bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Oxidative Stress and the Glutathione System in Loss of Dopamine Neurons Due to Impairment of Energy Metabolism
Authors: Zeevalk Gail D. , Bernard Laura P. , Nicklas William J.
Published: July 2010

Abstract: Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose‐dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high‐affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24‐h pretreatment with 10 µM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin‐trapping agents, α‐phenyl‐tert‐butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate‐induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin‐trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase‐positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin‐trapping agents. NMDA receptors have also been shown to play a role in malonate‐induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1‐h exposure to 50 µM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.
β‐Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons
Authors: Beffert Uwe , Aumont Nicole , Dea Doris , Lussier‐Cacan Suzanne , Davignon Jean , Poirier Judes
Published: July 2010

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late‐onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to β‐amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ1–42, Aβ1–40, and Aβ25–35, increase significantly apoE‐liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ25–35, was further shown to increase significantly the internalization of both apoE3‐ and apoE4‐liposomes, without affecting apoE degradation. Conversely, Aβ1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE‐liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.
Presynaptic GABAB Receptor Modulation of Glutamate Exocytosis from Rat Cerebrocortical Nerve Terminals: Receptor Decoupling by Protein Kinase C
Authors: Perkinton Michael S. , Sihra Talvinder S.
Published: July 2010

Abstract: GABA and the GABAB receptor agonist (−)‐baclofen inhibited 4‐aminopyridine (4AP)‐ and KCl‐evoked, Ca2+‐dependent glutamate release from rat cerebrocortical synaptosomes. The GABAB receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (−)‐Baclofen‐mediated inhibition of glutamate release was insensitive to 2 µg/ml pertussis toxin. As determined by examining the mechanism of GABAB receptor modulation of glutamate release, (−)‐baclofen caused a significant reduction in 4AP‐evoked Ca2+ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP‐mediated depolarization; thus, the inhibition of Ca2+ influx could not be attributed to GABAB receptor activation causing a decrease in synaptosomal excitability. Ionomycin‐mediated glutamate release was not affected by (−)‐baclofen, indicating that GABAB receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABAB receptors to voltage‐dependent Ca2+ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (−)‐baclofen‐mediated inhibition of 4AP‐evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine‐tune transmitter release.
Effects of Local Administration of L‐, N‐, and P/Q‐Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat
Authors: Bergquist F. , Jonason J. , Pileblad E. , Nissbrandt H.
Published: July 2010

Abstract: The pivotal role for voltage‐sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage‐sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω‐conotoxin‐GVIA, ω‐agatoxin‐IVA, and neomycin were administered locally through the dialysis probes and compared with calcium‐free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N‐ and P/Q‐type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium‐dependent mechanism, for example, calcium mobilization through T‐, O‐, or R‐type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5‐bisphosphate breakdown products.
Substance P and Histamine Induce Interleukin‐6 Expression in Human Astrocytoma Cells by a Mechanism Involving Protein Kinase C and Nuclear Factor‐IL‐6
Authors: Lieb Klaus , Schaller Helen , Bauer Joachim , Berger Mathias , Schulze‐Osthoff Klaus , Fiebich Bernd L.
Published: July 2010

Abstract: Interleukin‐6 (IL‐6) is a proinflammatory cytokine whose synthesis is induced by a variety of stimuli including interleukin‐1 (IL‐1), substance P (SP), and histamine. Because IL‐6 has been implicated in the etiopathology of different human diseases including multiple myeloma, rheumatoid arthritis, multiple sclerosis, acquired immunodeficiency syndrome dementia complex, and Alzheimer's disease, its inhibition may be of therapeutic interest. A main demand on an effective inhibitor of IL‐6 expression is that it inhibits IL‐6 synthesis independently of the inducing stimulus. We therefore used human astrocytoma cells to search for signal transduction cascades and transcription factors whose inhibition suppresses IL‐6 synthesis after stimulation with three different inductors, IL‐1β, SP, and histamine. Whereas the antioxidant pyrrolidinedithiocarbamate was only able to inhibit IL‐1β‐induced IL‐6 expression, inhibition of protein kinase C prevented IL‐6 expression induced by all three substances. Promoter deletion analysis revealed that IL‐1β‐induced IL‐6 expression required the transcription factor nuclear factor‐κB (NF‐κB), whereas SP‐ and histamine‐induced IL‐6 synthesis was essentially controlled by NF‐IL‐6. These findings suggest that inhibition of protein kinase C or a combinatory inhibition of NF‐IL‐6 and NF‐κB binding are strategies to effectively suppress IL‐6 synthesis. They therefore provide the basis for the development of antiinflammatory drugs used to treat disorders in which IL‐6 is pathogenically involved.
Expression, Purification, and Encephalitogenicity of Recombinant Human Myelin Oligodendrocyte Glycoprotein
Authors: Bettadapura Jayaram , Menon Krishna K. , Moritz Susanne , Liu Junliang , Bernard Claude C. A.
Published: July 2010

Abstract: Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N‐terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST‐MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS‐like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.
Decreased Expression of Calmodulin Kinase II and Calcineurin Messenger RNAs in the Mouse Hippocampus After Kainic Acid‐Induced Seizures
Authors: Solà Carme , Tusell Josep M. , Serratosa Joan
Published: July 2010

Abstract: The Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIα and CaMKIIβ being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aα is the predominant form in the brain. We studied the expression of CaMKIIα, CaMKIIβ, and CaN Aα subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIα and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid‐treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid‐treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down‐regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.
Identification, Characterization, Immunocytochemical Localization, and Developmental Changes in the Activity of Calcium/Calmodulin‐Dependent Protein Kinase II in the CNS of Bombyx mori During Postembryonic Development
Authors: Shanavas A. , Dutta‐Gupta Aparna , Murthy Ch. R. K.
Published: July 2010

Abstract: In the present investigation, in vitro phosphorylation of CNS proteins of the silkworm Bombyx mori during the postembryonic development have been studied. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography of phosphorylated proteins revealed the presence of major phosphoproteins of 59/60 kDa. Based on molecular mass, calcium/calmodulin‐dependent autophosphorylation, substrate specificity, KN‐62 inhibition, apparent Km for ATP and syntide‐2, these proteins were identified as calcium/calmodulin‐dependent protein kinase II (CaM kinase II). Anti‐rat CaM kinase II monoclonal antibody showed immunoreactivity with Bombyx CaM kinase II isoforms. This kinase showed a high degree of autophosphorylation in neural tissue. During postembryonic development of Bombyx, two distinct peaks of enzyme activity could be noticed, one at the late‐larval and another at the late‐pupal stage, which were associated with an increase in amount of the enzyme. These results suggested that the expression of CaM kinase II in the CNS of Bombyx was developmentally regulated.
Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH‐SY5Y Human Neuroblastoma Cells
Authors: Szekeres P. G. , Koenig J. A. , Edwardson J. M.
Published: July 2010

Abstract: Preexposure of SH‐SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol‐stimulated intracellular Ca2+ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca2+ response to 1 mM carbachol is ∼50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist‐induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.
Murine Glial Cells Regenerate NAD, After Peroxide‐Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates
Authors: Grant Ross S. , Kapoor Vimal
Published: July 2010

Abstract: The potential for regeneration of intracellular pyridine nucleotide levels from different precursors, after peroxide‐induced NAD depletion, in cultured glial cells was investigated. Cultured murine glial cells showed a decrease in intracellular NAD levels of >40% after treatment with H2O2 (100 µM). Removal of the H2O2 followed by a 2‐h incubation did not result in NAD recovery in the absence of precursors. However, NAD levels increased significantly in these cells after the following substrate additions, at minimum effective concentrations of 1 mM for quinolinic acid (QUIN), 500 µM for nicotinamide, and 2 µM for nicotinic acid. The regeneration of significant amounts of NAD from nicotinic acid at doses 250 and 500 times lower than either nicotinamide or QUIN indicates a preferred route for NAD biosynthesis in glial cells in vitro, probably via nicotinic acid phosphoribosylation.
CNS Drug Design Based on Principles of Blood‐Brain Barrier Transport
Authors: Pardridge William M.
Published: July 2010

Abstract: Lipid‐soluble small molecules with a molecular mass under a 400–600‐Da threshold are transported readily through the blood‐brain barrier in vivo owing to lipid‐mediated transport. However, other small molecules lacking these particular molecular properties, antisense drugs, and peptide‐based pharmaceuticals generally undergo negligible transport through the blood‐brain barrier in pharmacologically significant amounts. Therefore, if present day CNS drug discovery programs are to avoid termination caused by negligible blood‐brain barrier transport, it is important to merge CNS drug discovery and CNS drug delivery as early as possible in the overall CNS drug development process. Strategies for special formulation that enable drug transport through the blood‐brain barrier arise from knowledge of the molecular and cellular biology of blood‐brain barrier transport processes.
Evidence for κ‐ and μ‐Opioid Receptor Expression in C6 Glioma Cells
Authors: Bohn Laura M. , Belcheva Mariana M. , Coscia Carmine J.
Published: July 2010

Abstract: The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high‐affinity U69,593, endomorphin‐1, morphine, and β‐endorphin binding in desipramine (DMI)‐treated C6 cell membranes by performing homologous and heterologous binding assays with [3H]U69,593, [3H]morphine, or 125I‐β‐endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin‐1, morphine, nor β‐endorphin binding. Cross‐linking of 125I‐β‐endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT‐PCR analysis of opioid receptor expression in control and DMI‐treated C6 cells indicate that both κ‐ and μ‐opioid receptors are expressed. There does not appear to be a significant difference in the level of μ nor κ receptor expression in naive versus C6 cells treated with DMI over a 20‐h period. Collectively, the data indicate that κ‐ and μ‐opioid receptors are present in C6 glioma cells.
Up‐Regulation of NR2B Subunit of NMDA Receptors in Cerebellar Granule Neurons by Ca2+/Calmodulin Kinase Inhibitor KN93
Authors: Corsi Lorenzo , Li Jin Hong , Krueger Karl E. , Wang Yue Hua , Wolfe Barry B. , Vicini Stefano
Published: July 2010

Abstract: Recordings of NMDA‐activated currents from cerebellar granule neurons in culture revealed a developmental increase in current density accompanied by a slight decrease of the half‐maximal effective concentration. At the same time, a decrease of NMDA receptors comprising NR2B subunits was demonstrated by the reduction in the antagonism of NMDA currents by ifenprodil. Ifenprodil antagonism increased after treatment for 24 h with KN93‐ and KN62‐selective inhibitors of the Ca2+/calmodulin‐dependent protein kinases (CaM kinases), indicating a selective increase of receptor containing NR2B subunit. This increase was observed at all ages tested: 4 days in vitro (DIV4), DIV6, and DIV13. Western blot analysis with specific NMDA receptor antibodies performed at DIV6 confirmed the electrophysiological data. At this age, the negative control KN92 was ineffective. The increasing ifenprodil antagonism after KN93 treatment was proportionally greater in cells at DIV13 than at DIV4. Treatment with NMDA (100 µM) of cerebellar cultures for 24 h produced a decrease in the NMDA‐induced current density by almost 50% at all ages tested. Ifenprodil antagonism, however, was unchanged. We propose that the expression of NR2B subunits in cerebellar granule cells is selectively stimulated by the inhibition of CaM kinases.
Role of Potassium Channels in Amyloid‐Induced Cell Death
Authors: Colom Luis V. , Diaz Maria E. , Beers David R. , Neely Alan , Xie Wen‐jie , Appel Stanley H.
Published: July 2010

Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid β‐peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)‐sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K+ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48‐ and 96‐h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of Aβ. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in Aβ‐mediated toxicity for septal cholinergic cells.
Relationships Between the Catechol Substrate Binding Site and Amphetamine, Cocaine, and Mazindol Binding Sites in a Kinetic Model of the Striatal Transporter of Dopamine In Vitro
Authors: Wayment Hollie , Meiergerd Susan M. , Schenk James O.
Published: July 2010

Abstract: Experiments were conducted to determine how (−)‐cocaine and S(+)‐amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m‐tyramine and S(+)‐amphetamine caused release of dopamine from intracellular stores at concentrations ≥12‐fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m‐tyramine and S(+)‐amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S(+)‐Amphetamine and m‐tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (−)‐Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site (Kint = 583 nM). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (−)‐cocaine suggest that the (−)‐cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (−)‐cocaine binding may be developed to block (−)‐cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.
Neuronal Overexpression of Heme Oxygenase‐1 Correlates with an Attenuated Exploratory Behavior and Causes an Increase in Neuronal NADPH Diaphorase Staining
Authors: Maines M. D. , Polevoda B. , Coban T. , Johnson K. , Stoliar S. , Huang T. J. , Panahian N. , Cory‐Slechta D. A. , McCoubrey W. K.
Published: July 2010

Abstract: Heme oxygenase isozymes, HO‐1 (also known as hsp32) and HO‐2, are the source for the formation of the putative messenger molecule carbon monoxide (CO), reactive iron, and the in vitro antioxidant bilirubin. We have developed and characterized transgenic (Tg) mice that overexpress the stress protein in neurons in various brain regions. The Tg mice were generated by the use of rat HO‐1 cDNA under the control of the neuron‐specific enolase promoter. Except for a tendency to have an enlarged spleen, Tg mice did not show gross anatomical changes. Increase in HO‐1 mRNA, which was demonstrated by northern blot analysis and in situ hybridization, was accompanied by an increase in neuronal HO‐1 protein expression, shown by immunohistochemistry and western blotting, and an increase in HO activity. Expression of the transgene correlated with an attenuation of exploratory behavior and increased circling activity and coincided with enhanced neuronal NADPH diaphorase staining. Those changes were not accompanied by an increase in DNA damage or significant change in whole‐brain NO synthase activity. The HO‐1 Tg mice potentially represent a good model to examine the function of CO as a neuromodulator, iron as a gene regulator, and bile pigments as in vivo antioxidants.
Functional Coupling of the NK1 Tachykinin Receptor to Phospholipase D in Chinese Hamster Ovary Cells and Astrocytoma Cells
Authors: Torrens Yvette , Beaujouan Jean‐Claude , Saffroy Monique , Glowinski Jacques , Tencé Martine
Published: July 2010

Abstract: In [3H]myristic acid‐prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro9]substance P ([Pro9]SP) time and concentration dependently stimulated the formation of [3H]phosphatidylethanol in the presence of ethanol. This [Pro9]SP‐induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro9]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro9]SP‐evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a Gi/Go protein is not involved in these different signaling pathways. The activation of PLD by [Pro9]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31‐8220) or its down‐regulation (long‐term treatment with a phorbol ester) abolished the response. In contrast, a cAMP‐dependent process was not involved in the activation of PLD because the [Pro9]SP‐evoked response was neither affected by Rp‐8‐bromoadenosine 3′,5′‐cyclic monophosphorothioate nor mimicked by cAMP‐generating compounds (cholera toxin or forskolin) or by 8‐bromo‐cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro9]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors.
Implication of the First and Third Extracellular Loops of the μ‐Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ‐Opioid/Angiotensin Receptors
Authors: Dietrich Gilles , Gaibelet Gérald , Capeyrou Régine , Butour Jean‐Luc , Pontet Frédéric , Emorine Laurent J.
Published: July 2010

Abstract: Recent studies on chimeric μ/δ‐, μ/κ‐ and δ/κ‐opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100‐fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10‐fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.
Calcium Influx Through AMPA Receptors and Through Calcium Channels Is Regulated by Protein Kinase C in Cultured Retina Amacrine‐Like Cells
Authors: Carvalho Ana Luísa , Duarte Carlos B. , Faro Carlos J. , Carvalho Arsélio P. , Pires Euclides V.
Published: July 2010

Abstract: The functional modulation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine‐like cells. The kainate‐evoked [Ca2+]i increase is due to Ca2+ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3‐benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12‐myristate 13‐acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down‐regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 µM kainate. The effect of PKC activation or inhibition on voltage‐gated Ca2+‐channel activity was also investigated. Activation of PKC caused inhibition of Ca2+ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca2+ channels in cultured retina cells.
Peroxynitrite and Brain Mitochondria: Evidence for Increased Proton Leak
Authors: Brookes Paul S. , Land John M. , Clark John B. , Heales Simon J. R.
Published: July 2010

Abstract: Peroxynitrite has been reported to inhibit irreversibly mitochondrial respiration. Here we show that three sequential additions of 200 µM peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg of protein/ml) significantly stimulated state 4 respiration and that further additions progressively inhibited it. No stimulation of state 3 respiration or of the maximal enzymatic activities of the respiratory chain complexes was observed on identical peroxynitrite exposure. State 4 respiration is a consequence of the proton permeability of the mitochondrial inner membrane, and we demonstrate that the peroxynitrite‐induced stimulation of state 4 respiration is accompanied by a decreased mitochondrial membrane potential, suggesting an increase in this proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. The stimulation was prevented by the lipid‐soluble vitamin E analogue Trolox, suggesting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. Lipid peroxidation has previously been reported to increase membrane bilayer proton permeability. The high polyunsaturate content of brain mitochondrial phospholipids may predispose them to peroxidation, and thus a peroxynitrite‐induced, lipid peroxidation‐mediated increase in proton leak may apply particularly to brain mitochondria and to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.
Amyloid‐β Deposition in Alzheimer Transgenic Mice Is Associated with Oxidative Stress
Authors: Smith Mark A. , Hirai Keisuke , Hsiao Karen , Pappolla Miguel A. , Harris Peggy L. R. , Siedlak Sandra L. , Tabaton Massimo , Perry George
Published: July 2010

Abstract: Increased awareness for a role of oxidative stress in the pathogenesis of Alzheimer's disease has highlighted the issue of whether oxidative damage is a fundamental step in the pathogenesis or instead results from disease‐associated pathology. In vitro experiments support both possibilities: Oxidative stress increases amyloid‐β production, and, conversely, amyloid‐β increases oxidative damage. To address the relationship between amyloid‐β and oxidative stress in vivo, we examined, using an array of oxidative markers, transgenic mice that overexpress amyloid‐β precursor protein and, as in Alzheimer's disease, develop characteristic amyloid‐β deposits within the brain parenchyma. Transgenic animals show the same type of oxidative damage that is found in Alzheimer's disease, and it is important that this damage directly correlates with the presence of amyloid‐β deposits. The significance of these studies is twofold. First, they provide evidence that amyloid‐β and oxidative damage are inextricably linked in vivo. Second, they support the use of transgenic animals for the development of antioxidant therapeutic strategies.
Functional Analysis of a Voltage‐Gated Sodium Channel and Its Splice Variant from Rat Dorsal Root Ganglia
Authors: Dietrich Paul S. , McGivern Joseph G. , Delgado Stephen G. , Koch Bruce D. , Eglen Richard M. , Hunter John C. , Sangameswaran Lakshmi
Published: July 2010

Abstract: Neurons of the dorsal root ganglia (DRG) express a diversity of voltage‐gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin‐sensitive sodium channel α subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT‐PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low‐level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V1/2≅−100 mV for steady‐state inactivation and V1/2≅−40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50≅ 1 nM. Coexpression of the β1 subunit accelerated inactivation kinetics, but the β2 subunit was without effect.
Cloning and Characterization of a Mouse σ1 Receptor
Authors: Pan Ying‐Xian , Mei Jianfeng , Xu Jin , Wan Ban‐Lin , Zuckerman Amy , Pasternak Gavril W.
Published: July 2010

Abstract: A cDNA clone (S2‐1a) isolated from a mouse brain cDNA library, using a guinea pig σ1 cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ1 receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single‐copy gene in region A5‐B2 of chromosome 4. Expression of the clone in MCF‐7 and CHO cells led to a pronounced increase in (+)‐[3H]pentazocine binding with a selectivity profile consistent with σ1 receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2‐1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)‐[3H]‐Pentazocine binding to immunopurified HA‐tagged receptor demonstrated conclusively that S2‐1a.HA encodes a high‐affinity (+)‐[3H]pentazocine binding site with characteristics of a murine σ1 receptor. An antisense oligodeoxynucleotide designed from S2‐1a potentiated opioid analgesia in vivo.
Defining Responsiveness of Avian Cochlear Neurons to Brain‐Derived Neurotrophic Factor and Nerve Growth Factor by HSV‐1‐Mediated Gene Transfer
Authors: Garrido Juan José , Alonso Maria Teresa , Lim Filip , Carnicero Estela , Giraldez Fernando , Schimmang Thomas
Published: July 2010

Abstract: The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV‐1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV‐1‐mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF‐responsive by transducing them with the high‐affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.
Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor
Authors: McLaughlin B. A. , Nelson D. , Erecińska M. , Chesselet M.‐F.
Published: July 2010

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3‐nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose‐dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µM) nor dopamine (2.5 µM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µM) did not alter the ATP/ADP ratio after a 6‐h incubation. Methyl malonate, at 500 µM, reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.
Restoration of Norepinephrine and Reversal of Phenotypes in Mice Lacking Dopamine β‐Hydroxylase
Authors: Thomas Steven A. , Marck Brett T. , Palmiter Richard D. , Matsumoto Alvin M.
Published: July 2010

Abstract: Mice with a targeted disruption of the dopamine β‐hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, l‐threo‐3,4‐dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25–30% of normal. NE levels typically peak ∼5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S(−)‐Carbidopa, which does not cross the blood‐brain barrier, inhibits aromatic l‐amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind‐limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine β‐hydroxylase‐deficient mice are all reversed by DOPS injection.
Recycling of Synaptic Vesicles at the Frog Neuromuscular Junction in the Presence of Strontium
Authors: Guatimosim Cristina , Romano‐Silva Marco A. , Gomez Marcus V. , Prado Marco A. M.
Published: July 2010

Abstract: In these experiments, we followed the exocytosis and endocytosis of synaptic vesicles with the vital dye FM1‐43 and asked whether calcium is important for membrane retrieval at the frog neuromuscular junction. We replaced calcium with equimolar amounts of strontium and monitored the staining of recycling vesicles by inducing exocytosis with electrical stimulation. Trains of 2,400 (2 or 20 Hz) or 4,200 (20 Hz) pulses failed to induce FM1‐43 internalization in the presence of strontium, but they did in the presence of calcium. This effect of strontium was not due to a decrease in exocytosis, because FM1‐43 release was similar in the presence of calcium or strontium. The impairment in endocytosis, observed as inhibition of FM1‐43 internalization, could be overcome by longer periods of stimulation (6,000 pulses at 2 or 20 Hz) in the presence of strontium (1.8 mM) or by increasing the extracellular concentration of strontium to 10 mM (2,400 action potentials at 20 Hz). It is suggested that endocytosis is dependent on calcium influx and that strontium is much less effective in replacing calcium for endocytosis than it is for exocytosis.
Postischemic Reperfusion Induces α‐Fodrin Proteolysis by m‐Calpain in the Synaptosome and Nucleus in Rat Brain
Authors: Fukuda Shiro , Harada Kazuki , Kunimatsu Mitoshi , Sakabe Takefumi , Yoshida Ken‐ichi
Published: July 2010

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+‐dependent protease, calpain. It remains unknown whether μ‐calpain or m‐calpain is involved in the proteolysis of either α‐ or β‐fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ‐calpain or a significant change of m‐calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca2+‐dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE‐cellulose column. α‐ and β‐fodrins and m‐calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α‐fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µM, 1 ml, i.v.). The μ‐calpain‐specific fragment of β‐fodrin was not generated during ischemia‐reperfusion, supporting the possibility of the involvement of m‐calpain rather than μ‐calpain in the α‐fodrin proteolysis.
Regulation of Neuroprotective Action of Vasoactive Intestinal Peptide in the Murine Developing Brain by Protein Kinase C and Mitogen‐Activated Protein Kinase Cascades: In Vivo and In Vitro Studies
Authors: Gressens Pierre , Marret Stéphane , Martin Jean‐Luc , Laquerrière Annie , Lombet Alain , Evrard Philippe
Published: July 2010

Abstract: Intracerebral administration of the excitotoxin ibotenate to newborn mice induces white matter lesions mimicking periventricular leukomalacia, the most frequent brain lesion occurring in premature human babies. In this model, coinjection of vasoactive intestinal peptide prevents white matter lesions. In the present study, coadministration of ibotenate, vasoactive intestinal peptide, and selective transduction inhibitors showed that protein kinase C and mitogen‐associated protein kinase pathways were critical for neuroprotection. In vivo and in vitro immunocytochemistry revealed that vasoactive intestinal peptide activated protein kinase C in astrocytes and neurons, and mitogen‐associated protein kinase in neurons. In vitro neuronal transduction activation was indirect and required medium conditioned by astrocytes in which protein kinase C had been activated by vasoactive intestinal peptide. Although vasoactive intestinal peptide did not prevent the initial in vivo appearance of white matter lesion, it promoted a secondary repair of this lesion with axonal regrowth. Through protein kinase C activation, vasoactive intestinal peptide also prevented ibotenate‐induced white matter astrocyte death. These data support the following hypothetical model: Vasoactive intestinal peptide activates protein kinase C in astrocytes, which promotes astrocytic survival and release of soluble factors; these released factors activate neuronal mitogen‐associated protein kinase and protein kinase C, which will permit axonal regrowth.
Role of G Protein βγ Subunits in Muscarinic Receptor‐Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb
Authors: Olianas Maria C. , Ingianni Angela , Onali Pierluigi
Published: July 2010

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and Gs‐stimulated adenylyl cyclase activities and inhibiting the Ca2+/calmodulin‐ and forskolin‐stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγt) and blocked by putative βγ scavengers. Membrane incubation with βγt caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγt potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin‐releasing hormone. RT‐PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated Gs. In addition, βγt inhibited the Ca2+/calmodulin‐ and forskolin‐stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP‐bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.