bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfection of N‐Methyl‐d‐Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death
Authors: Anegawa Norifusa J. , Lynch David R. , Verdoorn Todd A. , Pritchett Dolan B.
Published: July 2010

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 mM) of the neurotransmitter l‐glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N‐methyl‐d‐aspartate (NMDA) receptors and the consequent Ca2+ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca2+ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca2+ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca2+ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron‐specific component of excitotoxicity.
Prolonged Time Course of Glutamate Release from Nerve Terminals: Relationship Between Stimulus Duration and the Secretory Event
Authors: Turner Timothy J. , Dunlap Kathleen
Published: July 2010

Abstract: The kinetics of synaptosomal [3H]glutamate release were measured on a subsecond time scale to study the relationship between the length of depolarization and the duration of the secretory event. The time course of release evoked by elevated K+ was complex, proceeding for several seconds after a 200‐ms depolarization. We developed a protocol for depolarizing excitable membranes on a millisecond time scale to deliver brief depolarizations, termed the synthetic action potential, by using batrachotoxin to activate Na+ channels. Depolarization is achieved by superfusing with solutions containing elevated concentrations of Na+, and the duration of the depolarization is limited by including tetrodotoxin (TTX) in the superfusion solution to block Na+ entry. Direct measurements of the time courses of Na+ current and membrane depolarizations were made in batrachotoxin‐treated sensory neurons using patch clamp recording methods. Rapid increases in Na+ and TTX concentrations produced transient increases in inward Na+ current that decayed with a time course proportional to TTX concentration. Current clamp measurements indicated that, with 10 µM TTX, depolarizations last ∼30 ms. Nonetheless, synaptosomal release of [3H]glutamate triggered by the synthetic action potential remained prolonged. Brief neuronal action potentials at some synapses may trigger transmitter release that persists for several seconds.
Adenosine A1 Agonists and the Ca2+ Channel Agonist Bay K 8644 Produce a Synergistic Stimulation of the GTPase Activity of Go in Rat Frontal Cortical Membranes
Authors: Sweeney M. I. , Dolphin A. C.
Published: July 2010

Abstract: The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)‐Phenylisopropyladenosine, 2‐chloroadenosine, and N‐ethylcarboxamideadenosine produced a concentration‐dependent stimulation of GTPase. At 10−5M, the increase above basal GTPase in frontal cortex was 25 ± 4, 20 ± 3, and 8 ± 1%, respectively, and in the cerebellum 55 ± 2, 41 ± 4, and 22 ± 2%, respectively. The effects of (R)‐phenylisopropyladenosine and 2‐chloroadenosine were inhibited by (1) A1 antagonists (76–96% reduction), (2) pretreatment with pertussis toxin (90–100% reduction), and (3) antibodies raised against the α‐subunit of Gi and Go (55–57% reduction by each), suggesting that A1 receptors interact equally with Gi and Go. (R)‐Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin‐sensitive manner, indicative of activation of Gi or Go. Previously, (±)‐Bay K 8644 enhanced GTP hydrolysis by Go but not Gi. Now we report a profound synergistic stimulation of GTPase in the presence of (R)‐phenylisopropyladenosine and (±)‐Bay K 8644 (10−7 to 10−5M). (±)‐Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate Go. It appears that a positive cooperative stimulation of Go occurs when it is first activated by A1 receptors and subsequently interacts with the L‐type Ca2+ channel.
Roles for Nitric Oxide as an Intra‐ and Interneuronal Messenger at NMDA Release‐Regulating Receptors: Evidence from Studies of the NMDA‐Evoked Release of [3H]Noradrenaline and d‐[3H]Aspartate from Rat Hippocampal Slices
Authors: Jones Nicole M. , Loiacono Richard E. , Beart Philip M.
Published: July 2010

Abstract: N‐Methyl‐d‐aspartate (NMDA) receptors regulating the release of [3H]noradrenaline ([3H]NA) and d‐[3H]aspartate (d‐[3H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg2+‐free Krebs‐Henseleit buffer, the NMDA‐evoked release of [3H]NA and d‐[3H]Asp was Ca2+ dependent and inhibited by the NMDA antagonist (±)‐3‐(2‐carboxypiperazin‐4‐yl)propenyl‐1‐phosphonic acid. NMDA‐stimulated release of [3H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d‐[3H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d‐[3H]Asp appear to be presynaptic, whereas those for [3H]NA are extrasynaptic in location. l‐Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA‐evoked release of [3H]NA (100%) and d‐[3H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3‐morpholinosyndnomine, and S‐nitroso‐N‐acetylpenicillamine (all 100 µM)—stimulated the NMDA‐evoked release. An exception was the inhibition by nitroprusside of NMDA‐evoked release of [3H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (NG‐nitro‐, NG‐methyl‐, and NG‐amino‐l‐arginine, all 100 µM) attenuated (50–80%) the NMDA‐stimulated release of [3H]NA and d‐[3H]Asp, as did KN‐62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release‐regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.
Involvement of Bidirectional Adenosine Transporters in the Release of l‐[3H]Adenosine from Rat Brain Synaptosomal Preparations
Authors: Gu J. G. , Foga I. O. , Parkinson F. E. , Geiger J. D.
Published: July 2010

Abstract: Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l‐[3H]adenosine, our objectives here were to determine (1) if l‐[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d‐adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l‐[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l‐[3H]Adenosine release was inhibited dose‐dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l‐[3H]adenosine alone or l‐[3H]adenosine plus unlabeled l‐adenosine, d‐adenosine, or uridine, l‐[3H]‐adenosine release was inhibited 42% by l‐adenosine, 69% by uridine, and 81% by d‐adenosine. The inhibition of l‐[3H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.
Differential Coupling of D1 and D5 Dopamine Receptors to Guanine Nucleotide Binding Proteins in Transfected GH4C1 Rat Somatomammotrophic Cells
Authors: Kimura Kazuhiro , Sela Shifra , Bouvier Claudia , Grandy David K. , Sidhu Anita
Published: July 2010

Abstract: D1 and D5 dopamine receptor genes, stably expressed in GH4C1 rat somatomammotrophic cells, display identical binding values and stimulate adenylate cyclase. Approximately 60% of D1 receptors were in the agonist high‐affinity state and were converted to the low‐affinity state by 100 µM guanyl‐5′‐ylimidodiphosphate [Gpp(NH)p]. Of the 48% of D5 receptors in the high‐affinity state, only half were modulated by 100 µM Gpp(NH)p; in the presence of the G protein activator, AlF4−, the high‐affinity sites of D5 receptors were abolished by Gpp(NH)p, suggesting tight coupling between D5 receptors and G proteins. The high‐affinity sites of D1, but not D5, receptors were reduced after pertussis toxin treatment of cells. Thus, whereas D1 receptors in GH4C1 cells couple to both Gs, the G stimulatory protein, and a pertussis toxin‐sensitive G protein, D5 receptors couple to Gs and a pertussis toxin‐insensitive G protein. Neither D1 nor D5 receptors were able to stimulate phosphoinositide metabolism in these cells. The ability of D5, but not D1, receptors to couple to novel G proteins may be significant in assigning a functional role for these receptors.
Ca2+/Calmodulin Kinase II Translocates in a Hippocampal Slice Model of Ischemia
Authors: Kolb Stephen J. , Hudmon Andy , Waxham M. Neal
Published: July 2010

Abstract: Rat hippocampal slices were exposed to conditions that simulate an ischemic insult, and the subcellular distribution and the enzymatic activity of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase) were monitored. Semiquantitative western blots using a monoclonal antibody to the 50‐kDa α subunit showed that there was a significant redistribution of the enzyme from a supernatant to a pellet fraction after 10 min of an anoxic/aglycemic insult. No significant change in the total amount of CaM kinase enzyme was detected in the homogenates for up to 20 min of exposure to the insult. Ca2+/CaM‐dependent enzyme activity did not significantly change in the pellet during the 20‐min insult. Supernatant activity decreased throughout the insult. The persistence of Ca2+/CaM‐dependent CaM kinase activity in the pellet fraction and the detected movement of enzyme from the supernatant to the pellet indicate that redistribution may be an important mechanism in regulating the cellular location of CaM kinase activity.
Changes of Respiratory Chain Activity in Mitochondrial and Synaptosomal Fractions Isolated from the Gerbil Brain After Graded Ischaemia
Authors: Allen Kathryn L. , Almeida Angeles , Bates Timothy E. , Clark John B.
Published: July 2010

Abstract: In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II–III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30‐min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD‐linked and FAD‐linked substrates, was initially affected at CBF values of ∼35 ml 100 g−1 min−1, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below ∼30 ml 100 g−1 min−1 (mitochondria and synaptosomes) and complex II–III activities decreased at blood flows below 20 ml 100 g−1 min−1 (mitochondria) and 35–30 ml 100 g−1 min−1 (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15–10 ml 100 g−1 min−1 (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.
Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study
Authors: Massieu Lourdes , Morales‐Villagrán Alberto , Tapia Ricardo
Published: July 2010

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high‐affinity sodium‐dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl‐threo‐β‐hydroxyaspartate and l‐aspartate‐β‐hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate or by intrastriatal coinjection of 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.
Regulation of Endogenous ADP‐Ribosylation by Acute and Chronic Lithium in Rat Brain
Authors: Nestler Eric J. , Terwilliger Rose Z. , Duman Ronald S.
Published: July 2010

Abstract: In the present study, we investigated the effects of lithium on endogenous ADP‐ribosylation in rat brain. It was found that addition of lithium in vitro inhibits endogenous ADP‐ribosylation activity in extracts of frontal cortex at therapeutically relevant concentrations. Inhibition is observed at concentrations as low as 0.3 mM and is maximal at 1 mM when 50% inhibition is obtained. A similar degree of inhibition of endogenous ADP‐ribosylation was observed for all substrate proteins identified, including Gsα, suggesting that lithium's effect may be achieved at the level of ADP‐ribosyltransferases and not specific substrate proteins. In contrast to lithium, chloride salts of sodium and potassium do not alter endogenous ADP‐ribosylation activity in frontal cortex. To assess the possible in vivo relevance of this in vitro action of lithium, we studied the effect of chronic lithium administration on levels of endogenous ADP‐ribosylation in frontal cortex. It was found that chronic lithium treatment, in contrast to the inhibitory effect of the drug in vitro, produced a >35% increase in endogenous ADP‐ribosylation activity. A similar degree of increase was observed for all of the substrate proteins identified. These novel findings raise the possibility that certain endogenous ADP‐ribosyltransferases are among the acute targets of lithium in the brain and that adaptations in these enzymes may be part of the mechanisms underlying lithium's long‐term effects on brain function.
Novel Reticular Calcium Binding Protein Is Purified on Taipoxin Columns
Authors: Dodds D'Nette , Schlimgen Anne K. , Lu Szu‐Yao , Perin Mark S.
Published: July 2010

Abstract: We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute‐phase proteins. Here we report the identification of the second protein as a 49‐kDa lumenal calcium binding protein that we have named taipoxin‐associated calcium binding protein 49 (TCBP‐49). This protein contains six EF‐hand putative calcium binding domains and the carboxyl‐terminal sequence His‐Asp‐Glu‐Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP‐49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP‐49 will allow direct tests of whether TCBP‐49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.
Purification, Properties, and Specificity of Rat Brain Cytosolic Fatty Acyl Coenzyme A Hydrolase
Authors: Broustas Constantinos G. , Hajra Amiya K.
Published: July 2010

Abstract: Rat brain cytosolic acyl‐CoA hydrolase has been purified 3,500‐fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high‐performance size‐exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl‐CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)‐CoA. For the purified enzyme the Km for palmitoyl‐CoA is 5.8 µM and the Vmax is 1,300 µmol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p‐hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine‐reacting agent, but not by phenylmethylsulfonyl fluoride (10 mM), a serine esterase inhibitor. The purified enzyme does not appear to possess any O‐ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.
The β‐Subunit of Caenorhabditis elegans Avermectin Receptor Responds to Glycine and Is Encoded by Chromosome 1
Authors: Laughton D. L. , Wheeler S. V. , Lunt G. G. , Wolstenholme A. J.
Published: July 2010

Abstract: A full‐length cDNA encoding the β subunit of the recently described avermectin receptor was amplified from Caenorhabditis elegans mRNA. When this cDNA was injected into Xenopus oocytes a dose‐dependent response to glycine was observed, together with a smaller response to 1 mM GABA. The EC50 of the glycine response was similar to that described previously for glutamate (0.38 mM). Hybridisation of the cDNA to polytene filters identified three yeast artificial chromosome clones that gave a positive signal, Y37B3, Y38E5, and Y24C9, all of which are mapped to chromosome 1. Hybridisation to a series of cosmid clones covering this area further mapped the gene encoding this subunit to the region −2,818 to −2,824.
Exposure to an Antisense Oligonucleotide Decreases Corticotropin‐Releasing Factor Receptor Binding in Rat Pituitary Cultures
Authors: Owens Michael J. , Mulchahey Jeff J. , Kasckow John W. , Plotsky Paul M. , Nemeroff Charles B.
Published: July 2010

Abstract: Corticotropin‐releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of “antisense knockdown” strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15‐mer antisense (5′ CTG‐CGG‐GCG‐CCG‐TCC 3′) and “scrambled” control (5′ CGT‐CCG‐CGC‐GCT‐GCG 3′) oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in 125I‐ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of “scrambled” oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30‐min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.
Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis
Authors: Bowling Allen C. , Barkowski Elizabeth E. , McKenna‐Yasek D. , Sapp Peter , Horvitz H. Robert , Beal M. Flint , Brown Robert H.
Published: July 2010

Abstract: Some cases of autosomal‐dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1, the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.
Activation of arc, a Putative “Effector” Immediate Early Gene, by Cocaine in Rat Brain
Authors: Fosnaugh Jennifer S. , Bhat Ratan V. , Yamagata Kanato , Worley Paul F. , Baraban Jay M.
Published: July 2010

Abstract: As immediate early genes (IEGs) are thought to play a critical role in mediating stimulus‐induced neuronal plasticity, several laboratories have characterized the IEG response induced by cocaine to help define the changes in gene expression that may underlie its long‐lasting behavioral effects. Although activation of several transcription factor IEGs has been described, little is known about which “effector” IEGs, if any, are also induced. In the present study, we have examined whether cocaine administration affects expression of a recently identified “effector” IEG, referred to as arc (activity‐regulated, cytoskeleton‐associated). This IEG encodes a protein with homology to spectrin that appears to be associated with the actin cytoskeleton. Using in situ hybridization, we have found that systemic cocaine administration elicits a robust, transient rise in arc mRNA levels in striatum, which is suppressed by D1 dopamine receptor blockade, reserpine treatment, or striatal 6‐hydroxydopamine lesions. D2 receptor antagonists triggered arc expression when administered alone. Immunohistochemical studies indicated that Arc protein induced by cocaine is expressed in neuronal cell bodies and dendrites. As Arc appears to be a component of the neuronal cytoskeleton, it may be involved in structural alterations underlying neuronal plasticity triggered by cocaine.
Expression of Proteolipid Protein Gene Is Directly Associated with Secretion of a Factor Influencing Oligodendrocyte Development
Authors: Nakao Junji , Yamada Masahisa , Kagawa Tetsushi , Kim Seung U. , Miyao Yasuyoshi , Shimizu Keiji , Mikoshiba Katsuhiko , Ikenaka Kazuhiro
Published: July 2010

Abstract: Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild‐type cells in vivo and in vitro. It is possible that the presence of PLP or DM‐20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM‐20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM‐20‐producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM‐20. By addition of the supernatants from the PLP/DM‐20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.
Hypoxia Alters Iron Homeostasis and Induces Ferritin Synthesis in Oligodendrocytes
Authors: Qi Yan , Jamindar Tejal M. , Dawson Glyn
Published: July 2010

Abstract: Both iron and the major iron‐binding protein ferritin are enriched in oligodendrocytes compared with astrocytes and neurons, but their functional role remains to be determined. Progressive hypoxia dramatically induces the synthesis of ferritin in both neonatal rat oligodendrocytes and a human oligodendroglioma cell line. We now report that the release of iron from either transferrin or ferritin‐bound iron, after a decrease in intracellular pH, also leads to the induction of ferritin synthesis. The hypoxic induction of ferritin synthesis can be blocked either with iron chelators (deferoxamine or phenanthroline) or by preventing intracellular acidification (which is required for the release of transferrin‐bound iron) with weak base treatment (ammonium chloride and amantadine). Two sources of exogenous iron (hemin and ferric ammonium citrate) were able to stimulate ferritin synthesis in both oligodendrocytes and HOG in the absence of hypoxia. This was not additive to the hypoxic stimulation, suggesting a common mechanism. We also show that ferritin induction may require intracellular free radical formation because hypoxia‐mediated ferritin synthesis can be further enhanced by cotreatment with hydrogen peroxide. This in turn was blocked by the addition of exogenous catalase to the culture medium. Our data suggest that disruption of intracellular free iron homeostasis is an early event in hypoxic oligodendrocytes and that ferritin may serve as an iron sequestrator and antioxidant to protect cells from subsequent iron‐catalyzed lipid peroxidation injury.
Rapid Desensitization of Serotonin 5‐HT2C Receptor‐Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5‐HT2C Receptors
Authors: Akiyoshi Jotaro , Nishizono Akira , Yamada Kenji , Nagayama Haruo , Mifune Kumato , Fujii Isao
Published: July 2010

Abstract: Serotonin 5‐HT2C receptor‐mediated intracellular Ca2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5‐HT2C receptors. Fura‐2 acetoxymethyl ester was used to investigate the regulation of 5‐HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5‐HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin‐sensitive [3H]mesulergine binding (KD = 0.49 nM). The addition of 5‐HT mobilized intracellular Ca2+ in a dose‐dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC50 value for 5‐HT of 0.029 µM. Exposure to 5‐HT, 1‐(3‐chlorophenyl)piperazine dihydrochloride (a 5‐HT2C agonist), and 1‐(4‐iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane (a 5‐HT2C and 5‐HT2A agonist) resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5‐HT‐mediated intracellular Ca2+ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5‐HT in a time‐ and concentration‐dependent manner. Mezerein and phorbol 12‐myristate 13‐acetate, protein kinase C activators, weakly inhibited the intracellular Ca2+ mobilization induced by 10 µM 5‐HT. Furthermore, the protein kinase C inhibitor H‐7 partially prevented the protein kinase C activator‐induced inhibition of the 5‐HT‐mediated increase in intracellular Ca2+ concentration. The desensitization induced by pretreatment with 5‐HT was blocked by W‐7, added in conjunction with 5‐HT, and partially inhibited by W‐5, a nonselective inhibitor of protein kinases and weak analogue of W‐7. Therefore, the 5‐HT2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5‐HT2C receptor activation mobilizes Ca2+ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase‐mediated feedback.
Pharmacology of Sodium‐Dependent High‐Affinity l‐[3H]Glutamate Transport in Glial Cultures
Authors: Garlin Amy B. , Sinor Amy D. , Sinor Jeroo D. , Jee Sandy H. , Grinspan Judith B. , Robinson Michael B.
Published: July 2010

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium‐dependent high‐affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l‐[3H]Glu transport were characterized using astrocyte‐enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium‐dependent transport were consistent with a single site with Km values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ∼40 excitatory amino acid analogues for inhibition of sodium‐dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l‐[3H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)‐2‐(carboxycyclopropyl)glycine and l‐α‐aminoadipate, all of the compounds examined were ∼10–200‐fold less potent as inhibitors of l‐[3H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.
The Relationship Between Plasma and Brain Quinolinic Acid Levels and the Severity of Hepatic Encephalopathy in Animal Models of Fulminant Hepatic Failure
Authors: Basile Anthony S. , Saito Kuniaki , Li Yong , Heyes Melvyn P.
Published: July 2010

Abstract: Quinolinic acid is an excitatory, neurotoxic tryptophan metabolite proposed to play a role in the pathogenesis of hepatic encephalopathy. This involvement was investigated in rat and rabbit models of fulminant hepatic failure at different stages of hepatic encephalopathy. Although plasma and brain tryptophan levels were significantly increased in all stages of hepatic encephalopathy, quinolinic acid levels increased three‐ to sevenfold only in the plasma, CSF, and brain regions of animals in stage IV hepatic encephalopathy. Plasma‐CSF and plasma‐brain quinolinic acid levels in rats and rabbits with fulminant hepatic failure were strongly correlated, with CSF and brain concentrations ∼10% those of plasma levels. Moreover, there was no significant regional difference in brain quinolinic acid concentrations in either model. Extrahepatic indoleamine‐2,3‐dioxygenase activity was not altered in rats in stage IV hepatic encephalopathy, but hepatic l‐tryptophan‐2,3‐dioxygenase activity was increased. These results suggest that quinolinic acid synthesized in the liver enters the plasma and then accumulates in the CNS after crossing a permeabilized blood‐brain barrier in the end stages of liver failure. Furthermore, the observation of low brain concentrations of quinolinic acid only in stage IV encephalopathy suggests that the contribution of quinolinic acid to the pathogenesis of hepatic encephalopathy in these animal models is minor.
Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line
Authors: Bold Richard J. , Ishizuka Jin , Rajaraman Srinivasan , Perez‐Polo J. Regino , Townsend Courtney M. , Thompson James C.
Published: July 2010

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75LNGFR and p140trkA) and membrane‐associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor‐linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K‐252a and specific anti‐NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.
Neuronal Cyclin‐Dependent Kinase‐5 Phosphorylation Sites in Neurofilament Protein (NF‐H) Are Dephosphorylated by Protein Phosphatase 2A
Authors: Veeranna  , Shetty K. T. , Link W. T. , Jaffe H. , Wang J. , Pant Harish C.
Published: July 2010

Abstract: Neurofilament (NF) protein [high molecular mass (NF‐H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys‐Ser‐Pro) repeat motifs. There are two major types of KSP motifs in the NF‐H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2‐like kinase [cyclin‐dependent kinase‐5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF‐H and lysine‐rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF‐H in a NF preparation, and a bacterially expressed C‐terminal tail domain of NF‐H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks—P1, P2, and P3—were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5‐phosphorylated 32P‐histone (H1), 32P‐NF‐H in the NF preparation, and 32P‐NF‐H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A‐like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF‐H.
Regulation of Multiple Effectors by the Cloned δ‐Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase
Authors: Tsu Rachel C. , Chan Joy S. C. , Wong Yung H.
Published: July 2010

Abstract: The δ‐opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ‐opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ‐selective agonist [d‐Pen2,d‐Pen5]enkephalin. The inhibitory response of [d‐Pen2,d‐Pen5]enkephalin was dependent on the expression of the δ‐opioid receptor and exhibited an EC50 of 1 nM. The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ‐opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ‐opioid receptor with type II adenylyl cyclase and an activated mutant of αs converted the δ‐opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk− fibroblasts, the cloned δ‐opioid receptor was able to stimulate the formation of inositol phosphates (EC50 = 8 nM). This response was sensitive to pertussis toxin. The opioid‐mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ‐opioid receptor to couple to G proteins other than Gi was also examined. Cotransfection studies revealed that the δ‐opioid receptor can utilize Gz to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.
Characterization and Subcellular Localization of l‐[3H]Carnitine Binding Sites in Rat Brain
Authors: Vesci Loredana , Tobia Patrizia , Corsico Nerina , Martelli Edoardo Arrigoni , Arduini Arduino
Published: July 2010

Abstract: In the present study, we investigated the existence of a binding site for l‐carnitine in the rat brain. In crude synaptic membranes, l‐[3H]carnitine bound with relatively high affinity (KD = 281 nM) and in a saturable manner to a finite number (apparent Bmax value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a Koff of 0.018 min−1 and a Kon of 0.187 × 10−3 min−1 nM−1. Binding was highest in spinal cord, followed by medulla oblongata‐pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l‐[3H]Carnitine binding was stereoselective for the l‐isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l‐[3H]carnitine binding was l‐carnitine followed by propionyl‐l‐carnitine. Acetyl‐l‐carnitine and isobutyryl‐l‐carnitine showed an affinity ∼500‐fold lower than that obtained for l‐carnitine. The precursor γ‐butyrobetaine had negligible activity at 0.1 mM. l‐Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5‐hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl‐carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l‐carnitine membrane binding sites whereas l‐carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l‐[3H]carnitine binding site should be essentially presynaptic.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase‐Activating Polypeptide Potentiate c‐fos Expression Induced by Glutamate in Cultured Cortical Neurons
Authors: Martin Jean‐Luc , Gasser Didier , Magistretti Pierre J.
Published: July 2010

Abstract: Previous reports have demonstrated that glutamate stimulates c‐fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c‐fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK‐801, therefore indicating that VIP stimulates c‐fos expression in a glutamate‐dependent manner. A similar effect was observed with pituitary adenylate cyclase‐activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate‐dependent stimulation of c‐fos expression evoked by VIP, because either H‐89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a “biochemical AND gate” mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c‐fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c‐fos expression, suggesting that both peptides can increase the “throughput” or “strength” of glutamate‐containing circuits in the cerebral cortex.
Differential Regulation of D1 Dopamine Receptor‐ and of A2a Adenosine Receptor‐Stimulated Adenylyl Cyclase by μ‐, δ1‐, and δ2‐Opioid Agonists in Rat Caudate Putamen
Authors: Noble Florence , Cox Brian M.
Published: July 2010

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D1 dopamine or A2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D1 dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ‐[Tyr‐d‐Ala‐Gly‐(N‐Me)Phe‐Gly‐ol (DAMGO)], δ1‐[Tyr‐d‐Pen‐Gly‐Phe‐d‐Pen (DPDPE)], and δ2‐ ([d‐Ala2]deltorphin‐II [DT‐II]) opioid agonists on the D1 dopamine receptor‐ and A2a adenosine receptor‐stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT‐II inhibit forskolin‐stimulated adenylyl cyclase [selectively antagonized by d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP; μ antagonist), 7‐benzylidenenaltrexone (BNTX; δ1 antagonist), and naltriben (NTB; δ2 antagonist), respectively], but only μ‐ and δ2‐opioid agonists inhibit D1 dopamine‐stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT‐II and DPDPE inhibit A2a adenosine‐stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A2a adenosine‐stimulated adenylyl cyclase activity. These results suggest that μ‐, δ1‐, and δ2‐opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ1 and δ2 subtypes. μ‐Opioid receptors may be preferentially expressed by striatonigral neurons, δ1‐ by striatopallidal neurons, and δ2‐ by these two striatal efferent neuron populations.
Ethanol Inhibition of Recombinant Heteromeric NMDA Channels in the Presence and Absence of Modulators
Authors: Chu Benson , Anantharam Vellareddy , Treistman Steven N.
Published: July 2010

Abstract: The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25–100 mM. We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady‐state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg2+ (3 and 12.5 µM), Zn2+ (1 and 10 µM), or the glycine antagonist 7‐chlorokynurenic acid (1.25 or 5 µM), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 mM) or 5,5′‐dithiobis(2‐nitrobenzoic acid) (1 mM) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor.