bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface Expression of the AMPA Receptor Subunits GluR1, GluR2, and GluR4 in Stably Transfected Baby Hamster Kidney Cells
Authors: Hall Randy A. , Hansen Annette , Andersen Peter H. , Soderling Thomas R.
Published: July 2010

Abstract: The surface expression of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)‐570 cells. Two methods were used to quantify surface expression: cross‐linking with the membrane‐impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane‐impermeant biotinylating reagent sulfosuccinimidyl 2‐(biotinamido)ethyl‐1,3‐dithiopropionate (NHS‐ss‐biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25–40% of the total GluR population is located in the plasma membrane of the BHK‐570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR‐expressing BHK‐570 cells performed via the biotinylation/precipitation method; these studies revealed that 30–40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10–20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.
Neuroprotective Effect of the Iron Chelator Desferrioxamine Against MPP+ Toxicity on Striatal Dopaminergic Terminals
Authors: Santiago M. , Matarredona E. R. , Granero L. , Cano J. , Machado A.
Published: July 2010

Abstract: Microdialysis was used to evaluate the effect of desferrioxamine (DES) against 1‐methyl‐4‐phenylpyridinium (MPP+) toxicity. The presence of DES (40 fmol‐40 nmol/15 min for a total of 90 min) in the Ringer solution, coperfused with MPP+ (40 nmol/15 min) on day 1, produced on day 2 a higher extracellular dopamine output after perfusion of MPP+ than in control MPP+ perfusion experiments, in which no DES was administered on day 1. Both Ringer perfusion alone (control Ringer) and coperfusion of 40 nmol DES with 40 nmol MPP+ on day 1 produced on day 2 similar increases in extracellular dopamine output after a second MPP+ perfusion. In the control Ringer experiment, note that the MPP+ on day 2 is the first MPP+ perfusion. Perfusion of 800 fmol FeCl3/15 min along with 40 nmol MPP+ and 400 fmol DES on day 1 completely abolished on day 2 the neuroprotective effect found with 40 nmol MPP+ and 400 fmol DES; 800 fmol FeCl3 did not increase the neurotoxic effect of 40 nmol MPP+ perfusion. The ability of DES to protect against MPP+ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.
Stimulation of [3H]GABA and β‐[3H]Alanine Release from Rat Brain Slices by cis‐4‐Aminocrotonic Acid
Authors: Chebib Mary , Johnston Graham A. R.
Published: July 2010

Abstract: cis‐4‐Aminocrotonic acid (CACA; 100 µM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β‐[3H]alanine from slices of cerebellum and spinal cord without influencing potassium‐evoked release. In contrast, CACA (100 µM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of d‐[3H]aspartate from slices of cerebellum and did not influence potassium‐evoked release of [3H]taurine from the cerebellum and spinal cord and d‐[3H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β‐alanine release are due to CACA acting as a substrate for a β‐alanine‐sensitive GABA transport system, consistent with CACA inhibiting the uptake of β‐[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against β‐[3H]alanine uptake in the cerebellum was 750 ± 60 µM. CACA appears to be 10‐fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and β‐alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β‐alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT‐3, cloned from rat CNS. The structural similarities of GABA, β‐alanine, CACA, and nipecotic acid are demonstrated by computer‐aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.
Cloning of the Human Puromycin‐Sensitive Aminopeptidase and Evidence for Expression in Neurons
Authors: Tobler A. R. , Constam D. B. , Schmitt‐Gräff A. , Malipiero U. , Schlapbach R. , Fontana A.
Published: July 2010

Abstract: The puromycin‐sensitive aminopeptidase (PSA) is thought to contribute to the degradation of enkephalins. Besides being the most abundant aminopeptidase in the brain, PSA is expressed in other organs as well. From a human fetal brain cDNA library, we have isolated cDNA encoding the human PSA (huPSA) protein. The isolated cDNA gave rise to a protein with a molecular mass of 99 kDa. Compared with mouse PSA, homology at the amino acid and cDNA level was 98 and 93%, respectively. Translation of the huPSA was found to be initiated at the second of two possible start codons, as shown by studies with antibodies directed against peptide sequences of both potential N‐terminal regions. Northern blot analysis with RNA isolated from different human organs demonstrated that the huPSA transcript is strongest but not exclusively expressed in the brain. Vesicular stomatitis virus epitope‐tagged huPSA protein was expressed in HeLa cells and found to be localized in the cytoplasm, especially in the perinuclear region. By in situ hybridization, huPSA transcript could be identified in cortical and cerebellar neurons, whereas glial cells and blood vessels remained negative.
Defective Herpes Simplex Virus Vectors Expressing the Rat Brain Stress‐Inducible Heat Shock Protein 72 Protect Cultured Neurons from Severe Heat Shock
Authors: Fink Sheri L. , Chang Louis K. , Ho Dora Y. , Sapolsky Robert M.
Published: July 2010

Abstract: Recently, preinduction of the heat shock response has been shown to protect CNS neurons undergoing various stressful insults, e.g., heat, ischemia, or exposure to excitotoxins. However, it is not known which of the proteins induced by the heat shock response mediate the protective effects. Previous correlative evidence points to a role for the highly stress‐induced 72‐kDa heat shock protein (hsp72). However, it is not known whether hsp72 expression alone can protect against a range of acute neuronal insults. We constructed a herpes simplex virus‐1 vector carrying the rat brain stress‐inducible hsp72 gene and the Escherichia coli lacZ (marker) gene. Infection with the vector caused hippocampal neurons to coexpress hsp72 and β‐galactosidase. Infection with a control vector led to marker gene expression only. Overexpression of hsp72 protected cultured hippocampal neurons against a heat shock but not against the metabolic toxin 3‐nitropropionic acid or the excitotoxin glutamate. This is the first published report of protection following heat shock protein transfection in CNS neurons.
Induction of Tyrosine Hydroxylase Protein and a Transgene Containing Tyrosine Hydroxylase 5′ Flanking Sequences by Stress in Mouse Adrenal Gland
Authors: Osterhout Cheryl A. , Chikaraishi Dona M. , Tank A. William
Published: July 2010

Abstract: Prolonged stress is associated with the induction of tyrosine hydroxylase (TH) gene expression in rat adrenal medulla. We have used transgenic mice expressing a transgene encoding 4.5 kb of rat TH gene 5′ flanking region fused upstream of the structural gene encoding chloramphenicol acetyltransferase (CAT) to test whether cold exposure or immobilization stress regulates TH gene expression in mouse adrenal gland. Exposure of mice to cold for 3 days increases adrenal TH protein and enzymatic activity. Cold exposure also increases adrenal TH‐CAT expression two‐ to threefold. Immobilization stress induces mouse adrenal TH‐CAT expression after either one immobilization or multiple immobilizations. TH‐CAT expression increases transiently after a single immobilization, but after multiple immobilizations the induction of TH‐CAT is sustained for at least 24 h. TH protein and TH enzymatic activity in mouse adrenal gland are elevated 2.8‐fold and 1.5‐fold, respectively, after seven immobilizations, but are not increased after either one, two, or three immobilizations. These results indicate that cold exposure and immobilization stress induce adrenal TH gene expression at least partially by stimulating the transcription rate of the TH gene. Furthermore, as observed in the rat, multiple mechanisms apparently regulate adrenal TH gene transcription rate and TH enzyme induction, depending on whether mice are subjected to a single immobilization or multiple immobilizations. Our results indicate that these transgenic mice are an excellent model system to study the molecular mechanisms regulating TH gene expression in adrenal medulla.
Ischemia‐Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase
Authors: Parsons J. T. , Churn S. B. , DeLorenzo R. J.
Published: July 2010

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg2+/Ca2+ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (45Ca2+) accumulation in the microsomes mediated by Mg2+/Ca2+ ATPase. Ischemia caused a statistically significant inhibition of presteady‐state and steady‐state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia‐induced inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase.
Angiotensin‐Converting Enzyme Modulates Dopamine Turnover in the Striatum
Authors: Jenkins Trisha A. , Mendelsohn Frederick A. O. , Chai Siew Yeen
Published: July 2010

Abstract: The effect of chronic inhibition of the angiotensin‐converting enzyme on dopamine content and release in the striatum was investigated using in vivo microdialysis in awake, freely moving rats. Rats were treated for 1 week with the angiotensin‐converting enzyme inhibitor perindopril (1 mg/kg) via the drinking water, whereas the controls were given water alone. One week after perindopril treatment, striatal dopamine dialysate levels in the treated group were markedly elevated compared with control values: control, 233 ± 43 pg/ml; perindopril, 635 ± 53 pg/ml (p < 0.001). These results were confirmed by a complementary study in which dopamine content was measured in striatal extracts (3.5 ± 0.4 µg of dopamine/g of tissue for controls compared with 9.2 ± 2.4 µg of dopamine/g of tissue for the treated group; p < 0.05). In the rats that were dialyzed, angiotensin‐converting enzyme levels in the striatum were decreased by 50% after perindopril treatment. Levels of dopamine D1 and D2 receptors and of preprotachykinin and tyrosine hydroxylase mRNAs were unchanged after angiotensin‐converting enzyme inhibition. A small, but significant, increase was detected in striatal preproenkephalin mRNA levels in the angiotensin‐converting enzyme inhibitor‐treated group. These results indicate that peripherally administered angiotensin‐converting enzyme inhibitors penetrate the blood‐brain barrier when given chronically and modulate extracellular dopamine and striatal neuropeptide levels.
Human Serotonin Transporter: Regulation by the Neuroprotective Agent Aurintricarboxylic Acid and by Epidermal Growth Factor
Authors: Kekuda Ramesh , Torres‐Zamorano Viviana , Leibach Frederick H. , Ganapathy Vadivel
Published: July 2010

Abstract: The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonin transporter activity. This effect was not observed, however, when the treatment was done for 1–2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis‐Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady‐state levels of serotonin transporter‐specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180‐kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady‐state levels of the transporter‐specific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.
Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l‐Amino Acid Decarboxylase: Neurochemical Effects
Authors: Kaddis Farida G. , Clarkson Edward D. , Weber Michel J. , Vandenbergh David J. , Donovan David M. , Mallet Jacques , Horellou Philippe , Uhl George R. , Freed Curt R.
Published: July 2010

Abstract: To study the possibility that increasing striatal activity of aromatic l‐amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l‐3,4‐dihydroxyphenylalanine (l‐DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos‐haadc cells) into 6‐hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14CO2 from l‐[14C]DOPA. The Km value for l‐DOPA in intact and disrupted cells was 0.60 and 0.56 mM, respectively. The cofactor, pyridoxal 5‐phosphate, enhanced enzymatic activity with maximal effect at 0.1 mM. The pH optimum for enzyme activity was 6.8. Grafting Cos‐haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l‐DOPA. When measured 2 h after l‐DOPA administration, the mean dopamine level in the striata of Cos‐haadc‐grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos‐ut cells) was 1 µg/g. At 6–8 h after l‐DOPA administration, striatal dopamine content in the Cos‐haadc‐grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos‐ut‐grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l‐DOPA.
Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells
Authors: Suh Byung‐Chang , Son Jin H. , Joh Tong H. , Kim Kyong‐Tai
Published: July 2010

Abstract: We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT‐β) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′‐O‐(3‐thiotriphosphate) ≥ UTP > 2‐chloro‐ATP > 3′‐O‐(4‐benzoyl)benzoyl ATP, GTP ≥ 2‐methylthio ATP, adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β‐methyleneadenosine 5′‐triphosphate, β,γ‐methyleneadenosine 5′‐triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP‐and UTP‐induced [Ca2+]i rise, because the responses depend on the ATP4− and UTP4− concentrations, respectively. The P2U purinoceptor‐selective agonist UTP and the P2Y purinoceptor‐selective agonist ADPβS induce inositol 1,4,5‐trisphosphate generation in a concentration‐dependent manner with maximal effective concentrations of ∼100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5‐trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12‐myristate 13‐acetate inhibits the ADPβS‐induced [Ca2+]i rise more effectively than the ATP‐ and UTP‐induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT‐β cells and that both receptors are linked to phospholipase C.
Definition of a Sequence Unique in βII Spectrin Required for Its Axon‐Specific Interaction with Fodaxin (A60)
Authors: Hayes Nandini V. L. , Phillips Gareth W. , Carden Martin J. , Baines Anthony J.
Published: July 2010

Abstract: Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon‐specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of αI, βI, αII, and βII polypeptides) but not erythrocyte spectrin (αI and βI). Because αII and βII spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain α chains. Bacterially expressed C‐terminal segments 18–19 of βII spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18–19 of the somatodendritic βIΣ2 spectrin showed no interaction with fodaxin. Within βII, fodaxin binding activity was localized to residues 2,087–2,198, which are unique to βII and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of βII and βIΣ2 are candidates to mediate the isotype‐specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of β spectrin isoforms.
Rapid Changes in Local Extracellular Rat Brain Glucose Observed with an In Vivo Glucose Sensor
Authors: Hu Yibai , Wilson George S.
Published: July 2010

Abstract: A needle‐type electrochemically based microsensor for glucose (110 µm o.d.) is described. This sensor, designed for monitoring transient glucose content changes in response to neural stimuli, has a response time of ∼5 s and has been shown to be free of interference from endogenous electroactive species such as ascorbate, urate, and various neurotransmitters. It exhibits linear response to glucose up to 10 mM. The usefulness of the sensor has been demonstrated by examining the time‐dependent interstitial glucose concentration in the rat hippocampus in response to KCl depolarization and by stimulation of glutamate neurons through a perforant pathway. Simultaneous monitoring of oxygen is also carried out and demonstrates that for both oxygen and glucose there is substantial local depletion of both species and that their pools are replenished by increased regional cerebral blood flow. The transient initial rapid (10–13 s) decrease up to 20–34%, observed on a time scale comparable to that for neurotransmitter release, may be involved in a recently suggested astrocytic uptake for glutamate‐stimulated aerobic glycolysis possibly needed to meet energy homeostasis in brain. These studies demonstrate the importance of microsensors in monitoring transient events linked to neuronal stimulation.
Differential Agonist Regulation of the Human κ‐Opioid Receptor
Authors: Blake Allan D. , Bot George , Li Shuixing , Freeman John C. , Reisine Terry
Published: July 2010

Abstract: Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human κ‐opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor‐expressing cells showed specific high‐affinity membrane binding for a κ‐selective opioid, 3H‐labeled (+)‐(5α,7α,8β)‐N‐methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐oxaspiro[4,5]dec‐8‐yl]benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5′‐O‐(3‐thiotriphosphate) reduced [3H]69,593 binding, indicating that the human κ receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor‐mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a κ‐selective antagonist, norbinal‐torphimine. A 3‐h pretreatment with a κ‐selective agonist, (±)‐trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488), caused receptor down‐regulation, whereas no receptor down‐regulation was found after levorphanol pretreatment. U50,488 or dynorphin A1–17 pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A1–17 to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin‐stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human κ receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.
Apoptosis Regulates the Number of Schwann Cells at the Premyelinating Stage
Authors: Nakao Junji , Shinoda Jun , Nakai Yoko , Murase Shin‐ichi , Uyemura Keiichi
Published: July 2010

Abstract: At the premyelinating stage, the Schwann cells of peripheral nerves are able to recognize the axon, to arrange themselves along it in a nonoverlapping manner, and finally to establish a one‐to‐one cell‐axon relationship. The mechanism that regulates these processes is not known in detail. We found the existence of a significant Schwann cell apoptosis in vivo of rat postnatal sciatic nerve, peaking around postnatal day 3. More than 50% of the neonatal Schwann cells cultured in axon‐free medium undergo a rapid apoptosis. The apoptosis can be suppressed by addition of survival factors such as Neu differentiation factors or by increasing the adhesion of Schwann cells to substratum. We suggest that in neonatal nerves in vivo, Schwann cells are highly susceptible to apoptosis, but they are saved from death by contact with axons. The dramatic increase in number of Schwann cells between postnatal day 0 and 3 overcomes the number of axons available for them. Consequently the Schwann cells that fail to contact an axon undergo apoptosis. In conclusion, the number of Schwann cells in the developing nerves is regulated by the apoptosis and clearly depends on the survival signals from axons.
Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation
Authors: Atlante A. , Gagliardi S. , Minervini G. M. , Ciotti M. T. , Marra E. , Calissano P.
Published: July 2010

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2′,7′‐dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µM glutamate pulse administered to 7‐day‐old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O2•−) production are completely inhibited by concomitant incubation of glutamate with MK‐801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate‐triggered, NMDA‐mediated, massive Ca2+ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.
Kainic Acid and Decorticating Lesions Stimulate the Synthesis of C1q Protein in Adult Rat Brain
Authors: Goldsmith Sara K. , Wals Pat , Rozovsky Irina , Morgan Todd E. , Finch Caleb E.
Published: July 2010

Abstract: The first component of the classic complement cascade, C1q, was increased in whole rat brain after lesioning by intraperitoneally injected kainic acid (KA) (20‐fold, 3 days after KA) and in the striatum ipsilateral to unilateral decortication (fivefold, 10 days after decortication). C1q was measured after purification by chromatography and electrophoresis. De novo biosynthesis of C1q 3 days after KA was increased >10‐fold, as measured by the incorporation of [35S]methionine into C1q after incubation of brain slices from KA‐treated rats for 2 h. In parallel with these responses, KA induced fivefold increase of C1q bioactivity, as evaluated with C1q‐dependent hemolysis. The contribution of C1q from entrapped cerebrovascular blood was evaluated by the effects of perfusion and was minor relative to the increases of C1q in response to KA lesioning. These findings support the hypothesis that the C1q protein detected by immunocytochemistry in senile plaques of Alzheimer brains and in the hippocampus after deafferenting lesions is synthesized by resident brain cells.
Overexpression of Adhesion Molecule L1 in NG108‐15 Neuroblastoma × Glioma Hybrid Cells Enhances Dibutyryl Cyclic AMP‐Induced Neurite Outgrowth and Functional Synapse Formation with Myotubes
Authors: Zhong Zhen‐Guo , Yokoyama Shigeru , Noda Mami , Higashida Haruhiro
Published: July 2010

Abstract: The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma × glioma hybrid NG108‐15 cells. L1 overexpression was found in ∼50% of the transfected NG108‐15 cell population. Neurite outgrowth induced by 0.25 mM dibutyryl cyclic AMP (cAMP) was much greater in L1‐transfected NG108‐15 cells than that in nontransfected and mock‐transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1‐transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4–7) in L1‐transfected cells than in control cells. The miniature end‐plate potential frequency in myotubes was the same for the three types of NG108‐15 cells. These results show that overexpression of L1 in NG108‐15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.
Regulation of Corticotropin‐Releasing Factor Receptor Function in Human Y‐79 Retinoblastoma Cells: Rapid and Reversible Homologous Desensitization but Prolonged Recovery
Authors: Hauger R. L. , Dautzenberg F. M. , Flaccus A. , Liepold T. , Spiess J.
Published: July 2010

Abstract: Homologous receptor desensitization is an important regulatory response to continuous activation by agonist that involves the uncoupling of a receptor from its G protein. When human retinoblastoma Y‐79 cells expressing corticotropin‐releasing factor (CRF) receptors were preincubated with CRF for 10 min‐4 h, a time‐dependent reduction in both the peak and sensitivity of CRF‐stimulated intracellular cyclic AMP (cAMP) accumulation developed with a t1/2 of 38 min and an EC50 of 6–7 nM CRF. CRF receptor desensitization was slowly reversible after a 4‐h CRF preincubation with a t1/2 of 13 h and a full restoration of cAMP responsiveness to CRF at 24 h following the removal of 10 nM CRF. Because the ability of vasoactive intestinal peptide, forskolin, or (−)‐isoproterenol to stimulate cAMP accumulation was not diminished in Y‐79 cells desensitized with 10 nM CRF, the observed desensitization was considered to be a specific homologous action of CRF. CRF receptor desensitization was markedly attenuated by CRF receptor antagonists, which alone did not produce any appreciable reduction in CRF‐stimulated cAMP accumulation. Although recent reports have demonstrated a rapid decline in steady‐state levels of CRF receptor type 1 (CRF‐R1) mRNA in anterior pituitary cells during several hours of exposure to CRF, there was no observed reduction in CRF‐R1 mRNA levels when Y‐79 cells were preincubated with 10 nM CRF for 10 min‐24 h despite a rapid time‐ and concentration‐dependent loss of CRF receptors from the retinoblastoma cell surface.
A Constitutively Internalizing and Recycling Mutant of the μ‐Opioid Receptor
Authors: Segredo Veronica , Burford Neil T. , Lameh Jelveh , Sadée Wolfgang
Published: July 2010

Abstract: Internalization and recycling of G protein‐coupled receptors (GPCRs), such as the μ‐opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in μ‐opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr‐rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C‐terminus of the μ‐opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these μ‐receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N‐terminus of the wildtype and mutant receptors. Both the wild‐type μ‐opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin‐coated vesicles and recycled constitutively. Despite agonist‐independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C‐terminus, particularly the Ser/Thr domain, appears to suppress μ‐receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist‐dependent process, can occur spontaneously without concomitant G protein activation.
Neurosteroids Modulate Nicotinic Receptor Function in Mouse Striatal and Thalamic Synaptosomes
Authors: Bullock Amy E. , Clark Amy L. , Grady Sharon R. , Robinson Scott F. , Slobe Brian S. , Marks Michael J. , Collins Allan C.
Published: July 2010

Abstract: Progesterone and its A‐ring reduced metabolites are allosteric activators of GABAA receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an 86Rb+ efflux assay that likely measures the function of α4β2‐type nicotinic receptors and [3H]dopamine release, which may be modulated by an α3‐containing nicotinic receptor. Both of the A‐ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the 86Rb+ efflux assay. The 86Rb+ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays (t1/2 = 0.4 min) and was reversed even more slowly (t1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [3H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.
The Globin Fragment LVV‐Hemorphin‐7 Is an Endogenous Ligand for the AT4 Receptor in the Brain
Authors: Moeller Ingrid , Lew Rebecca A. , Mendelsohn Frederick A. O. , Smith A. Ian , Brennan Michelle E. , Tetaz Timothy J. , Chai Siew Yeen
Published: July 2010

Abstract: Angiotensin IV (Val‐Tyr‐Ile‐His‐Pro‐Phe) has been reported to interact with specific high‐affinity receptors to increase memory retrieval, enhance dopamine‐induced stereotypy behavior, and induce c‐fos expression in several brain nuclei. We have isolated a decapeptide (Leu‐Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln‐Arg‐Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using 125I‐angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30–39 of sheep βA‐ and βB‐globins and has previously been named LVV‐hemorphin‐7. Pharmacological studies demonstrated that LVV‐hemorphin‐7 and angiotensin IV were equipotent in competing for 125I‐angiotensin IV binding to sheep cerebellar membranes and displayed full cross‐displacement. Using in vitro receptor autoradiography, 125I‐LVV‐hemorphin‐7 binding to sheep brain sections was identical to 125I‐angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV‐hemorphin‐7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.
Pretreatment of Astrocytes with Interferon‐α/β Impairs Interferon‐γ Induction of Nitric Oxide Synthase
Authors: Stewart Victoria C. , Giovannoni Gavin , Land John M. , McDonald W. Ian , Clark John B. , Heales Simon J. R.
Published: July 2010

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Exposure of astrocytes, in primary culture, to interferon‐γ results in stimulation of nitric oxide synthase activity and increased nitric oxide release. In contrast to interferon‐γ, interferon‐α/β had a minimal effect on astrocytic nitric oxide formation. Furthermore, pretreatment of astrocytes with interferon‐α/β inhibited (∼65%) stimulation by interferon‐γ of nitric oxide synthase activity and nitric oxide release. Treatment with interferon‐α/β at a concentration as low as 10 U/ml caused inhibition of mitochondrial cytochrome c oxidase. Furthermore, the damage to cytochrome c oxidase was prevented by the putative interferon‐α/β receptor antagonist oxyphenylbutazone. In view of these observations, our current hypothesis is that the mitochondrial damage caused by exposure to interferon‐α/β may impair the ability of astrocytes to induce nitric oxide synthase activity on subsequent interferon‐γ exposure. These results may have implications for our understanding of the mechanisms responsible for the therapeutic effects of interferon‐α/β preparations in multiple sclerosis.
GABAA Receptor Blockade in the Anterior Ventral Tegmental Area Increases Extracellular Levels of Dopamine in the Nucleus Accumbens of Rats
Authors: Ikemoto Satoshi , Kohl Robert R. , McBride William J.
Published: July 2010

Abstract: Previously, it was shown that microinfusion of the GABAA antagonist picrotoxin into the anterior ventral tegmental area (VTA) is reinforcing. It was hypothesized that this reinforcing effect of picrotoxin in the anterior VTA is mediated, at least in part, by the activation of the mesoaccumbens dopamine (DA) system. The objective of the present study was to determine if blockade of GABAA receptors in the anterior VTA can increase extracellular levels of DA in the nucleus accumbens (ACB), using an in vivo microdialysis technique in freely moving rats. Concentrations of picrotoxin (40, 80, and 160 µM) that had previously been shown to produce a reinforcing effect increased the extracellular levels of DA and its major metabolites in the ACB. The increased extracellular DA levels induced by intra‐VTA injection of picrotoxin was markedly attenuated by coadministration with the GABAA agonist muscimol, whereas intra‐VTA injection of muscimol alone did not have an apparent effect on extracellular DA levels in the ACB. Microinjection of another GABAA antagonist, bicuculline, into the anterior VTA also increased the extracellular release of DA in the ACB. These results suggest that DA neurons projecting from the anterior VTA to the ACB are tonically inhibited by GABA through its actions at the GABAA receptors.
Impact of Corticotropin‐Releasing Hormone on Extracellular Norepinephrine in Prefrontal Cortex After Chronic Cold Stress
Authors: Finlay Janet M. , Jedema Hank P. , Rabinovic Ariel D. , Mana Michael J. , Zigmond Michael J. , Sved Alan F.
Published: July 2010

Abstract: We have previously demonstrated that exposing rats to cold (5°C) for 3–4 weeks potentiates the increase in extracellular norepinephrine (NE) in the medial prefrontal cortex produced by acute tail shock. In the present study, we used microdialysis to determine the duration of cold exposure required to produce this sensitization and explored the mechanism of the phenomenon. Tail shock elicited a twofold greater increase in extracellular NE in the medial prefrontal cortex of rats exposed to cold for 2 weeks than in naive control rats or in rats exposed to cold for 1 week and tested either immediately or after a 2‐week delay. Local infusion of 10 µMd‐amphetamine or 30 mM K+ increased extracellular NE in the medial prefrontal cortex (∼350 and 190%, respectively) comparably in control rats and rats exposed to cold for 3 weeks. In contrast, intraventricular administration of 3.0 µg of corticotropin‐releasing hormone increased extracellular NE in the medial prefrontal cortex by 65% in rats exposed to cold for 2 weeks, but only 35% in control rats. These results indicate that an enhanced responsiveness of noradrenergic neurons to acute tail shock (1) requires ∼2 weeks of cold exposure to develop and (2) may be mediated by a change at the level of the noradrenergic cell bodies rather than the nerve terminals.
Delayed Phospholipid Degradation in Rat Brain After Traumatic Brain Injury
Authors: Homayoun P. , Rodriguez de Turco E. B. , Parkins N. E. , Lane D. C. , Soblosky J. , Carey M. E. , Bazan N. G.
Published: July 2010

Abstract: Lipid second messengers such as arachidonic acid and its metabolites and diacylglycerols (DAGs) are affected in brain injury. Therefore, changes in the pool size and the fatty acid composition of free fatty acids (FFAs) and DAGs were analyzed in different rat brain areas 4 and 35 days after traumatic injury. Cortical impact injury of low‐grade severity was applied in the right frontal somatosensory cortex. Four days after injury, FFAs and DAGs were increased by three‐ and twofold, respectively, in the injured cortex and to a lesser extent in the contralateral cortex compared with sham‐operated animals. Docosahexaenoic acid followed by stearic acid, and arachidonic acid, displayed the greatest changes in both FFAs and DAGs. By day 35, free stearic, oleic, and arachidonic acids remained elevated in the damaged cortex (1.5‐fold each). DAGs showed the greatest change, reaching values 2.7‐fold higher than sham in all frontal and occipital cortical areas, including brainstem. Oleoyl‐ and arachidonoyl‐DAGs (four‐ and threefold increase, respectively) followed by docosahexaenoyl‐DAGs (twofold) contributed to the DAG accumulation. These results reveal that traumatic brain injury triggers a sustained and time‐dependent activation of phospholipase‐mediated signaling pathways leading to membrane phospholipid degradation and targeting, early on, docosahexaenoyl phospholipid‐enriched excitable membranes.
Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death
Authors: Chen Jun , Jin Kunlin , Chen Minzhi , Pei Wei , Kawaguchi Kenji , Greenberg David A. , Simon Roger P.
Published: July 2010

Abstract: Using in situ DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT) and terminal deoxynucleotidyl‐transferase‐mediated dUTP nick end‐labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single‐strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double‐strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB‐containing cells was greater than that of DSB‐containing cells at all time points tested. SSB‐containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB‐ and DSB‐containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.
Brain‐Specific Modulation of Kynurenic Acid Synthesis in the Rat
Authors: Gramsbergen Jan Bert P. , Hodgkins Paul S. , Rassoulpour Arash , Turski Waldemar A. , Guidetti Paolo , Schwarcz Robert
Published: July 2010

Abstract: This study was designed to investigate modulatory mechanisms that control the synthesis of the neuroprotective endogenous excitatory amino acid receptor antagonist kynurenate. De novo kynurenate formation was examined in vitro using tissue slices from rat brain, liver, and kidney. In slices from adult cerebral cortex, veratridine, quisqualate, and l‐α‐aminoadipate decreased kynurenate synthesis substantially. Glucose removal or changes in the ionic milieu, too, influenced kynurenate formation significantly, suggesting that demands on cellular energy interfere with kynurenate production in the adult rat brain. The effects of quisqualate and l‐α‐aminoadipate were also observed in the immature brain, in the quinolinate‐lesioned adult striatum, and, to a lesser extent, in peripheral organs. In contrast, the effect of veratridine was not seen in the lesioned brain or in kidney and liver tissue, indicating its dependency on intact neuron‐glia interactions. Compared with the normal adult brain, ionic manipulations yielded qualitatively distinct results in the developing brain and in the periphery, but their effects remained unchanged in the lesioned striatum. Glucose deprivation was less consequential in the immature than in the adult brain and was entirely ineffective in the lesioned striatum and in the periphery. These results further link cellular, especially astrocytic, energy metabolism to kynurenate formation in the brain. More generally, the existence of brain‐specific mechanisms for the regulation of kynurenate production is suggestive of a modulatory role of this metabolite in excitatory amino acid receptor function and dysfunction.
Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum
Authors: Dixon Alistair K. , Widdowson Leon , Richardson Peter J.
Published: July 2010

Abstract: The influence of the adenosine A2A receptor on the A1 receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low‐affinity binding site for the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA). This effect was blocked by the A2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C‐mediated heterologous desensitisation of the A1 receptor by the A2A receptor.