bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies
Authors: Friedl Waltraut , Lentes Klaus‐Ulrich , Schmitz Elke , Propping Peter , Hebebrand Johannes
Published: October 2006

Abstract: Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoamnity‐labeled with [3H]flun‐itrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.
Galanin Inhibits Noradrenaline‐Induced Accumulation of Cyclic AMP in the Rat Cerebral Cortex
Authors: Nishibori Masahiro , Oishi Ryozo , Itoh Yoshinori , Saeki Kiyomi
Published: October 2006

Abstract: The effect of galanin on poradrenaline (NA)‐induced accumulation of cyclic AMP was investigated in slices of rat cerebral cortex. NA (10−4M) increased cyclic AMP levels during a 20‐min observation period. Galanin (3 × 10−7M) significantly inhibited this response at all time points examined, although it did not change the basal levels of cyclic AMP. Galanin (10−3× 10−6M) inhibited the cyclic AMP response to NA (10−4M) in a dose‐dependent manner, with an IC50 of ∼5.6 × 10−5M and a maximum inhibition of 59%. These results suggest that galanin, devoid of any detectable effects by itself, modulates the cyclic AMP response to NA in the rat cerebral cortex.
Cellular and Subcellular Distribution of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase and Its mRNA in the Rat Central Nervous System
Authors: Trapp Bruce D. , Bernier Lise , Andrews S. Brian , Colman David R.
Published: October 2006

Abstract: The 2′,3′‐cyclic nucleotide 3′‐phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the‘“incisure‐like” membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.
Thyroxine Injections Do Not Cause Premature Induction of Thymidine Kinase in sg/sg Mice
Authors: Messer Anne
Published: October 2006

Abstract: Severe hyperthyroidism from the time of birth causes a premature induction and termination of thymidine kinase activity in the cerebella of wild‐type mice. This leads to elevated enzyme levels at postnatal days 5 and 6, with significantly lower levels by postnatal day 7 (which is actually the time of peak activity in normal animals). In this study, neonatal hyperthyroidism does not have significant effects on postnatal day 5, 6, or 7 enzyme levels in the neurological mutant staggerer. This is consistent with the hypothesis that thyroid hormone exerts its effects via the Purkinje cells, which are reduced in number and grossly stunted in the mutant.
Chronic Membrane Depolarization Regulates the Level of the Guanine Nucleotide Binding Protein Goα in Cultured Neuronal Cells
Authors: Luetje Charles W. , Nathanson Neil M.
Published: October 2006

Abstract: Chronic membrane depolarization results in an increase in muscarinic acetylcholine receptor (mAChR) number in N1E‐115 neuroblastoma cells. Because the mAChR interacts with the guanine nucleotide binding regulatory (G) proteins, Gi and Go, the effect of chronic membrane depolarization on the levels of subunits of these G proteins was examined. Quantitation of G protein subunit levels was performed using affinity‐purified, monospecific antibodies in a quantitative immunoblot assay. Incubation with 50 μM veratridine (VTN), an activator of voltage‐sensitive Na+ channels, induced a 48 ± 15% increase in the level of the α subunit of Go. The effect of VTN was blocked by tetrodotoxin. On removal of VTN, the level of Goα decreased to control levels within 24 h. The levels of the α subunit of Gi and the common β subunit were not affected by VTN treatment. These results show that in N1E‐115 cells, the level of the α subunit of Go is regulated in a manner similar to the level of mAChR in response to chronic membrane depolarization.
GABAA Receptor Populations Bind Agonists and Antagonists Differentially and with Opposite Affinities
Authors: Maksay Gábor
Published: October 2006

Abstract: Pretreatment of synaptosomal membranes with a diazo‐coupling reagent and the presence of CI− ions were used to differentiate high‐ and low‐affinity populations of postsynaptic γ‐aminobutyric acid (GABAA) receptors. The super‐low‐affinity GABAA receptors were characterized by the enhancing effect of GABA on [3H]diazepam binding. The GABA antagonists 2–(3‐carboxypropyl)‐3‐amino‐4‐methyl‐6‐phenylpyridazinium chloride (SR 95103) and 3‐α‐hydroxy‐16‐imino‐5β‐17‐aza‐androstan‐11‐one (R 5135) shifted and suppressed the dose‐response curve of GABA on diazepam binding. SR 95103 displaced the lower affinity [3H]GABA binding with higher potency. Dissociation of the binding of the antagonist 2–(3‐carboxypropyl)‐3‐amino‐6‐p‐methoxyphenylpyridazinium bromide ([3H]SR 95531) was polyphasic. Displacing potencies of SR 95531 and GABA were examined on the major (85%) rapid and minor slower phases of dissociation separated kinetically. The slower phase corresponded to higher affinity binding of SR 95531 which was displaced by GABA with about 10 times less potency. Photoaffinity labeling with muscimol decreased the number of [3H]muscimol binding sites by 27%. It decreased the displacing potency of GABA by 72%, but not that of bicuculline methiodide. These findings can be explained by a preferential binding of antagonists to hydrophobic accessory sites around low‐affinity GABAA receptors.
Amphetamine‐Induced Dopamine Release in the Rat Striatum: An In Vivo Microdialysis Study
Authors: Butcher Steven P. , Fairbrother Iain S. , Kelly John S. , Arbuthnott Gordon W.
Published: October 2006

The effects of a number of biochemical and pharmacological manipulations on amphetamine (AMPH)‐induced alterations in dopamine (DA) release and metabolism were examined in the rat striatum using the in vivo brain microdialysis method. Basal striatal dialysate concentrations were: DA, 7 nM; dihydroxyphenylacetic acid (DOPAC), 850 nM; homovanillic acid (HVA), 500 nM; 5‐hydroxyindoleacetic acid (5‐HIAA), 300 nM; and 3‐methoxytyramine (3‐MT), 3 nM. Intraperitoneal injection of AMPH (4 mg/kg) induced a substantial increase in DA efflux, which attained its maximum response 20–40 min after drug injection. On the other hand, DOPAC and HVA efflux declined following AMPH. The DA response, but not those of DOPAC and HVA, was dose dependent within the range of AMPH tested (2–16 mg/kg). High doses of AMPH (>8 mg/kg) also decreased 5‐HIAA and increased 3‐MT efflux. Depletion of vesicular stores of DA using reserpine did not affect significantly AMPH‐induced dopamine efflux. In contrast, prior inhibition of catecholamine synthesis, using α‐methyl‐p‐tyrosine, proved to be an effective inhibitor of AMPH‐evoked DA release (<35% of control). Moreover, the DA releasing action of AMPH was facilitated in pargyline‐pretreated animals (220% of control). These data suggest that AMPH releases preferentially a newly synthesised pool of DA. Nomifensine, a DA uptake inhibitor, was an effective inhibitor of AMPH‐induced DA efflux (18% of control). On the other hand, this action of AMPH was facilitated by veratrine and ouabain (200–210% of control). These results suggest that the membrane DA carrier may be involved in the actions of AMPH on DA efflux.
Levels and Distribution of the Calcium‐Modulated Proteins S100 and Calmodulin in Rat C6 Glioma Cells
Authors: Zimmer Danna B. , Van Eldik Linda J.
Published: October 2006

To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium‐modulated proteins, we have examined the calcium‐modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100β polypeptide. The level of S100β in C6 cells increases approximately 20‐fold from subconfluency to postconfluency whereas the level of calmodulin increases only about twofold. The subcellular distribution of S100β and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100– and calmodulin‐binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100β is the predominant S100 polypeptide in C6 cells and suggest that changes in S100β and S100β‐binding proteins may be involved in regulating S100‐mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.
Requirement of ATP for Exocytotic Release of Catecholamines from Digitonin‐Permeabilized Adrenal Chromaffin Cells
Authors: Morita Kyoji , Ishii Shuta , Uda Hiroshi , Oka Motoo
Published: October 2006

Cultured chromaffin cells were preincubated with digitonin to deplete endogenous ATP from the cell cytoplasm. Catecholamine release from these digitonin‐pretreated cells was then studied in the presence and absence of exogenous ATP to elucidate a possible involvement of the cytoplasmic ATP in the exocytotic process. The preincuba‐tion of the cells with digitonin in the ATP‐free permeabilizing medium resulted in a marked decline of the releasing response to a calcium challenge. Furthermore, the declined activity of catecholamine release caused by digitonin pre‐treatment was restored by the presence of ATP, but not by other adenine nucleotides, and this recovery was observed in a manner dependent on the concentration of ATP. These findings, therefore, seem to indicate that a decrease in the releasing activity of the digitonin‐pretreated cells may be due to the removal of endogenous ATP from the cytoplasmic space of the cells, thus suggesting that the cytoplasmic ATP may be involved in the exocytotic mechanism of catecholamine secretion.
Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis
Authors: Pocock Jennifer M. , Murphie Helen M. , Nicholls David G.
Published: October 2006

Kainate inhibits the exchange of d‐aspartate into guinea‐pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+‐independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+‐independent net efflux of endogenous and labelled glutamate and aspartate in the ab‐sence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate‐sensitive Na+‐cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+‐cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+‐dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.
Subcellular Localization of Rat Brain Insulin Binding Sites
Authors: Marks J. L. , Maddison J. , Eastman C. J.
Published: October 2006

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, γ‐aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mi‐tochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r= 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.
Characteristics of Phorbol Ester‐ and Agonist‐Induced Down‐Regulation of Astrocyte Receptors Coupled to Inositol Phospholipid Metabolism
Authors: Pearce Brian , Morrow Christine , Murphy Sean
Published: October 2006

We have examined some of the characteristics of phorbol ester‐ and agonist‐induced down‐regulation of astrocyte receptors coupled to phosphoinositide metabolism. Our results show that preincubation of [3H]inositol‐labelled astrocyte cultures with phorbol 12‐myristate 13‐acetate (PMA) resulted in a time‐ (t1/2, 1–2 min) and concentration‐dependent (IC50, 1 nM) decrease in the accumulation of [3H]inositol phosphates (IP) evoked by muscarinic receptor stimulation. Much longer (30–40 min) preincubation periods with higher concentrations (IC50, 600 μM) were required to elicit the same effect with the receptor agonist carbachol. Following preincubation, agonist‐stimulated [3H]IP accumulation recovered with time; in both cases pretreatment levels of inositol lipid metabolism were attained within 2 days. Both phorbol ester and agonist pre‐treatments were also effective in reversing the carbachol‐evoked mobilisation of 45Ca2+ in these cells. However, their effects on phosphoinositide metabolism were found not to be additive. Although neither pretreatment affected the incorporation of [3H]inositol into phosphoinositides, both resulted in a loss of membrane muscarinic receptors as assessed by [3H]N‐methylscopolamine binding. In washed membranes prepared from [3H]inositol‐labelled cultures, the guanine nucleotide analogue, guanosine 5′‐O‐thiotri‐phosphate (GTP‐γ‐S), caused a dose‐dependent increase in [3H]IP formation. This response was enhanced when carbachol was also included in the incubation medium, although the agonist alone was without effect. Pretreatment with either PMA or carbachol had no effect on GTP‐γ‐S‐stimulated [3H]IP accumulation but did reduce the ability of carbachol to augment this response. Similar findings were obtained when membranes were exposed directly to PMA. Phorbol ester pretreatment was also effective in reversing the increase in [3H]IP accumulation and 45Ca2+ mobilisation evoked by noradrenaline. However, following preincubation with carbachol there was no loss of nor‐adrenaline‐stimulated phosphoinositide breakdown although its ability to mobilise 45Ca2+ was blocked.
Two Developmentally Regulated Isoenzymes of Calmodulin‐Stimulated Protein Kinase II in Rat Forebrain
Authors: Rostas John A. P. , Seccombe Margaret , Weinberger Ron P.
Published: October 2006

Soluble calmodulin‐stimulated protein kinase II has been purified from adult and 10‐day‐old rat forebrain. By autoradiography, the α/β subunit ratios of the 10‐day and adult enzymes were 0.67 ± 0.03 and 2.20 ±0.15, respectively. By silver staining, the α/β subunit ratios were 1.02 ± 0.06 and 2.36 ±0.10, respectively. The apparent holoenzyme molecular masses of the purified 10‐day and adult enzymes were 500,000 daltons and 700,000 daltons. However, varying the purification conditions revealed higher and lower molecular mass forms at both ages and suggested that the form of the kinase that is usually purified is merely that which has the highest affinity for calmodulin‐Sepharose and may not be the form of the kinase that exists in vivo. The subunits of the adult and 10‐day enzymes were indistinguishable by one‐ and two‐dimensional electrophoresis and one‐dimensional proteolytic peptide maps. These results are consistent with the suggestion that at least two developmentally regulated isoenzymes of this kinase exist in rat forebrain.
Analysis of the 5’Region of the Human Tyrosine Hydroxylase Gene: Combinatorial Patterns of Exon Splicing Generate Multiple Regulated Tyrosine Hydroxylase Isoforms
Authors: Bourdellès B. Le , Boularand S. , Boni C. , Horellou P. , Dumas S. , Grima B. , Mallet J.
Published: October 2006

A single human gene has been described to encode multiple tyrosine hydroxylase (TH) mRNAs. The study of this variation has been extended by S1 mapping experiments and by analysis of the 5’region of the TH gene. Four different mRNAs were found to originate solely from alternative splicing of two exons. Comparison of the 5’flanking regions of human and rat genes discloses several highly conserved segments, likely to play an important role in the regulation of TH gene expression.
Depletion of Brain Glutathione in Preweanling Mice by L‐Buthionine Sulfoximine
Authors: Slivka Adam , Spina Mary Beth , Calvin Harold I. , Cohen Gerald
Published: October 2006

Abstract: Previous studies indicated that DL‐buthionine sulfoximine (dl‐BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of l‐BSO with marked declines (79.6–86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8–29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L‐BSO represents a tool for studying the role of GSH in the CNS.
The Intact Human Neuroblastoma Cell (SH‐SY5Y) Exhibits High‐Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols
Authors: Serra Mariangela , Mei Lin , Roeske William R. , Lui George K. , Watson Mark , Yamamura Henry I.
Published: October 2006

Abstract: The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH‐SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k‐1) rate constants of 5.2 ± 1.4 × 106 M−1 min−1 and 1.1 ± 0.06 × 10−1 min−1, respectively. Saturation experiments revealed a single class of binding sites (nH= 1.1) for the radioligand with a total binding capacity of 160 ± 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency vas atropine sulfate > pirenzepine > AF‐DX116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](‐)‐quinuclidinyl benzilate and its quaternary derivative [3H](‐)‐methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF‐DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high‐affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.
Detection and Characterization of the Serotonin 5‐HT1D Receptor in Rat and Human Brain
Authors: Herrick‐Davis Katharine , Titeler Milt
Published: October 2006

Abstract: In the presence of 1 μM (±)‐pindolol [to block 5‐hydroxytryptamine (5‐HT, serotonin) 5‐HT1A and 5‐HT1B receptors] and 100 nM mesulergine (to block 5‐HT1C receptors), 2.0 nM [3H]5‐HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5‐HT binding, in the presence of 1 μM (±)‐pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5‐HT receptor subtypes. Compounds with high affinity for 5‐HT1A (8‐hydroxydipropylaminotetralin), 5‐HT1B (trifluoromethylphenylpiperazine), 5‐HT1C (mesulergine), 5‐HT2 (4‐bromo‐2,5‐dimethoxyphenylisopropylamine), and 5‐HT3 (ICS 205–930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5‐HT binding site in rat and human brain tissue. This putative novel 5‐HT receptor has a similar pharmacology to the “5‐HT1D” site detected in bovine brain by Heuring and Peroutka.
A Two‐Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus
Authors: Gutierrez Luis M. , Ballesta Juan J. , Hidalgo Maria J. , Gandia Luis , García Antonio G. , Reig Juan A.
Published: October 2006

Abstract: Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two‐dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two‐dimensional electrophoretograms were studied with the aid of computer‐assisted image analysis (CAIA). A database map of 32P‐labeled proteins was constructed; ∼500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and iso‐electric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+‐dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC 12 ceils.
ATP‐Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice
Authors: Fischer‐Bovenkerk Carolyn , Kish Phillip E. , Ueda Tetsufumi
Published: October 2006

Abstract: The ATP‐dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP‐dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP‐dependent glutamate uptake system is reduced by 68% in the staggerer and 57–67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP‐dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.
Immunohistochemical Localization of Ca2+/Calmodulin‐Dependent Protein Kinase II in Rat Brain and Various Tissues
Authors: Fukunaga Kohji , Goto Satoshi , Miyamoto Eishichi
Published: October 2006

Abstract: Polyclonal antibodies against Ca2+/calmodulin‐de‐pendent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen‐affinity column. The antibodies which recognized both sub‐units of the enzyme with MrS 49K and 60K were used for the study on the distribution of CaM kinase II in formalin‐fixed, paraffin‐embedded tissues. In the brain, a light‐microscopic study demonstrated strong immunoreactivity in neu‐ronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cer‐ebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as as‐trocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.
Source and Physiological Significance of Plasma 3,4‐Dihydroxyphenylalanine in the Rat
Authors: Eisenhofer Graeme , Goldstein David S. , Ropchak Torn G. , Kopin Irwin J.
Published: October 2006

Abstract: To elucidate the source and physiological significance of plasma 3,4‐dihydroxyphenylalanine, the immediate product of the rate‐limiting step in catecholamine biosynthesis, plasma 3,4‐dihydroxyphenylalanine was quantified in conscious rats after administration of reserpine, desipramine, clorgyline, or forskolin, treatments that affect tyrosine hydroxylase activity. Plasma 3,4‐dihydroxyphenylalanine was also examined during infusions of norepinephrine with or without clorgyline, reserpine, or desipramine pretreatment. After reserpine, the plasma 3,4‐dihydroxyphenylalanine level decreased by 22% and then increased by 40%, a result consistent with modulation of tyrosine hydroxylase activity first by an increased axoplasmic norepinephrine content and then by depletion of norepinephrine stores. After desipramine, the plasma 3,4‐dihydroxyphenylalanine level decreased by 20%, reflecting the depressant effect of neuronal uptake blockade on norepinephrine turnover. Forskolin increased the plasma 3,4‐dihydroxyphenylalanine level by 30%, consistent with activation of tyrosine hydroxylase by cyclic AMP‐dependent phosphorylation. Acute administration of clorgyline was without effect on the plasma 3,4‐dihydroxyphenylalanine level. Norepinephrine infusions decreased the plasma 3,4‐dihydroxyphenylalanine concentration, as expected from end‐product inhibition of tyrosine hydroxylase. Pretreatment with desipramine prevented the norepinephrine‐induced decrease in plasma dihydroxyphenylalanine content, indicating that inhibition of tyrosine hydroxylase required neuronal uptake of norepinephrine. Both reserpine and clorgyline augmented the norepinephrine‐induced decrease in plasma 3,4‐dihydroxyphenylalanine level, suggesting that retention of norepinephrine in the axoplasm—due to inhibition of norepinephrine sequestration into storage vesicles or catabo‐lism—caused further inhibition of tyrosine hydroxylase. Changes in plasma 3,4‐dihydroxyphenylalanine concentration during norepinephrine infusions were negatively correlated with those in plasma 3,4‐dihydroxyphenylglycol level, a finding consistent with modulation of tyrosine hydroxylase activity by axoplasmic norepinephrine. In reserpinized animals, clorgyline and norepinephrine infusion together decreased the plasma 3,4‐dihydroxyphenylalanine content by 50%, a result demonstrating that hydroxylation of tyrosine was depressed by at least half. The results indicate that quantification of plasma 3,4‐dihydroxyphenylalanine can provide a simple and direct approach for examination of the rate‐limiting step in catecholamine biosynthesis.
Acute Uteroplacental Ischemic Embryo: Lactic Acid Accumulation and Prostaglandin Production in the Fetal Rat Brain
Authors: Magal Ella , Goldin Ehud , Harel Shaul , Yavin Ephraim
Published: October 2006

A new experimental model for studying the effects of acute ischemia on brain development in the near‐term fetal rat has been devised. Ischemic conditions are achieved by complete clamping of blood vessels branching from the uterine vasculature into each individual fetus for designated times followed by removal of the clamps to permit reperfusion. Accumulation of lactic acid in the fetal orain depends on the length of the restriction period, reaching a plateau level of 29 μmol/g tissue at about 30 min. It also depends on the reperfusion time. Thus after a period of 15 min of restriction lactate levels show an increase over the next 30‐min reperfusion to a value of 25.5 μmol/g followed by a rapid decrease to normal values by 3 h of reperfusion. Restriction of 15 min followed by reperfusion of 45 min causes an elevation of prostaglandin E2 (PGE2) level from 12.4 ± 0.86 ng/g to 21.1 ± 0.6 ng/g (p < 0.001). This elevation in PGE2 level is less apparent after 20 min of restriction. No effects are seen on the level of PGF2α. Both PGE2 and PGF2α accumulate in vitro in a time‐dependent manner by brain paniculate fraction. In vitro synthesis of both PGE2 and PGF2α is inhibited by indomethacin (100% and 68%, respectively) and AA861 (94% and 76%, respectively). BW755c and nordihydro‐guaiaretic acid do not affect PGE2 formation but enhance PGF2a production by 112% and 152%, respectively. Paniculate fractions from restricted brain produce less PGF2α than control brains (6.38 ± 1.62 versus 11.43 ± 2.2, p < 0.01). The new experimental model should be adequate for studying the consequences of perinatal ischemia on the developing brain.
Partial Purification and Characterization of Detergent‐Solubilized N‐Sulfotransferase Activity Associated with Calf Brain Microsomes
Authors: Miller R. Robert , Waechter Charles J.
Published: October 2006

Calf brain 3′‐phosphoadenosine 5′‐phosphosul‐fate (PAPS):proteoheparan sulfate (PHS) N‐sulfotransfer‐ase activity is solubilized by extracting salt‐washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)‐Sephacel, (2) heparin‐Sepharose CL‐6B, and (3) 3′,5′‐ADP‐agarose as chromato‐graphic supports. Sulfotransferase activity was followed by using 3′‐phosphoadenosine 5′‐phospho[35S]sulfate and endogenous acceptors in heat‐inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE‐Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N‐sulfotransferase fractions of 22– to 29‐fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89‐fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5–8. Apparent Km values for PAPS of 2.3 μM and 0.9 μM have been determined for ST1 and ST2, respectively. ST1 exhibits N‐sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N‐ and O‐sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and ly‐sophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N‐sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N‐sulfation and O‐sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.
Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle
Authors: Tsim Karl W. K. , Randall William R. , Barnard Eric A.
Published: October 2006

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1‐day‐old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110‐kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72‐kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58‐kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110‐kDa subunit were reactive with it in im‐munoblots. Sucrose density gradient analysis of the antibody‐enzyme complexes showed that the anti‐110‐kDa subunit mAbs cross‐link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2–3S increase in the sedimentation constant with the mAbs specific for the 72‐kDa or for the 58‐kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross‐linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross‐reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.
Regulation of Transmitter γ‐Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ‐Vinyl GABA and Hypoglycemia
Authors: Paulsen R. E. , Fonnum F.
Published: October 2006

Abstract: The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of γ‐aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of γ‐vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half‐maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.
[3H]Desipramine Binding to Rat Brain Tissue: Binding to Both Noradrenaline Uptake Sites and Sites Not Related to Noradrenaline Neurons
Authors: Bäckström Inger T. , Ross Svante B. , Marcusson Jan O.
Published: October 2006

Abstract The pharmacological and biochemical characteristics of [3H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [3H]desipramine binding site. Most of the noradrenaline‐sensitive binding represented a high‐affinity site, and this site appeared to be the same as the high‐affinity site of nisoxetine‐sensitive binding. The [3H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 μM nisoxetine, the binding was saturable and fitted a single‐site binding model with a binding affinity of ∼1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromo‐benzylamine (DSP4). In contrast, when 10 μM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two‐site binding model with KD values of 0.5 and >100 nM for the high‐ and low‐affinity components, respectively. The high‐affinity site was abolished after DSP4 lesioning, whereas the low‐affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 μM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60‐90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high‐affinity sites correlated significantly with the regional distribution of [3H]noradrenaline uptake but not with 5‐[3H]hydroxytryptamine uptake. The low‐affinity sites did not correlate with the regional distribution of [3H]noradrenaline uptake. Drug inhibition studies showed that noradrenaline inhibits the binding defined by 0.1 μM nisoxetine in a competitive manner. Together, these findings suggest that only a small fraction of the [3H]desipramine binding can be regarded as “specific” binding, and this binding fraction may represent the substrate recognition site for noradrenaline uptake. Assuming that one molecule of desipramine binds to each carrier molecule, the turnover number for the noradrenaline carrier was calculated to be 20/min, i.e., the duration of one transport cycle was 3 s.
Use of Calcium Antagonism for the Characterization of Drug‐Evoked Dopamine Release from the Brain of Conscious Rats Determined by Microdialysis
Authors: Westerink B. H. C. , Hofsteede R. M. , Tuntler J. , de Vries J. B.
Published: October 2006

Abstract: Dopamine was determined by microdialysis of the striatum of conscious rats. We investigated whether the release of dopamine, induced by nine different pharmacological treatments, was sensitive to calcium antagonism. Calcium antagonism was determined by Mg2+ or Cd2+ infusion. The following conditions were investigated: haloperidol, haloper‐idol plus GBR 12909, nomifensine, (+)‐amphetamine (all administered intraperitoneally), KC1, l‐methyl‐4‐phenyl‐pyridinium ion (MPP+), glutamate, ouabain, and 120 mmol/ L magnesium (all applied by infusion through the dialysis membrane). The results on calcium antagonism were combined with data on tetrodotoxin (TTX) sensitivity. With the combined data, three different types of dopamine release were characterized. First, action potential‐dependent dopamine release was observed in animals treated with saline, haloperidol, haloperidol plus GBR 12909, nomifensine, and ouabain. Second, action potential‐independent release was established in the case of (+)‐amphetamine, glutamate, MPP+, and 120 mmol/L Mg2+. Finally, K+‐induced dopamine release was classified as TTX independent and calcium dependent. It is concluded that brain dialysis is a powerful method for differentiating between different types of neurotransmitter release.
In Vivo and In Vitro Expression of Gangliosides in Chick Retina Müller Cells
Authors: Gravotta Diego , Landa Carlos A. , Panzetta Pedro , Maccioni Hugo J. F.
Published: October 2006

Abstract: The expression of gangliosides of the lactosylcer‐amide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double‐color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl‐GTC) was detected using the cholera toxin‐cholera toxin antibody experimental approach, after conversion of polysialosyl‐GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6‐day‐old embryos (R6), most cells (∼80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron‐like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13‐day‐old embryo (R13) or 1‐day‐old hatched chick retina (Rpl) expressed GD3, GM1, and polysialosyl‐GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC. In addition, it was concluded that in contrast to retinal neurons, which express GTC in vitro, flat, Müller‐derived cells either lack (R6 cell cultures) or are unable to maintain (R13 cell cultures) the expression of GTC, observations suggesting a dependence of these cells on environmental conditions for expression of these gangliosides.
Decreased Fatty Acylation of Myelin Proteolipid Protein in the Twitcher Mouse
Authors: Yoshimura Takeo , Kobayashi Takuro , Mitsuo Kunihiko , Goto Ikuo
Published: October 2006

Abstract: We examined chronological changes of myelin proteins of the brainstem and spinal cord of the twitcher mouse (15, 20, and 30 days old), a murine model of human globoid cell leukodystrophy caused by a genetic deficiency of galactosylceramidase I activity. The yield of myelin was normal until postnatal day 20, whereas galactosylsphingosine (psychosine) accumulated with age in myelin. The protein profiles of myelin and the activity of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in the myelin remained normal throughout the experimental period. Fatty acylation of proteolipid protein (PLP) was examined in a cell‐free system by incubation of myelin with [3H]palmitic acid, CoA, and ATP, and was normal at postnatal day 15, but decreased after postnatal day 20. Decreased fatty acylation of PLP was also observed in the twitcher mouse at postnatal day 20 when the isolated myelin was incubated with [14C]palmitoyl‐CoA in the absence of ATP and CoA, or the slices of brainstem and spinal cord were incubated with [3H]palmitic acid. The activity of fatty acid: CoA ligase was reduced in myelin. These data suggest that decreased acylation of PLP in twitcher mouse myelin is probably due to reduced activities for both activation and transfer of fatty acid into PLP and that metabolic disturbance is present in myelin because acylation of PLP has been shown to occur in myelin membrane. Although psychosine (200 μM) inhibited only 17% of the acylation in vitro, it may be responsible for the reduced acylation of PLP in vivo.
Concentrations of Tryptoline and Methtryptoline in Rat Brain
Authors: Peura Pekka , Johnson Jodie V. , Yost Richard A. , Faull Kym F.
Published: October 2006

Abstract: Combined gas chromatography‐mass spectrometry and gas chromatography‐tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5‐hydroxytryptoline, and 5‐hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptohne were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neu‐roanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5‐hydroxytryptoline and 5‐hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated‐5‐hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5‐hydroxytryptoline and 5‐hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.
Catecholamine Content Changes in Brain Regions of Spontaneously Hypertensive Rats Under Immobilization Stress
Authors: Nomura Masahiko , Okamura Kunihiro
Published: October 2006

Abstract: We compared the effect of immobilization stress on noradrenaline (NA) and 3‐methoxy‐4‐hydroxyphenyleth‐yleneglycol (MHPG) content in two brain regions–diencephalon and pons‐medulla oblongata–in young and adult spontaneously hypertensive rats (SHR) and Wistar‐Kyoto rats (WKY). In SHR, NA content decreased with time after the onset of the stress, whereas levels of its metabolite MHPG increased. In WKY, NA and MHPG showed no change. The MHPG/NA ratio in both regions increased relative to the duration of the stress in SHR, whereas it remained almost constant in WKY. The rate of increase in the ratio was much higher in the diencephalon of adult (12‐week‐old) than of young (4‐week‐old) SHR. In SHR, NA turnover in the brain is readily affected by environmental stress, and these changes in the noradrenergic system may induce or sustain hypertension.
Characterization of [3H]Quipazine Binding to 5‐Hydroxytryptamine3 Receptors in Rat Brain Membranes
Authors: Milburn Christina M. , Peroutka Stephen J.
Published: October 2006

Abstract: [3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5‐hydroxytryptamine3 (5‐HT3) receptor. The radioligand binds with high affinity (KD, 1.2 ± 0.1 nM) to a saturable population of sites (Bmax, 3.0 ± 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205–930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5‐HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5‐HT3 receptor in the rat CNS.