bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Fructose‐1,6‐Bisphosphate on Glutamate Release and ATP Loss from Rat Brain Slices During Hypoxia
Authors: Bickler Philip E. , Buck Leslie T.
Published: July 2010

Abstract: Fructose‐1,6‐bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase)‐catalyzed conversion of glutamate to α‐ketoglutarate) during hypoxia (Po2 15 mm Hg) or hypoxia plus 100 µM cyanide. FBP (3.5 mM, with glucose 20 mM) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% (p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose‐free conditions with 20 but not 3.5 mM FBP, and fell to <20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 mM FBP without glucose decreased hypoxia‐evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia‐evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.
Amyloid β‐Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation
Authors: Zhang Zhiyuan , Rydel Russell E. , Drzewiecki Gary J. , Fuson Kimberly , Wright Sarah , Wogulis Mark , Audia James E. , May Patrick C. , Hyslop Paul A.
Published: July 2010

Abstract: H2O2 and free radical‐mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2‐scavenging enzyme catalase protects neurons in culture against Aβ‐mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. Aβ‐mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2‐mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde‐3‐phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24‐h exposure of neurons to Aβ. Our results challenge a pivotal role for H2O2 generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.
Molecular Evolution of τ Protein: Implications for Alzheimer's Disease
Authors: Nelson Peter T. , Stefansson Kari , Gulcher Jeffrey , Saper Clifford B.
Published: July 2010

Abstract: The brains of patients with Alzheimer's disease contain deposits of hyperphosphorylated τ proteins that have polymerized into insoluble fibrils. These deposits, in neurofibrillary tangles and dystrophic neurites, correlate with loss of cells and synapses, and consequently with dementia. Neurofibrillary pathology occurs in humans, as well as certain ungulates, including goats, sheep, and cows, but not in nonhuman primates. We hypothesize that the differences among species in the propensity to develop neurofibrillary pathology may be attributable to variations in the amino acid sequence of τ proteins. To investigate this hypothesis, we sequenced τ‐encoding mRNA transcripts from the brains of rhesus monkey and domesticated goat and compared them with the known sequences of τ mRNAs from humans. The major difference we observed was that some τ mRNAs from rhesus monkey neocortex contain exon 8, whereas this exon has not been found in cortical τ from human or goat. Cows express very low levels of exon 8, and they tend to develop sparse neurofibrillary pathology with aging. We also found a transcribed τ‐related pseudogene in rhesus monkey, which may be present in humans. We propose that differences in the expression of τ and τ‐related protein sequences may underlie the predilection of human but not monkey brains to develop neurofibrillary degeneration.
Chronic Activation of the Cyclic AMP Signaling Pathway Promotes Development and Long‐Term Survival of Mesencephalic Dopaminergic Neurons
Authors: Michel Patrick P. , Agid Yves
Published: July 2010

Abstract: Dibutyryl cyclic AMP (dbcAMP), a permeant analogue of cyclic AMP (cAMP), prevented, for at least 3 weeks, the death of tyrosine hydroxylase (TH)‐immunopositive dopaminergic neurons, which occurred spontaneously by apoptosis in mesencephalic cultures. Treatment with the cyclic nucleotide analogue also led to a significant increase in the uptake of [3H]dopamine, attesting that the rescued TH+ neurons were fully functional and differentiated. dbcAMP was most effective when added immediately after plating, but delayed treatment could still arrest the ongoing degenerative process. Trophic/survival effects were long‐lasting, declining only progressively after withdrawal of dbcAMP from the culture medium. They were independent of cell density and still detectable in the absence of serum proteins. The effects of dbcAMP were mimicked by depolarizing concentrations of potassium and by agents that increase endogenous production of cAMP, such as forskolin or 3‐isobutyl‐1‐methylxanthine, but not by native cAMP, which cannot cross cell membranes. Elimination of glial cells by arabinoside‐C did not reduce the activity of dbcAMP. GABAergic neurons, also present in these cultures, were much less dependent on the cyclic nucleotide analogue for their survival, and serotoninergic cells were not dependent at all. Therefore, cAMP‐dependent signaling may be particularly crucial for the maturation and long‐term survival of mesencephalic dopaminergic neurons.
Delivery of Human Fibroblast Growth Factor‐1 Gene to Brain by Modified Rat Brain Endothelial Cells
Authors: Johnston P. , Nam M. , Hossain M. A. , Indurti R. R. , Mankowski J. L. , Wilson M. A. , Laterra J.
Published: July 2010

Abstract: Fibroblast growth factor (FGF) is an endothelial cell mitogen and serves as a mitogen and/or differentiating factor that can be neuroprotective for other cell types within the CNS. We established brain microvascular endothelial cell lines that secrete FGF‐1 with the ultimate goal of examining their usefulness as a cellular platform for FGF gene delivery to brain. A chimeric gene consisting of the secretory sequence of FGF‐4 linked at the 5′ end of human FGF‐1 (sp‐hst/KS3:FGF‐1) was transfected into rat microvascular endothelial cells previously altered to express the lacZ reporter gene (RBEZ), and numerous clones were found to secrete FGF‐1 (RBEZ‐FGF). Immunoblotting of conditioned medium demonstrated an 18‐kDa protein corresponding to FGF‐1. Conditioned medium from RBEZ‐FGF cells enhanced [3H]thymidine incorporation in BALB/c3T3 fibroblasts by up to sevenfold when compared with conditioned medium of control cell lines, corresponding to as much as 110 ng of active FGF‐1/mg of cell protein/24 h. RBEZ‐FGF cell lines remained contact‐inhibited and proliferated independent of exogenous endothelial mitogens, in contrast to control lines that are mitogen‐dependent. Incubation of PC12 cells with RBEZ‐FGF cells or their conditioned medium induced neurite outgrowth by PC12 cells. RBEZ‐FGF cells survived following implantation to neonatal and adult rat caudate‐putamen for at least 21 days based on 5‐bromo‐4‐chloro‐3‐indolyl β‐d‐galactopyranoside (X‐gal) histochemistry, and FGF‐1 gene expression by these cells in vivo was demonstrated by in situ hybridization and reverse transcriptase‐PCR. These findings suggest that endothelial cells may be useful for FGF gene delivery to the CNS.
Calcium‐Dependent Self‐Association of Synaptotagmin I
Authors: Damer Cynthia K. , Creutz Carl E.
Published: July 2010

Abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium‐triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1–5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium‐dependent manner. p65 1–5 may be able to aggregate membrane vesicles by a self‐association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1–5, in the absence of membranes, was able to self‐associate to form large aggregates in a calcium‐dependent manner as shown by light‐scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self‐associate and aggregate phospholipid vesicles in a calcium‐dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self‐associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium‐dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium‐dependent manner in vitro suggests that multimerization may have an important function in vivo.
Specific Delay in the Degradation of Mitochondrial ATP Synthase Subunit c in Late Infantile Neuronal Ceroid Lipofuscinosis Is Derived from Cellular Proteolytic Dysfunction Rather than Structural Alteration of Subunit c
Authors: Ezaki Junji , Wolfe Leonhard S. , Kominami Eiki
Published: July 2010

Abstract: Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell‐free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn2+‐superoxide dismutase (SOD). In contrast, E‐64‐d stimulates greater lysosomal storage of Mn2+‐SOD than of subunit c. Incubation of mitochondrial‐lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn2+‐SOD, β subunit of ATP synthase, and subunit IV of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E‐64‐c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin‐sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial‐lysosomal fractions from patient cells is degraded on incubation with mitochondrial‐lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.
Depletion of Neuronal Endoplasmic Reticulum Calcium Stores by Thapsigargin: Effect on Protein Synthesis
Authors: Paschen Wulf , Doutheil Jens , Gissel Cornelia , Treiman Marek
Published: July 2010

Abstract: We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+‐ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 µM caused a permanent inhibition of protein synthesis in hippocampal slices from 3‐week‐old rats but no inhibition in slices prepared from 2‐month‐old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3‐week‐ and 2‐month‐old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24‐h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with ∼5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.
Accelerated Myelinogenesis by Dietary Lipids in Rat Brain
Authors: Salvati Serafina , Sanchez Massimo , Campeggi Lorenzo Malvezzi , Suchanek Gerda , Breitschop Helene , Lassmann Hans
Published: July 2010

Abstract: Our previous work showed an early development of behavioral reflexes in rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n‐alkanes (which contain 50% odd‐chain fatty acids) in comparison with controls fed a margarine diet. To clarify whether the observed changes might be linked to an early myelination, we have investigated mRNAs involved in myelin synthesis in the brains of offspring at 5 days of age by northern blot and in situ hybridization. Northern blot analysis showed that proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) mRNAs were higher in animals on the lipid diet compared with controls. In situ hybridization with probes specific for PLP, myelin basic protein, and MOG mRNA showed significantly higher numbers of positive cells in test animals compared with controls in all brain regions. This study shows an acceleration of myelinogenesis induced by dietary lipids. These data can give a new insight in the therapeutical approaches involved to promote repair in demyelinating diseases.
Differential Effects of Ipsapirone on 5‐Hydroxytryptamine Release in the Dorsal and Median Raphe Neuronal Pathways
Authors: Casanovas Josep M. , Artigas Francesc
Published: July 2010

Abstract: Serotonergic neurons of the dorsal and median raphe nuclei are morphologically dissimilar. Recent results challenge previous evidence indicating a greater inhibition of dorsal raphe neurons after 5‐hydroxytryptamine1A (5‐HT1A) autoreceptor activation. As both nuclei innervate different forebrain territories, this issue is critical to understanding the changes in brain function induced by anxiolytic and antidepressant drugs. Using microdialysis, we examined the modifications of 5‐HT release induced by the selective 5‐HT1A agonist ipsapirone in both neuronal pathways. Maximal and minimal basal 5‐HT values (in the presence of 1 µM citalopram) were 45.0 ± 4.8 fmol/fraction in the median raphe nucleus and 8.4 ± 0.4 fmol/fraction in the dorsal hippocampus. Ipsapirone (0.3, 3, and 10 mg/kg s.c.) reduced dose‐dependently 5‐HT in the two raphe nuclei and four forebrain areas. Maximal reductions (to ∼25% of predrug values) were observed in cortex and striatum and in median raphe nucleus. The effects were more moderate in dorsal and ventral hippocampus (to 66 and 50% of baseline, respectively). These results are consistent with a higher sensitivity of dorsal raphe neurons to 5‐HT1A autoreceptor activation. Yet the differential reduction of 5‐HT release in the median raphe nucleus and hippocampus suggests the presence of complex mechanisms of control of 5‐HT release in these neurons.
Multiple Determinants of Dihydro‐β‐Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits
Authors: Harvey Scott C. , Maddox Floyd N. , Luetje Charles W.
Published: July 2010

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro‐β‐erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro‐β‐erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro‐β‐erythroidine by ∼120‐fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro‐β‐erythroidine sensitivity to be distributed throughout the N‐terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro‐β‐erythroidine sensitivity by 4.3‐fold.
Brain Polyamine Stress Response: Recurrence After Repetitive Stressor and Inhibition by Lithium
Authors: Gilad Gad M. , Gilad Varda H.
Published: July 2010

Abstract: We recently demonstrated that, unlike in peripheral tissues, the increase in activity of polyamine synthesizing enzymes observed in the brain after acute stress can be prevented by long‐term, but not by short‐term, treatment with lithium. In the present study we sought to examine the effects of chronic intermittent stress on two key polyamine synthesizing enzymes, ornithine decarboxylase and S‐adenosylmethionine decarboxylase, and their modulation by lithium treatment. Adult male rats were subjected to 2 h of restraint stress once daily for 5 days and to an additional delayed stress episode 7 days later. Enzyme activities were assayed 6 h after the beginning of each stress episode. In contrast to the liver, where ornithine decarboxylase activity was increased (300% of the control) only after the first stress episode, the enzyme activity in the brain was increased after each stress episode (to ∼170% of the control). Unlike ornithine decarboxylase activity, S‐adenosylmethionine decarboxylase activity was slightly reduced after the first episode (86% of the control) but remained unchanged thereafter. After cessation of the intermittent stress period, an additional stress episode 7 days later led again to an increase in ornithine decarboxylase activity in the brain (225% of the control) but not in the liver, whereas S‐adenosylmethionine decarboxylase activity remained unchanged. The latter increase in ornithine decarboxylase activity was blocked by lithium treatment during the intervening 7‐day interval between stressors. The results warrant the following conclusions: (a) Repetitive application of stressors results in a recurrent increase in ornithine decarboxylase activity in the brain but to habituation of this response in the liver. (b) This brain polyamine stress response can be blocked by long‐term (days) lithium treatment. (c) The study implicates an overreactive polyamine response as a component of the adaptive, or maladaptive, brain response to stressful events and as a novel molecular target for lithium action.
Inosine Mediates the Protective Effect of Adenosine in Rat Astrocyte Cultures Subjected to Combined Glucose‐Oxygen Deprivation
Authors: Haun Steven E. , Segeleon Joseph E. , Trapp Victoria L. , Clotz Michael A. , Horrocks Lloyd A.
Published: July 2010

Abstract: Preliminary evidence suggests adenosine, a neuromodulator, has neuroprotective properties during cerebral ischemia. It is unclear, however, if adenosine has glioprotective effects. We studied the effect of adenosine on cellular injury in astroglial cultures subjected to combined glucose‐oxygen deprivation. Adenosine (100–1,000 µM) dramatically reduced astroglial injury, whereas the adenosine agonists 2‐chloroadenosine (10 nM–100 µM), N6‐cyclopentyladenosine (1 nM–10 µM), 5′‐N‐ethylcarboxamidoadenosine (10 nM–100 µM), and N6‐2‐(4‐aminophenyl)ethyladenosine (10 nM–100 µM) had no effect. Furthermore, the adenosine antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (1 nM–1 µM), xanthine amine congener (10 nM–10 µM), and 8‐(p‐sulfophenyl)‐theophylline (10–300 µM) failed to reverse the protective effect of 200 µM adenosine. Next, adenosine degradation products were studied. Inosine proved to be glioprotective at concentrations nearly identical to those of adenosine, but hypoxanthine and ribose had no effect. The protective effect of 200 µM inosine was not reversed by 8‐(p‐sulfophenyl)theophylline (10–300 µM). Adenosine deaminase (1 unit/ml) had no effect on protection produced by adenosine, whereas erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine hydrochloride (10 µM) reversed the protective effect of adenosine. Dipyridamole (4 µM) inhibited the protective effect of both adenosine and inosine. We conclude that adenosine dramatically decreases astroglial injury during combined glucose‐oxygen deprivation and that this protective effect appears to be mediated by inosine.
Expression, Catalytic Activity, and Inducibility of Cytochrome P450 2E1 (CYP2E1) in the Rat Central Nervous System
Authors: Tindberg Niclas , Ingelman‐Sundberg Magnus
Published: July 2010

Abstract: Cytochrome P450 2E1 (CYP2E1) metabolizes several neuroactive substrates, including exogenous compounds such as anesthetics, organic solvents, and muscle relaxants as well as endogenous substrates such as arachidonic acid. CYP2E1 and its mRNA were found to be expressed in the rat hippocampus, where the enzyme was localized mainly to the microsomal fraction. Chlorzoxazone (CZN), a CYP2E1 substrate, was 6‐hydroxylated in hippocampal homogenates with a Km of 25.5 µM and a Vmax of 0.22 pmol/mg/min. CYP2E1 was also expressed in vitro in cortical glial cultures, where CYP2E1 mRNA levels were found to be 1,000‐fold lower than in rat liver. Exposure of cortical glial cultures to 25 or 100 mM ethanol for 24 h caused a fourfold and sixfold increase, respectively, in the rate of CYP2E1‐dependent 6‐hydroxylation of CZN. After a continuous exposure to 100 mM ethanol for 48 or 72 h, however, the hydroxylation rate was down‐regulated. Chlormethiazole, a potent inhibitor of hepatic CYP2E1 transcription, inhibited the ethanol‐dependent induction of CYP2E1 by 50%. In vivo, acute ethanol treatment of rats (24 h, 3 g/kg) resulted in a 1.8‐fold increase in the rate of CZN 6‐hydroxylation in hippocampal homogenates. It is concluded that CYP2E1 is expressed and catalytically active in the rat CNS, and that CYP2E1 can be induced by a relatively low concentration of ethanol in cortical glial cultures. It is suggested that CYP2E1 substrates may be metabolically activated in situ in the CNS.
Attenuation of Cyclic AMP Production by Carbamazepine
Authors: Chen Guang , Pan Baishen , Hawver David B. , Wright Clinton B. , Potter William Z. , Manji Husseini K.
Published: July 2010

Abstract: The anticonvulsant carbamazepine is an effective treatment both for epilepsy and for bipolar affective disorder, but the molecular mechanism(s) underlying its therapeutic effects have not been identified. We have found that carbamazepine exerts significant inhibitory effects on the cyclic AMP (cAMP) generating system. Within the clinical therapeutic range (∼50 µM), carbamazepine inhibited both basal and forskolin‐stimulated cAMP production, without having any significant effects on phosphodiesterase activity. Carbamazepine also exerted its inhibitory effects on the cAMP generating system in pertussis toxin‐treated cells, suggesting that the action of carbamazepine was likely mediated through an inhibitory guanine nucleotide binding protein‐independent mechanism. A forskolin affinity purification column was used to purify adenylyl cyclases from rat cerebral cortex, and we found that carbamazepine inhibited both basal and forskolin‐stimulated activity of purified adenylyl cyclase. We also investigated the effects of carbamazepine on the levels of the transcription factor, cAMP response element binding protein in the phosphorylated (active) state, and found that carbamazepine significantly inhibited forskolin‐induced phosphorylation of the cAMP response element binding protein. The data indicate that carbamazepine inhibits adenylyl cyclase activity as well as the downstream effects of activation of adenylyl cyclase.
Novel ω‐Conotoxins Block Dihydropyridine‐Insensitive High Voltage‐Activated Calcium Channels in Molluscan Neurons
Authors: Kits Karel S. , Lodder Johannes C. , Van Der Schors Roel C. , Li Ka Wan , Geraerts Wijnand P. M. , Fainzilber Michael
Published: July 2010

Abstract: We have identified two novel peptide toxins from molluscivorous Conus species that discriminate subtypes of high voltage‐activated (HVA) calcium currents in molluscan neurons. The toxins were purified using assays on HVA calcium currents in the caudodorsal cells (CDCs) of the snail Lymnaea stagnalis. The CDC HVA current consists of a rapidly inactivating, transient current that is relatively insensitive to dihydropyridines (DHPs) and a slowly inactivating, DHP‐sensitive L‐current. The novel toxins, designated ω‐conotoxins PnVIA and PnVIB, completely and selectively block the transient HVA current in CDCs with little (PnVIA) or no (PnVIB) effect on the sustained L‐type current. The block is rapid and completely reversible. It is noteworthy that both PnVIA and PnVIB reveal very steep dose dependences of the block, which may imply cooperativity in toxin action. The amino acid sequences of PnVIA (GCLEVDYFCGIPFANNGLCCSGNCVFVCTPQ) and of PnVIB (DDDCEPPGNFCGMIKIGPPCCSGWCFFACA) show very little homology to previously described ω‐conotoxins, although both toxins share the typical ω‐conotoxin cysteine framework but have an unusual high content of hydrophobic residues and net negative charge. These novel ω‐conotoxins will facilitate selective analysis of the functions of HVA calcium channels and may enable the rational design of drugs that are selective for relevant subtypes.
Preferential Release of ATP and Its Extracellular Catabolism as a Source of Adenosine upon High‐ but Not Low‐Frequency Stimulation of Rat Hippocampal Slices
Authors: Cunha Rodrigo A. , Vizi E. Sylvester , Ribeiro J. Alexandre , Sebastião Ana M.
Published: July 2010

Abstract: The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high‐frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long‐term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low‐frequency (5 Hz) train stimulation, to mimic a long‐term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high‐frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low‐frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used; α,β‐methylene ADP (AOPCP), an inhibitor of ecto‐5′‐nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low‐frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high‐frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high‐frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.
Combined Administration of a 5‐Hydroxytryptamine (5‐HT)1D Antagonist and a 5‐HT Reuptake Inhibitor Synergistically Increases 5‐HT Release in Guinea Pig Hypothalamus In Vivo
Authors: Rollema Hans , Clarke Thomas , Sprouse Jeffrey S. , Schulz David W.
Published: July 2010

Abstract: In vivo microdialysis in guinea pig hypothalamus was used to study the effect of serotonin [5‐hydroxytryptamine (5‐HT)] subtype 1D autoreceptor blockade on the increase in extracellular 5‐HT levels produced by a selective 5‐HT reuptake inhibitor (SSRI). Administration of the selective 5‐HT1D antagonist GR127935 at 0.3 mg/kg had no effect, but 5 mg/kg significantly increased extracellular levels of 5‐HT and 5‐hydroxyindoleacetic acid to 135% of basal values. Moreover, at these doses GR127935 significantly attenuated the decrease in extracellular 5‐HT levels following local perfusion with the selective 5‐HT1D agonist CP‐135,807. The SSRI sertraline at 2 mg/kg increased 5‐HT levels to 130% of basal levels. The combination of this low dose of sertraline with either dose of GR127935 resulted in a pronounced, long‐lasting increase in 5‐HT levels to 230% of basal values. These results indicate that the effects of an SSRI on terminal 5‐HT are significantly enhanced by coadministration of a 5‐HT1D antagonist and confirm that in addition to somatodendritic 5‐HT1A autoreceptors, terminal 5‐HT1D autoreceptors mitigate the effect of SSRIs on terminal 5‐HT. As such, antagonists of the 5‐HT1D autoreceptor could be useful as rapidly acting antidepressants and may shorten the onset of antidepressant action when combined with SSRIs.
AP‐1‐Related Proteins Bind to the Enkephalin CRE‐2 Element in Adrenal Chromaffin Cells
Authors: MacArthur Linda
Published: July 2010

Abstract: Chromaffin cells of the adrenal medulla are neural crest‐derived neuroendocrine cells that express neuropeptide genes in vivo and in vitro. As such these cells are useful for examining tissue‐ and cell‐specific regulation of the enkephalin gene. We previously demonstrated that the chromatin configuration of the enkephalin gene correlated with its tissue‐specific expression in the adrenal medulla and primary chromaffin cell cultures. In this study we examine and characterize binding of transcription factors to the enkephalin promoter/enhancer region. Gel shift analyses of this region with extracts from chromaffin cells and PC12 cells (a pheochromocytoma cell line that does not express the enkephalin gene) demonstrate that all detectable binding is to ENKCRE‐2, a cyclic AMP response‐like element, and that the binding is cell specific. Gel shift and supershift analyses show that, unlike reports demonstrating that binding activity in the CNS is composed of the cyclic AMP response element binding protein, CREB, the majority of protein binding in chromaffin cells is from the AP‐1 family of transcription factors. This binding is composed of c‐Jun, JunD, and possibly a novel Fos‐related protein(s). These data suggest enkephalin gene expression in the adrenal gland is controlled by cell‐specific binding of transcription factors from the Fos/Jun families to the enkephalin CRE‐2 element. Furthermore, these data suggest at least two different modes of enkephalin gene regulation exist between endocrine and neuronal tissues.
Binding of Erythroagglutinating Phytohemagglutinin Lectin from Phaseolus vulgaris to the Epidermal Growth Factor Receptor Inhibits Receptor Function in the Human Glioma Cell Line, U373 MG
Authors: Rebbaa Abdelhadi , Yamamoto Hirotaka , Moskal Joseph R. , Bremer Eric G.
Published: July 2010

Abstract: Little is known about the role of the N‐linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF‐R). In a human glioma cell line, U373 MG, EGF‐Rs contain the bisecting N‐linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E‐PHA). Incubation of E‐PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF‐induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF‐R, phenotypic events that depend on EGF‐R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L‐PHA), an isolectin of E‐PHA, had no effect on EGF‐R activity or the biological functions of these cells even though L‐PHA was able to bind to the EGF‐R. These findings suggest the presence of an important bisecting N‐linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E‐PHA lectin binding may provide an additional approach to blocking EGF‐dependent glioma cell growth.
Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells
Authors: Budd Samantha L. , Nicholls David G.
Published: July 2010

Abstract: Exposure of cultured cerebellar granule cells to 100 µM glutamate plus glycine in the absence of Mg2+ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca2+ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca2+ concentration), and extensive cell death. Glutamate‐evoked Ca2+ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca2+ transport, allows glutamate‐exposed cells to maintain a high ATP/ADP ratio while accumulating little 45Ca2+ and maintaining a low bulk cytoplasmic free Ca2+ concentration determined by fura‐2. It is concluded that mitochondrial Ca2+ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca2+ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca2+ accumulation in a microdomain accessible to the mitochondria.
Acute and Chronic Ethanol Consumption Effects on the Immunolabeling of Gq/11α Subunit Protein and Phospholipase C Isozymes in the Rat Brain
Authors: Pandey Subhash C.
Published: July 2010

Abstract: The goal of this investigation was to examine whether postreceptor sites [Gq/11 protein and phospholipase C (PLC) isozymes] of the phosphoinositide signal transduction system are involved in neuroadaptational mechanisms in the brain during chronic ethanol consumption. It was observed that acute ethanol treatment has no effect on the immunolabeling of PLC‐β1, ‐γ1, and ‐δ1 and the α subunit of Gq/11 protein in the rat cortex as determined by western blotting using specific monoclonal antibodies. On the other hand, chronic ethanol consumption (15 days) resulted in a significant decrease in the immunolabeling of PLC‐β1, whereas under identical conditions, the immunolabeling of PLC‐γ1 and ‐δ1 isozymes was not significantly altered. The decreased immunolabeling of PLC‐β1 during chronic ethanol consumption was not altered by 24 h of withdrawal after 15 days of ethanol consumption. The immunolabeling of the α subunit of Gq/11 protein was significantly decreased after 15 days of ethanol consumption but had returned to normal levels after 24 h of ethanol withdrawal. Also, chronic ethanol treatment resulted in a significant decrease in phosphatidylinositol 4,5‐bisphosphate‐specific PLC activity, which remained the same after 24 h of ethanol withdrawal. These results suggest that decreased PLC activity during ethanol consumption and its withdrawal may be due to decreased protein levels of the Gq/11 protein‐coupled PLC‐β1 isozyme but not the PLC‐γ1 or ‐δ1 isozyme in the rat cortex. It is possible that changes in the protein levels of the Gq/11 protein‐coupled PLC‐β1 isozyme and in PLC activity in the brain may be involved in the cellular adaptation to chronic ethanol exposure.
Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy‐Terminal Region, but Is Not Caused by Receptor Internalization
Authors: Sanders Matthew A. , LeVine Harry
Published: July 2010

Abstract: The carboxy‐terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy‐terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5‐trisphosphate response) when preexposed for 1 min to 1 µM neurokinin, indicating a role for the carboxy‐terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 nM) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µM substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31‐8220, staurosporine, and Zn2+ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate‐limiting in neurokinin 1 receptor desensitization in this system.
Influence of Fluoxetine on Regional Serotonin Synthesis in the Rat Brain
Authors: Mück‐Šeler Dorotea , Jevric‐Causevic Adlija , Diksic Mirko
Published: July 2010

Abstract: The aim of the present study was to test the hypothesis that there should be a difference between the effects of an acute and an 8‐day (chronic) administration of fluoxetine (10 mg/kg) on the rate of serotonin [5‐hydroxytryptamine (5‐HT)] synthesis. The 5‐HT synthesis rate was measured in discrete regions of the rat brain using the α‐[14C]methyl‐l‐tryptophan autoradiographic method. The results show that the acute and chronic fluoxetine treatments influence the 5‐HT synthesis rate in different ways. A single dose of fluoxetine induced a significant increase in 5‐HT synthesis in the visual, auditory, and parietal cortices, substantia nigra, hypothalamus, ventral thalamus, and dorsal hippocampus. In contrast, after a chronic treatment a decrease was observed in the substantia nigra, caudate, and nucleus accumbens, the auditory, parietal, sensorimotor, and frontal cortices, and ventral tegmental area. A significant decrease in the rate of 5‐HT synthesis was observed in the dorsal raphe after both the single and chronic treatments. The results suggest that extracellular 5‐HT has a delayed influence on the brain 5‐HT synthesis rate in structures with serotonergic terminals. The findings from the acute study could be important for patients who have just started receiving fluoxetine treatment, as an increase in the 5‐HT synthesis rate might occur in the acute phase of their treatment. In addition, the findings from the chronic treatment study might give us a better understanding of how the brain serotonergic system adapts during a prolonged exposure to extracellular 5‐HT.
Cytoskeletal Breakdown and Apoptosis Elicited by NO Donors in Cerebellar Granule Cells Require NMDA Receptor Activation
Authors: Bonfoco Emanuela , Leist Marcel , Zhivotovsky Boris , Orrenius Sten , Lipton Stuart A. , Nicotera Pierluigi
Published: July 2010

Abstract: We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S‐nitrosocysteine (SNOC) or S‐nitroso‐N‐acetylpenicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA‐R) activation leading to intracellular Ca2+ overload. Early dissolution of actin filaments followed by breakdown of microtubules and nuclear lamins preceded the appearance of typical apoptotic features. NO donors induced tyrosine nitration in neurons, in a small population of contaminating astrocytes, and in cultures of cerebellar astroglial cells. However, astrocytes neither displayed cytoskeletal alterations nor underwent apoptosis. Competitive and uncompetitive NMDA receptor antagonists, such as d‐aminophosphonovaleric acid and MK‐801, did not influence tyrosine nitration but prevented the accumulation of intracellular Ca2+, cytoskeletal breakdown, and apoptosis induced by either SNOC or SNAP in CGCs. Taken together, these data strongly suggest that Ca2+ influx through NMDA‐R‐gated ion channels is a critical event in CGC apoptosis induced by NO donors.
Dopamine Inhibits Melatonin Synthesis in Photoreceptor Cells Through a D2‐Like Receptor Subtype in the Rat Retina: Biochemical and Histochemical Evidence
Authors: Nguyen‐Legros Jeanine , Chanut Evelyne , Versaux‐Botteri Claudine , Simon Axelle , Trouvin Jean‐Hugues
Published: July 2010

Abstract: An intrinsic oscillator, using dopamine and melatonin as antagonist signals, controls rhythmic events in the retina of nonmammals. The purpose of the present work was to localize and characterize a dopamine receptor responsible for the nocturnal inhibition of melatonin synthesis in photoreceptor cells in a mammalian retina. An antibody against the D2 receptor stained photoreceptor cell inner segments of the rat retina. α‐Methyl‐p‐tyrosine, a competitive inhibitor of tyrosine hydroxylase, enhanced the nocturnal content of melatonin, suggesting the dopamine control of melatonin synthesis as in non‐mammals. Clozapine, a D2C/D4 antagonist, also enhanced the nocturnal level of melatonin, whereas raclopride, a D2A antagonist, did not. Taken together, these results support the control of melatonin levels by dopamine through a D2C/D4 receptor in photoreceptor cells of the rat retina. The presence of D4 receptors in the rat retina was confirmed by reverse transcription‐PCR.
Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP
Authors: Griffon Nathalie , Pilon Catherine , Sautel François , Schwartz Jean‐Charles , Sokoloff Pierre
Published: July 2010

Abstract: As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108‐15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole‐induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6–16 h, was reproduced by cyclic AMP‐permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of >1 µM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor‐mediated mitogenic response, through phosphorylation by a cyclic AMP‐dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.
Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor‐Mediated Calmodulin Redistribution in SK‐N‐SH Neuroblastoma Cells
Authors: Shariat‐Madar Z. , Goldsmith A. M. , Gnegy M. E.
Published: July 2010

Abstract: Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK‐N‐SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol‐induced calmodulin translocation, we treated cells for 17 h with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) to down‐regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK‐N‐SH cells for 17 h with 70 nM TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol‐mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31‐8220 at 10 µM. In the 72‐h TPA‐treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31‐8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the α‐isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17‐ and 72‐h TPA‐treated cells, the ε‐isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine‐rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol‐mediated increases in cytosolic CaM levels were inhibited by Ro 31‐8220 in retinoic acid‐differentiated SK‐N‐SH cells. This study demonstrates that continuous TPA treatment, although initially down‐regulating the protein kinase C‐mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.