bluuebunny/crossref_metadata_2025 huggingface dataset:
Coupling of Corticotropin‐Releasing Hormone Receptors to Adenylyl Cyclase in Human Y‐79 Retinoblastoma Cells
Authors: Olianas Maria C. , Lampis Giorgio , Onali Pierluigi
Published: July 2010

Abstract: In human Y‐79 retinoblastoma cells, corticotropin‐releasing hormone (CRH) stimulates adenylyl cyclase activity and increases cyclic AMP accumulation. Different CRH analogues mimic the CRH stimulation of adenylyl cyclase and show similar sensitivity to the CRH receptor antagonist α‐helical CRH9–41. Vasoactive intestinal peptide (VIP) also increases the enzyme activity but less potently than CRH, and its effect is counteracted by the VIP receptor antagonist [d‐p‐Cl‐Phe6,Leu17]VIP. The VIP antagonist does not affect the response to CRH. The CRH‐stimulated adenylyl cyclase activity is amplified by Mg2+, is inhibited by submicromolar concentrations of Ca2+, and requires GTP. Moreover, the CRH stimulation is reduced by pretreatment of cells with cholera toxin and by incubation of membranes with the RM/1 antibody, which recognizes the C‐terminus of the α subunit of Gs. In immunoblots, the RM/1 antibody identifies a doublet of 45 and 52 kDa. Two proteins of similar molecular weights are ADP‐ribosylated by cholera toxin. These data demonstrate that in human Y‐79 retinoblastoma cells, specific CRH receptors stimulate cyclic AMP formation by interacting with Gs and by affecting a Ca2+‐inhibitable form of adenylyl cyclase.
Nerve Regeneration and Cholesterol Reutilization Occur in the Absence of Apolipoproteins E and A‐I in Mice
Authors: Goodrum Jeffry F. , Bouldin Thomas W. , Zhang Sunny H. , Maeda Nobuyo , Popko Brian
Published: July 2010

Abstract: Apolipoproteins have been implicated in the salvage and reutilization of myelin cholesterol during Wallerian degeneration and the subsequent nerve regeneration. Current evidence suggests that myelin cholesterol complexes with apolipoproteins E and A‐I to form lipoproteins that are taken up via low‐density lipoprotein receptors on myelinating Schwann cells. We recently reported, however, that apolipoprotein E is not required for nerve regeneration or reutilization of myelin cholesterol. We have now investigated nerve regeneration and the reutilization of cholesterol in mutant mice deficient in both apolipoproteins E and A‐I. Morphologic examination of nerves 4 and 12 weeks after crush injury revealed that regeneration proceeded at a normal rate in the absence of these apolipoproteins. Autoradiography of regenerating nerves indicated that prelabeled myelin lipid was reutilized in the regenerating myelin. 3‐Hydroxy‐3‐methylglutaryl‐CoA reductase, the rate‐limiting enzyme in cholesterol synthesis, was down‐regulated in the regenerating nerves, indicative of cholesterol uptake via lipoproteins. Prelabeled myelin cholesterol was present in lipoprotein fractions isolated from crushed nerves of mutant mice. These data suggest that there is considerable redundancy in the process of cholesterol reutilization within nerve, and that apolipoproteins other than apolipoproteins E and A‐I may be involved in the recycling of myelin cholesterol.
Elevated Levels of the Endosomal‐Lysosomal Proteinase Cathepsin D in Cerebrospinal Fluid in Alzheimer Disease
Authors: Schwagerl A. L. , Mohan P. S. , Cataldo A. M. , Vonsattel J. P. , Kowall N. W. , Nixon R. A.
Published: July 2010

Abstract: Lysosomal hydrolases are normally intracellular enzymes but are abundant extracellularly within senile plaques in Alzheimer disease and in other conditions where β‐amyloid accumulates. To examine whether acid hydrolases released from abnormal hydrolase‐laden neurons are detectable in CSF, we measured levels of the major aspartic proteinase of lysosomes, cathepsin D (Cat D), in ventricular CSF collected after death from 30 patients with Alzheimer disease, 14 patients with Huntington disease, and seven patients with other neurodegenerative diseases. The levels of Cat D‐immunoreactive protein, expressed as micrograms per milliliter of protein, determined by western blot immunoassay using a polyclonal antiserum against human brain Cat D, were more than fourfold higher in the Alzheimer patients than in the other patient groups (p < 0.0005). Cat D activity, assayed separately against [14C]methemoglobin at pH 3.2, was also significantly elevated but less than Cat D content. The lower specific activity of Cat D in Alzheimer CSF therefore indicated that the abnormally accumulated Cat D included a high proportion of inactive enzyme. These results indicate that abnormal Cat D release from affected neurons into the extracellular space is an active, ongoing process in Alzheimer brain. In addition, the levels of this enzyme and possibly other lysosomal hydrolases in CSF may prove to be useful biological markers of Alzheimer disease.
Structural Organization of the Murine D3 Dopamine Receptor Gene
Authors: Park Bae‐Hang , Fishburn C. Simone , Carmon Sara , Accili Domenico , Fuchs Sara
Published: July 2010

Abstract: We have cloned the gene encoding the murine D3 dopamine receptor and have analyzed its intron‐exon structural organization, to gain a better understanding of the detailed architecture of the D2 dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D3 receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron‐exon organization directly analogous to that found in the D2 receptor gene. In addition, we have sequenced the 5′ and 3′ nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D2 receptor, suggesting that the control of D3 receptor expression may vary more between species than the control of D2 receptor expression.
Analysis of the Alternative Promoters that Regulate Tissue‐Specific Expression of Human Aromatic l‐Amino Acid Decarboxylase
Authors: Sumi‐Ichinose Chiho , Hasegawa Seiko , Ichinose Hiroshi , Sawada Hirohide , Kobayashi Kazuto , Sakai Masao , Fujii Tetsuya , Nomura Hiroko , Nomura Takahide , Nagatsu Ikuko , Hagino Yasumichi , Fujita Keisuke , Nagatsu Toshiharu
Published: July 2010

Abstract: Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5′ untranslated regions of human aromatic l‐amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue‐specific manner. To determine the cis‐acting element regulating the tissue‐specific expression of human AADC, we produced three kinds of transgenic mice harboring 5′ flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis‐regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.
Inhibition by Lamotrigine of the Generation of Nitric Oxide in Rat Forebrain Slices
Authors: Lizasoain Ignacio , Knowles Richard G. , Moncada Salvador
Published: July 2010

Abstract: Depolarization of adult rat forebrain slices with veratrine induced the release of excitatory amino acids (glutamate and aspartate), the synthesis of nitric oxide (NO), and increases in cyclic GMP (cGMP). The NO synthase inhibitors Nω‐monomethyl‐l‐arginine and Nω‐nitro‐l‐arginine methyl ester decreased the release of NO and the levels of cGMP without affecting the release of excitatory amino acids. In contrast, the antiepileptic drug lamotrigine inhibited the release of excitatory amino acids and of NO, and decreased the levels of cGMP without causing a significant direct inhibition of the NO synthase. Furthermore, the synthesis of NO and the increases in cGMP induced by veratrine were partially blocked by the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801 but not by 6‐nitro‐7‐sulphamobenzo(f)quinoxaline‐2,3‐dione, a non‐NMDA receptor antagonist. Neither of these compounds inhibited directly the NO synthase or the release of excitatory amino acids. Thus, these three types of compound act as an inhibitor of voltage‐sensitive sodium channels (lamotrigine), as a receptor antagonist (MK‐801), or as direct inhibitors of the NO synthase, to block the pathway leading to increased cGMP after veratrine depolarization. It is likely that some of the pharmacological and therapeutic actions shared by these three types of compound are, at least in part, a consequence of inhibition of the synthesis of NO.
Coupling of the Human Y2 Receptor for Neuropeptide Y and Peptide YY to Guanine Nucleotide Inhibitory Proteins in Permeabilized SMS‐KAN Cells
Authors: Freitag Christian , Svendsen Anja B. , Feldthus Nya , Løssl Kristine , Sheikh Søren P.
Published: July 2010

Abstract: Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH‐terminals of the α‐subunits of Gi1/2, Gi3, and Go, the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS‐KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin‐stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against αi1/2 and αi3 blocked the functional response of PYY, with anti‐αi3 being the most potent; whereas anti‐αo failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS‐KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different Gis (but not Go).
NMDA and Non‐NMDA Receptor‐Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb
Authors: García Y. , Ibarra C. , Jaffé E. H.
Published: July 2010

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N‐methyl‐d‐aspartate (NMDA), kainate, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) on [3H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [3H]GABA release and the release evoked by a high K+ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine‐2,4‐dicarboxylate (2,4‐PDC) inhibited by 50% the glutamate‐induced [3H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [3H]GABA release. The presence of glycine and the absence of Mg2+ have no potentiating effect on NMDA‐stimulated release; however, when the tissue was previously depolarized with a high K+ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2‐amino‐5‐phosphonoheptanoic acid (AP‐7). The kainate and AMPA effects were antagonized by the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not by AP‐7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2‐amino‐5‐phosphonopentanoic acid (AP‐5); nevertheless, in the presence of glycine, [3H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP‐5. Tetrodotoxin inhibited glutamate‐ and kainate‐stimulated [3H]GABA release but not the NMDA‐stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor‐mediated mechanism as a mixed agonist on NMDA and non‐NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.
Dopamine Receptor Blockade Inhibits the Amphetamine‐Induced Release of Diadenosine Polyphosphates, Diadenosine Tetraphosphate and Diadenosine Pentaphosphate, from Neostriatum of the Conscious Rat
Authors: Pintor Jesús , Porras Alberto , Mora Francisco , Miras‐Portugal M. Teresa
Published: July 2010

Abstract: The diadenosine polyphosphates diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) are costored with ATP and released in a calcium‐dependent manner from neural preparations in vitro. By means of a push‐pull perfusion system, samples from conscious rat were collected from the caudate putamen area, and nucleotide compounds were analyzed by HPLC. The adenine dinucleotides were not detectable before systemic amphetamine injection. The maximal levels were reached 20 min after injection, independently of the dose. The EC50 values for amphetamine‐induced release of dinucleotides were 2.04 ± 0.15 and 2.43 ± 0.36 mg/kg for Ap4A and Ap5A, respectively. Amphetamine doses higher than 5 mg/kg did not increase the dinucleotide release, the maximal values being 12.9 ± 0.9 and 11.5 ± 0.9 pmol/fraction for Ap4A and Ap5A, respectively, which corresponds with 64.5 and 57.5 nM in the samples. Adenosine and AMP were present in push‐pull samples from rat brain under basal conditions. Their levels were 15 pmol/fraction (75 nM) and 50 pmol/fraction (250 nM) for adenosine and AMP, respectively. A significant increase was obtained for both compounds after amphetamine injection. The adenosine increase reached 45 pmol/sample (225 nM), which was 200% of the basal value 20 min after the stimulant administration. The increase at other times was not significant. The AMP levels increased significantly from 10 to 50 min. The maximal level was reached 20 min after amphetamine injection, with 150 pmol/fraction (750 nM), which represents a 200% increase with respect to the basal level. The adenine dinucleotide release was blocked by the dopamine receptor antagonist haloperidol, which returned the levels to the control basal values. It is suggested that dopamine, released in a nonexocytotic way by the action of amphetamine, induces the release of the dinucleotides Ap4A and Ap5A in the neostriatum area through dopaminergic receptors.
Characterization of Ligand Binding to the Cannabinoid Receptor of Rat Brain Membranes Using a Novel Method: Application to Anandamide
Authors: Hillard Cecilia J. , Edgemond William S. , Campbell William B.
Published: July 2010

Abstract: Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [3H]CP 55,940 and the Multiscreen Filtration System. Binding of [3H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 µg of membrane protein/well, the KD for [3H]CP 55,940 is 461 pM and the Bmax is 860 fmol/mg of protein. The apparent KD of [3H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 µg of membrane protein. Binding of [3H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The Ki of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 µM in buffer alone and 143 nM in the presence of 0.15 mM phenylmethylsulfonyl fluoride. When [14C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 mM phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high‐affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.
Identification of D3 and σ Receptors in the Rat Striatum and Nucleus Accumbens Using (±)‐7‐Hydroxy‐N,N‐Di‐n‐[3H]Propyl‐2‐Aminotetralin and Carbetapentane
Authors: Wallace David R. , Booze Rosemarie M.
Published: July 2010

Abstract: Cross‐reactions between dopamine D3 and σ receptor ligands were investigated using (±)‐7‐hydroxy‐N,N‐di‐n‐[3H]propyl‐2‐aminotetralin [(±)‐7‐OH‐[3H]DPAT], a putative D3‐selective radioligand, in conjunction with the unlabeled σ ligands 1,3‐di(2‐tolyl)guanidine (DTG), carbetapentane, and R(−)‐N‐(3‐phenyl‐1‐propyl)‐1‐phenyl‐2‐aminopropane [R(−)‐PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D3 receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)‐7‐OH‐[3H]DPAT binding. R(−)‐PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)‐7‐OH‐[3H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)‐7‐OH‐[3H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high‐ and low‐affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(−)‐PPAP identified two sites in equal proportion. Carbetapentane and R(−)‐PPAP (0.1 µM) displaced ∼20–50% of total (±)‐7‐OH‐[3H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)‐7‐OH‐[3H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D3 receptors with 1 µM dopamine. Under these conditions (+)‐7‐OH‐[3H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)‐7‐OH‐[3H]DPAT binds D3 receptors with high affinity in rat brain and (b) a significant proportion of (±)‐7‐OH‐[3H]DPAT binding consists of σ1 sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.
Dopamine Neurotoxicity: Inhibition of Mitochondrial Respiration
Authors: Ben‐Shachar Dorit , Zuk Roza , Glinka Yelena
Published: July 2010

Abstract: Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen‐derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose‐dependent manner with LD50 = 90 µg. Norepinephrine was less effective with LD50 = 141 µg. The iron chelator desferrioxamine completely protected against dopamine‐induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine‐induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron‐induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC50 = 8 µM, and that of norepinephrine was twice as much (IC50 = 15 µM). Dopamine‐induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine‐induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine‐induced inhibition of NADH dehydrogenase activity in a dose‐dependent manner.
Release of Glutamate from Striatum of Freely Moving Rats by pros‐Methylimidazoleacetic Acid
Authors: Blandina Patrizio , Cherici Giovanna , Moroni Flavio , Prell George D. , Green Jack Peter
Published: July 2010

Abstract: The effect of pros‐methylimidazoleacetic acid (p‐MIAA) was measured on the release of glutamate and aspartate from cerebral cortex, hippocampus, and striatum of freely moving rats, and on the uptake of 14C by striatal slices incubated in the presence of l‐[14C]‐glutamate. Twenty‐four hours after implantation of a dialysis fiber, striatum, hippocampus, or cerebral cortex spontaneously released both glutamate and aspartate in the micromolar range. p‐MIAA (1 µM to 1 mM), added to the dialysis perfusate, elicited a concentration‐dependent increase of glutamate release from striatum with a maximal increase of about threefold. This effect did not occur in hippocampus or cortex. In none of these regions did p‐MIAA increase aspartate release significantly. The p‐MIAA effect was not mimicked by its isomer tele‐methylimidazoleacetic acid. p‐MIAA did not influence the uptake of glutamate by striatal slices. The glutamate‐releasing action of p‐MIAA may affect striatal function and explain the positive correlation between levels of p‐MIAA in CSF and the severity of Parkinson's disease.
The Sodium Channel Blocker and Glutamate Release Inhibitor BW1003C87 and Magnesium Attenuate Regional Cerebral Edema Following Experimental Brain Injury in the Rat
Authors: Okiyama Koichi , Smith Douglas H. , Gennarelli Thomas A. , Simon Roger P. , Leach Michael , McIntosh Tracy K.
Published: July 2010

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor‐associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline‐treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex (p < 0.001), the cortical area adjacent to the site of maximal injury (p < 0.001), left hippocampus (p < 0.001), and left thalamus (p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury (p < 0.02) and left hippocampus (p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus (p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre‐ or post‐synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.
Characterization of Monoclonal Antibodies that Inhibit the Catalytic Activity of Acetylcholinesterases
Authors: Gentry Mary K. , Moorad Deborah R. , Hur Regina S. , Saxena Ashima , Ashani Yacov , Doctor Bhupendra P.
Published: July 2010

Abstract: Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7‐(methylethoxyphosphinyloxy)‐1‐methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to >98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross‐reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at >98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.
Aging‐Associated Changes in the Pharmacological Properties of the Benzodiazepine (ω) Receptor Isotypes in the Rat Hippocampus
Authors: Ruano Diego , Benavides Jesus , Machado Alberto , Vitorica Javier
Published: July 2010

Abstract: The aging‐associated changes in hippocampal benzodiazepine (ω) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [3H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3‐month‐old) Wistar strain rats, high (type I; ω1)‐, intermediate (type IIM; ω2)‐, and low (type IIL; ω5)‐ affinity sites for this imidazopyridine account for 27.1 ± 7.5, 44.2 ± 7.5, and 28.8 ± 5.1%, respectively. In hippocampal membranes from aged (24‐month‐old) rats of the same strain, the relative abundance of these sites was 42.8 ± 9.3, 26.3 ± 4, and 36.0 ± 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (ω) binding site density between adult and aged rats. The increase in type I (ω1) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [3H]zolpidem. This aging‐induced increase in [3H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [3H]zolpidem (5 nM) binding to type I (ω1) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [3H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging‐associated increases in the density of type I (ω1) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields.
Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ
Authors: Richardson C. Mark , Dowdall Michael J. , Green A. Chris , Bowman David
Published: July 2010

Abstract: The presynaptic terminals of skate (Raja montagui) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca2+ concentrations triggered by K+ depolarisation were studied. ACh release was measured as 3H efflux from slices of organ prelabelled with [3H]choline. Depolarisation caused a marked, Ca2+‐dependent increase in 3H efflux that was completely blocked by 100 µM Cd2+ and by 300 nMω‐conotoxin‐MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC50 of ∼20 nM) and reversible. No inhibition was seen with nifedipine (5 µM) or the two other peptide antagonists studied: ω‐conotoxin‐GVIA (GVIA) at 5 µM and ω‐agatoxin‐IVA (Aga‐IVA) at 1 µM. In a “nerve plate” preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca2+ concentrations were measured by microfluorimetry using fluo‐3. An increase in fluorescence, indicating a rise in the free [Ca2+], rapidly followed K+ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µM), GVIA (5 µM), and Aga‐IVA (300 nM) but almost completely abolished by MVIIC (1 µM). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.
IgH Intronic Enhancer Element HE2 (μB) Functions as a cis‐Activator in Choroid Plexus Cells at the Cellular Level as well as in Transgenic Mice
Authors: Enjoji Munechika , Iwaki Toru , Nawata Hajime , Watanabe Takeshi
Published: July 2010

Abstract: Immunoglobulin heavy‐chain (IgH) gene expression is regulated largely by the IgH gene intronic enhancer (ENHiH), which is composed of multiple protein‐binding motifs. These motifs are DNA elements that are important for the regulation of IgH gene transcription. It has been reported that the HE2 (μB) and μA motifs within the ENHiH affect B cell‐specific gene expression. To examine the function of the HE2 and μA elements in vivo, we established transgenic mouse lines. A deletion mutant of the human ENHiH that contains the HE2 and μA motifs, but lacks the motifs corresponding to murine E5, E3, and octamer, functioned not only in B lymphocytes but also in choroid plexus cells, which secrete CSF. As a result, we obtained choroid plexus tumor‐bearing transgenic mice and could establish choroid plexus carcinoma cell lines. In addition, using the luciferase assay, we confirmed at the cellular level that the HE2 motif shows a fair degree of enhancer activity in cultured choroid plexus carcinoma cells. These results suggest that existence of a trans‐acting factor for the HE2 motif in choroid plexus cells. Actually, in this cultured cell line, the existence of a protein binding to the HE2 motif was demonstrated by a gel retardation assay. Due to the sequence homology between the HE2 motif and the Ets‐binding sites, an Ets‐related protein is a highly probable candidate for being the binding factor.
Cultured Astrocytes Release a Factor that Decreases Endothelin‐1 Secretion by Brain Microvessel Endothelial Cells
Authors: Fédérici C. , Camoin L. , Créminon C. , Chaverot N. , Strosberg A. D. , Couraud P. O.
Published: July 2010

Abstract: Endothelin‐1 (ET‐1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood‐brain barrier, have been shown to secrete ET‐1, whereas specific ET‐1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET‐1 and its precursor big ET‐1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET‐1 production was purified by gel‐exclusion and ion‐exchange chromatography as a 280‐Da iron‐containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron‐nitrogen oxide complex, may be involved in a regulatory loop of ET‐1 production at the level of the blood‐brain barrier.
Cellular Expression, Developmental Regulation, and Phylogenic Conservation of PEA‐15, the Astrocytic Major Phosphoprotein and Protein Kinase C Substrate
Authors: Danziger Nicolas , Yokoyama Midori , Jay Therese , Cordier Jocelyne , Glowinski Jacques , Chneiweiss Hervé
Published: July 2010

Abstract: PEA‐15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15‐kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity‐purified antibodies were used for immunoblotting. PEA‐15 was found mainly in the cytosol, but its protein kinase C‐phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA‐15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA‐15 also increased during development. This paralleled results obtained in primary cultures, where PEA‐15 levels increase with cell maturation. Finally, physiological importance of PEA‐15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, amphibians, and fish.
Localization of Glycine Neurotransmitter Transporter (GLYT2) Reveals Correlation with the Distribution of Glycine Receptor
Authors: Jursky Frantisek , Nelson Nathan
Published: July 2010

Abstract: We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N‐terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal‐rostral gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunoreactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.
Modulation of Glutamate and Aspartate Release from Slices of Hippocampal Area CA1 by Inhibitors of Arachidonic Acid Metabolism
Authors: Peterson Cathleen L. , Thompson Michael A. , Martin David , Nadler J. Victor
Published: July 2010

Abstract: Slices of hippocampal area CA1 were used to test inhibitors of arachidonic acid metabolism for their effects on glutamate/aspartate release from the CA3‐derived Schaffer collateral, commissural, and ipsilateral associational terminals. Test compounds [3 µM nordihydroguaiaretic acid (NDGA) and 1 µM 3‐[3‐(4‐chlorobenzyl)‐3‐tert‐butylthio‐5‐isopropylindol‐2‐yl]‐2,2‐dimethyl‐propanoic acid (MK‐886)] that reduced the production and release of 5‐lipoxygenase metabolites also selectively reduced the K+‐evoked release of aspartate. In contrast, the cyclooxygenase inhibitor indomethacin (100 µM) selectively enhanced the release of glutamate. At a concentration (100 µM) that nonselectively depressed the release of arachidonic acid and its metabolites, NDGA markedly depressed the release of aspartate, glutamate, and GABA. An inhibitor of the 12‐lipoxygenase and an inhibitor of nitric oxide synthase did not affect the K+‐evoked release of any transmitter amino acid. These results suggest that a 5‐lipoxygenase product selectively enhances aspartate release and a cyclooxygenase product selectively depresses glutamate release. They are also consistent with previous evidence that arachidonic acid and/or platelet‐activating factor enhances the release and depresses the uptake of glutamate and aspartate. The K+‐evoked release of excitatory amino acids is much more sensitive to modulation by lipid mediators than is GABA release. Activation of NMDA receptors may enhance the K+‐evoked release of glutamate and aspartate from CA1 slices by stimulating the production and release of lipid modulators.
Effects of Amphetamine on Carrier‐Mediated and Electrically Stimulated Dopamine Release in Slices of Rat Caudate Putamen and Nucleus Accumbens
Authors: Iravani M. M. , Kruk Z. L.
Published: July 2010

Abstract: The effects of (+)‐amphetamine on carrier‐mediated and electrically stimulated dopamine release were investigated using fast cyclic voltammetry in rat brain slices incorporating the nucleus accumbens, and in the caudate putamen. In the caudate putamen, dopamine release either increased with increasing frequency of local electrical stimulation (hot spots) or did not increase significantly (cold spots); dopamine release increased with increasing frequency of electrical stimulation in the nucleus accumbens. Local pressure application of (+)‐amphetamine from a micropipette caused dopamine efflux at all sites examined, and this was not affected by sulpiride, indicating that efflux of dopamine caused by (+)‐amphetamine is not regulated by dopamine D2 autoreceptors. (+)‐Amphetamine reduced single‐pulse electrically stimulated dopamine release at all sites; sulpiride reversed this decrease, indicating that endogenous dopamine released by (+)‐amphetamine activates dopamine D2 autoreceptors. In nucleus accumbens and hot spots, (+)‐amphetamine did not affect 20‐pulse 50‐Hz‐stimulated dopamine release, whereas in cold spots it potentiated 20‐pulse 50‐Hz‐stimulated dopamine release. We conclude that (+)‐amphetamine modifies electrically stimulated dopamine release by uptake inhibition or by indirect activation of D2 autoreceptors; the precise mechanism is determined by site and duration of electrical stimulation.
Intracellular Ascorbic Acid Inhibits the Na+‐Ca2+ Exchanger in Cultured Rat Astrocytes
Authors: Takuma Kazuhiro , Matsuda Toshio , Asano Shoichi , Baba Akemichi
Published: July 2010

Abstract: The effect of ascorbic acid on Ca2+ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na+‐Ca2+ exchanger in the reverse mode. Ascorbic acid at 0.1–1 mM inhibited Na+‐dependent Ca2+ uptake significantly but not Na+‐dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca2+ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na+‐dependent Ca2+ uptake was also inhibited by isoascorbate at 1 mM but not by ascorbate 2‐sulfate, dehydroascorbate, and sulfhydryl‐reducing reagents such as glutathione and 2‐mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o‐phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na+‐free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na+‐dependent Ca2+ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na+‐Ca2+ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.
Release of Neuropeptide FF, an Anti‐Opioid Peptide, in Rat Spinal Cord Slices Is Voltage‐ and Ca2+‐Sensitive: Possible Involvement of P‐Type Ca2+ Channels
Authors: Devillers J.‐P. , Labrouche S. A. , Castes E. , Simonnet G.
Published: July 2010

Abstract: Neuropeptide FF (NPFF), an FMRFamide‐like peptide with antiopioid properties, inhibits morphine‐induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage‐sensitive Na+ channels with veratridine (20 µM) induced calcium‐dependent NPFF release, which was abolished by tetrodotoxin (1 µM), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 mM K+‐induced release was blocked by Co2+ or Ni2+ (2.5 mM) but was unaffected by Ca2+ channel blockers of the L type—Cd2+ (100 µM), nifedipine or nimodipine (10 µM), diltiazem (20 µM), or verapamil (50 µM)—or the N type—ω‐conotoxin GVIA (1 µM). In contrast, ω‐agatoxin IVA (1 µM) led to a 65% reduction in NPFF release, suggesting that P‐type Ca2+ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF‐like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.
Synergistic Effects of Acetylcholine and Glutamate on the Release of Arachidonic Acid from Cultured Striatal Neurons
Authors: Tencé Martine , Murphy Nuala , Cordier Jocelyne , Prémont Joël , Glowinski Jacques
Published: July 2010

Abstract: The activation of muscarinic and NMDA receptors by carbachol and NMDA, respectively, stimulated the release of [3H]arachidonic acid ([3H]AA) from cultured striatal neurons. Striking synergistic effects were observed when both agonists were coapplied. This synergistic response was suppressed by atropine or (5R, 10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate and inhibited by magnesium. It was markedly reduced in the absence of external calcium and suppressed by mepacrine. NMDA strongly elevated the intracellular calcium concentration ([Ca2+]i), but carbachol was ineffective. Ionomycin, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate, or potassium depolarization, which increased [Ca2+]i but was ineffective on [3H]AA release, also potentiated the carbachol response. Sphingosine and Ro 31‐8220 suppressed the responses evoked by carbachol, NMDA, or both agonists. However, no synergistic responses could be observed when phorbol 12‐myristate 13‐acetate was associated with either carbachol or NMDA. Together, these results suggest that both the massive influx of calcium induced by NMDA and the coupling of muscarinic receptors with a putative phospholipase A2 are required for the strong synergistic effects of carbachol and NMDA on [3H]AA release. Synergistic effects were also observed with acetylcholine and glutamate in the presence of magnesium, further revealing the physiological relevance of this process.
Classification of Brain Tumors by Ex Vivo 1H NMR Spectroscopy
Authors: Rutter Allison , Hugenholtz Herman , Saunders John K. , Smith Ian C. P.
Published: July 2010

Abstract: Ex vivo biopsy samples (n = 42) from human brain tumors and normal brain have been examined by high‐resolution proton magnetic resonance spectroscopy. Parameters from one‐dimensional 1H spectra, two‐dimensional COSY spectra, and transverse relaxation time (T2) data were used to classify the tumors according to the histopathological diagnoses. The ratio of the area between 3.4 and 3.1 ppm to that between 1.5 and 1.1 ppm distinguished glioblastomas from astrocytomas and normal brain, and appeared to be indicative of malignant potential. In support of the one‐dimensional data, cross‐peaks in the COSY spectra of brain specimens classified glioblastomas and metastases into one group and the more benign tumors, meningiomas, astrocytomas, and normal brain into a second group. The transverse relaxation of the resonance at 1.3 ppm was fitted by a model with two T2 values. The longer T2 value could be used to distinguish glioblastomas from normal brain, the latter having a much longer long T2 value. Astrocytomas showed a continuum of T2 values between glioblastomas and normal brain, with the grade of the astrocytoma correlating roughly with the value of the long T2 component.
Antigens Associated with N‐ and L‐Type Calcium Channels in Lambert‐Eaton Myasthenic Syndrome
Authors: El Far Oussama , Marquèze Béatrice , Leveque Christian , Martin‐Moutôt Nicole , Lang Bethan , Newsom‐Davis John , Yoshida Akira , Takahashi Masami , Seagar Michael J.
Published: July 2010

Abstract: In Lambert‐Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N‐type (125I‐ω conotoxin GVIA) and L‐type ([3H]PN200‐110) calcium channels. Sera with a high antibody titer (>3 nM) against rat brain N‐type channels contained autoantibodies that immunoprecipitated neuronal and muscle L‐type channels. These IgG fractions stained a 55‐kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65‐kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.
Free Radical‐Generated Neurotoxicity of 6‐Hydroxydopamine
Authors: Kumar Raj , Agarwal Ashok K. , Seth Prahlad K.
Published: July 2010

Abstract: Albino rats were lesioned bilaterally with 6‐hydroxydopamine (6‐OHDA) hydrochloride (4 µg/µl, dissolved in saline with 0.1% ascorbic acid) into the striatum, and 72 h after the injection, levels of lipid peroxidation, GSH, superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), lipid class, membrane fluidity, and intracellular calcium concentrations were studied and the results were compared with those in the sham‐operated controls. The malonaldialdehyde level and the level of conjugated dienes were increased by 43 and 40%, respectively, in corpus striatum, and GSH, SOD, and GSH‐Px levels were decreased (24–30%) following 6‐OHDA treatment. Total phospholipid content was also decreased (18%), whereas cholesterol content remained unaffected. Among the different phospholipids only phosphatidylcholine and phosphatidylinositol were decreased in level. Membrane fluidity was decreased (23%), whereas the intracellular calcium concentration was elevated (100%) in corpus striatum compared with control rats. The results suggest that these alterations in membrane‐related events by 6‐OHDA could be due to free radical generation.