented by tetrodotoxin, was antagonized by atropine (1.5 μM), and was mimicked by addition of physostigmine (3.8 μM) to the superfusion fluid. Atropine alone did not affect the evoked release, and none of the drugs modified the basal tritium efflux. The results indicate that under our stimulation conditions, the 3H‐labeled purine release is mostly dependent on propagated nervous activity. The data also show a muscarinic modulation of the evoked purine release from rat cerebral cortex.
Monoclonal Antibodies with High Affinity for Spiroperidol
Authors: Luedtke Robert R. , Korner Mira , Neve Kim A. , Molinoff Perry B.
Published: October 2006

Abstract: A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N‐amino‐phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to > 100 nM. The antigen binding sites of the anti‐NH2SPD and anti‐NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]‐SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti‐NH2SPD and four anti‐NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2‐selective antagonists of the benzamide class, or the agonist dopamine.