bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Calcitriol in Combination with Corticosterone, Interleukin‐1β, and Transforming Growth Factor‐β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line
Authors: Hahn Martina , Lorez Hanspeter , Fischer Günther
Published: July 2010

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin‐1β (IL‐1β) and transforming growth factor‐β1 (TGF‐β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25‐dihydroxyvitamin D3). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL‐1β, calcitriol, and TGF‐β1. Calcitriol had an additive effect when applied together with IL‐1β and a synergistic effect when applied with TGF‐β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF‐β1 but even augmented it to a level more than threefold higher than that reached with TGF‐β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL‐1β, or TGF‐β1 are elevated.
Expression of Bcl‐2 in Adult Human Brain Regions with Special Reference to Neurodegenerative Disorders
Authors: Vyas Sheela , Javoy‐Agid France , Herrero María‐Trinidad , Strada Ornella , Boissiere Florence , Hibner Urzula , Agid Yves
Published: July 2010

Abstract: The expression of the protooncogene bcl‐2, an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl‐2 protein was detected by western blot analysis and immunohistochemistry. Two bcl‐2 mRNA species were identified in the human brain. The pattern of distribution of bcl‐2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl‐2 mRNA was detected in some but not all neurons. In the regions examined for Bcl‐2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimer's and Parkinson's diseases, quantification of bcl‐2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.
Amyloid β Protein (25–35) Stimulation of Phospholipase C in LA‐N‐2 Cells
Authors: Singh Indrapal N. , Sorrentino Giuseppe , Kanfer Julian N.
Published: July 2010

Abstract: The amyloid β protein (25–35) stimulated appearance of 3H‐inositol phosphates from [3H]inositol‐prelabeled LA‐N‐2 cells was investigated. This stimulation was unaltered by extra‐ and intracellular calcium chelators in a calcium‐free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid β protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid β protein is a receptor‐mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC50 values were 12 µM for propranolol, 15 µM for AP‐3, and 25 nM for [Tyr4,d‐Phe12]bombesin. Additional support comes from results of densensitization and resensitization experiments. Amyloid β protein stimulation of phospholipase C was absent from LA‐N‐2 cells previously treated with norepinephrine, trans‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (t‐ACPD), bombesin, or amyloid β peptide. In a similar manner, LA‐N‐2 cells previously treated with amyloid β protein were no longer responsive to norepinephrine, t‐ACPD, or bombesin. The responsiveness to amyloid β protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid β protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.
Impairment of Glucose and Glutamate Transport and Induction of Mitochondrial Oxidative Stress and Dysfunction in Synaptosomes by Amyloid β‐Peptide: Role of the Lipid Peroxidation Product 4‐Hydroxynonenal
Authors: Keller Jeffrey N. , Pang Zheng , Geddes James W. , Begley James G. , Germeyer Ariane , Waeg Georg , Mattson Mark P.
Published: July 2010

Abstract: Deposits of amyloid β‐peptide (Aβ), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimer's disease. Synapse loss is closely correlated with cognitive impairments in Alzheimer's disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that Aβ causes oxyradical‐mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. Aβ induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to Aβ and decreased further with time. The lipid peroxidation product 4‐hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented Aβ‐induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of Aβ. FeSO4 (an initiator of lipid peroxidation), Aβ, and 4‐hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration‐dependent decreases in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. Aβ also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that Aβ induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4‐hydroxynonenal appears to be a mediator of these actions of Aβ. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimer's disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.
An Endogenous Dopaminergic Neurotoxin, N‐Methyl‐(R)‐Salsolinol, Induces DNA Damage in Human Dopaminergic Neuroblastoma SH‐SY5Y Cells
Authors: Maruyama Wakako , Naoi Makoto , Kasamatsu Toshio , Hashizume Yoshio , Takahashi Tsutomu , Kohda Kohfuku , Dostert Philippe
Published: July 2010

Abstract: Recently, an endogenous neurotoxin, 1(R),2(N)‐dimethyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline [N‐methyl‐(R)‐salsolinol], was found to elicit parkinsonism in rats with selective depletion of dopamine neurons in the substantia nigra without necrotic tissue reaction. The mechanism of the cell death was examined by detection of DNA damage using a single‐cell gel electrophoresis (comet) assay in human dopaminergic neuroblastoma SH‐SY5Y cells. Only N‐methylsalsolinol was found to induce DNA damage, whereas other catechol isoquinolines, such as (R)‐salsolinol, (S)‐salsolinol, and 1,2‐dimethyl‐6,7‐dihydroxyisoquinolinium ion, did not. The (R)‐enantiomer of N‐methylsalsolinol damaged DNA much more profoundly than the (S)‐enantiomer. Cycloheximide protected the cells from DNA damage, suggesting that an apoptotic process may account for the DNA damage. Morphological changes indicating apoptotic cell death were also confirmed. Antioxidants and deprenyl reduced DNA damage, indicating that the damage was initiated by oxidative stress and that neuroprotection by deprenyl may be partially ascribed to its prevention of DNA damage. Apoptosis induced by neurotoxins may be a mechanism underlying the cell death of dopamine neurons in the substantia nigra of Parkinson's disease.
Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+‐ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration
Authors: Kawai Hiromichi , Yasuda Hitoshi , Terada Masahiko , Omatsu‐Kanbe Mariko , Kikkawa Ryuichi
Published: July 2010

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na+,K+‐ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na+,K+‐ATPase was highly resistant to ouabain. The ouabain‐resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na+,K+‐ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na+,K+‐ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na+,K+‐ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.
Hypoxia‐Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells
Authors: Mochizuki‐Oda Noriko , Takeuchi Yuko , Matsumura Kiyoshi , Oosawa Yoshio , Watanabe Yasuyoshi
Published: July 2010

Abstract: Hypoxia (5% O2) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca2+ concentration ([Ca2+]i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca2+. Nifedipine and ω‐conotoxin decreased the population of the cells that showed the hypoxia‐induced [Ca2+]i increase, showing that the Ca2+ influx was attributable to L‐ and N‐type voltage‐dependent Ca2+ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O2). Membrane resistance increased twofold under the hypoxic condition. The current‐voltage relationship showed a hypoxia‐induced decrease in the outward K+ current. Among the K+ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP‐sensitive K+ channels, inhibited the hypoxia‐induced [Ca2+]i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP‐sensitive K+ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K+ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia‐induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen‐sensing mechanisms.
Interactions Between Neuronal Histamine and Halothane Anesthesia in Rats
Authors: Mammoto Tadanori , Yamamoto Yumiko , Kagawa Kiyokazu , Hayashi Yukio , Mashimo Takashi , Yoshiya Ikuto , Yamatodani Atsushi
Published: July 2010

Abstract: Using an in vivo microdialysis method, we measured the release of histamine in the anterior hypothalamic area (AHy) of rats under several concentrations of halothane anesthesia (1, 0.5, and 0.2%). The release of histamine increased to 341 and 325% at halothane concentrations of 0.5 and 0.2%, compared with the basal level at anesthesia induced by 1% halothane. α‐Fluoromethylhistidine (100 mg/kg i.v.), a specific and irreversible inhibitor of histidine decarboxylase, reduced the histamine release to <35% of the basal value at 1% halothane anesthesia in the AHy, and also decreased the anesthetic requirement for halothane, evaluated as the minimum alveolar concentration (MAC), by 26%. Furthermore, pyrilamine (20 mg/kg i.v.), a brain‐penetrating H1 antagonist, and zolantidine (20 mg/kg i.v.), a brain‐penetrating H2 antagonist, reduced the MAC for halothane by 28.5 and 16%, respectively. Although thioperamide (5 mg/kg i.v.), an antagonist of presynaptic H3 autoreceptor, induced an approximate twofold increase in the level of histamine release in conscious freely moving rats, the same dose of thioperamide had little effect on the release of histamine under 1% halothane anesthesia in the AHy. Furthermore, thioperamide did not change the anesthetic requirement (MAC) for halothane. The present findings indicate that halothane anesthesia inhibits the release of neuronal histamine and that histaminergic neuron activities change the anesthetic requirement (MAC) for halothane through H1 as well as H2 receptors.
Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5‐Hydroxytryptamine2C Receptors
Authors: Marcoli Manuela , Maura Guido , Tortarolo Massimo , Raiteri Maurizio
Published: July 2010

Abstract: Previous studies have shown that 5‐hydroxytryptamine (5‐HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5‐HT receptors. The aim of this study was to subclassify the 5‐HT2 receptor mediating inhibition of the cyclic GMP response elicited by N‐methyl‐d‐aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5‐HT2A, 5‐HT2B, and 5‐HT2C subtypes, differentially affected the inhibition by (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5‐HT2C subtype.
Transport of Iron in the Blood‐Brain‐Cerebrospinal Fluid System
Authors: Bradbury M. W. B.
Published: July 2010

Abstract: Iron is an important constituent in brain and, in certain regions, e.g., the basal nuclei, reaches concentrations equivalent to those in liver. It has a role in electron transfer and is a cofactor for certain enzymes, including those involved in catecholamine and myelin synthesis. Iron in CSF is likely to be representative of that in interstitial fluid of brain. Transferrin in CSF is fully saturated, and the excess iron may be loosely bound as Fe(II). Brain iron is regulated in iron depletion, suggesting a role for the blood‐brain barrier (BBB). Iron crosses the luminal membrane of the capillary endothelium by receptor‐mediated endocytosis of ferric transferrin. This results in an initial linear uptake of radioactive iron into brain at an average rate relative to serum of about 3.3 × 10−3 ml·g of brain−1·h−1 in the adult rat. This corresponds to about 80 nmol·kg−1·h−1. Much higher rates occur in the postnatal rat. These increase during the first 15 days of life and decline thereafter. Within the endothelium, most of the iron is separated from transferrin, presumably by the general mechanism of acidification within the endosome. Iron appears to be absorbed from the vesicular system into cytoplasm and transported across the abluminal plasma membrane into interstitial fluid as one or more species of low molecular weight. There is some evidence that ionic Fe(II) is involved. Certainly Fe(II) ions presented on the luminal side rapidly cross the complete BBB, i.e., luminal and abluminal membranes. Within interstitial fluid, transported iron will bind with any unsaturated transferrin synthesized or transported into the brain‐CSF system. Oligodendrocytes are one site of synthesis. From interstitial fluid, ferric transferrin is taken up by neurones and glial cells by the usual receptor‐mediated endocytosis. Calculations of the amount of iron leaving the system with the bulk flow of CSF indicate that most iron entering brain across the capillary endothelium finally leaves the system with the bulk outflow of CSF through arachnoid villi and other channels. A system in which influx of iron into brain is by regulated receptor‐mediated transport and in which efflux is by bulk flow is ideal for homeostasis of brain iron.
Cloning and Localization of Exon 5‐Containing Isoforms of the NMDAR1 Subunit in Human and Rat Brains
Authors: Nash Norman R. , Heilman Craig J. , Rees Howard D. , Levey Allan I.
Published: July 2010

Abstract: Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N‐terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1‐3b (hNMDA1‐1) and NMDAR1‐4b (hNMDA1‐2), both bearing the insert, is reported here. A monoclonal antibody generated against the N‐terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of ∼115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21‐amino acid N‐terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5‐containing isoforms of the NMDAR1 subunit in both rat and human brain.
Brain Tissue Acidosis: Effects on the Extracellular Concentration of N‐Acetylaspartate
Authors: Gotoh M. , Davies S. E. C. , Obrenovitch T. P.
Published: July 2010

Abstract: N‐Acetylaspartate (NAA) is characterized by a high tissue‐to‐extracellular concentration ratio under normal conditions and is released from neurons during hyposmotic cell swelling. As cell volume regulation and acid‐base homeostasis share common processes, we have examined by microdialysis whether the extracellular concentration of NAA is altered by various acidotic challenges. Twenty‐minute perfusion of 50 mM NH4+ through the microdialysis probe progressively lowered dialysate pH by 0.18, followed by a sudden, additional reduction after NH4+ removal. The latter effect indicated extrusion of cellular H+ because it was suppressed by blockade of Na+/H+ exchange with 5‐(N,N‐dimethyl)amiloride (1 or 5 mM in perfusion medium). NH4+ increased dialysate levels of NAA and lactate by approximately two‐ and threefold their initial values, respectively. These data demonstrate that pronounced intracellular acidosis is associated with NAA efflux, presumably from neurons. Whether this effect is linked directly to acid‐base homeostasis or is secondary to acidosis‐induced cell swelling remains to be clarified. Hypercapnia and perfusion of acid medium failed to increase dialysate NAA, probably because acidosis was not severe enough or the associated cellular swelling was not followed by regulatory volume decrease. As cellular swelling and acidosis are key features of cerebral ischaemia, further investigations into the role of NAA, and the development of sophisticated magnetic resonance spectroscopic methods capable of resolving intra‐/extracellular NAA redistribution, would be especially relevant to clinical practice.
Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain
Authors: Oda Haruko , Murayama Toshihiko , Nomura Yasuyuki
Published: July 2010

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF‐α/IL‐1β. The inhibitory effect of ET on TNF‐α/IL‐1β‐stimulated iNOS expression appears to be mediated by ETB receptors, because (1) both ET‐1 and ET‐3 inhibited the effects of TNF‐α/IL‐1β on iNOS expression and nitrite accumulation, (2) a selective ETB receptor agonist, Suc‐[Glu9,Ala11,15]‐ET‐1 (8–21) (IRL1620), decreased the effects of TNF‐α/IL‐1β, and (3) a selective ETB receptor antagonist, N‐cis‐2,6‐dimethylpiperidinocarbonyl‐l‐γ‐methylleucyl‐d‐1‐methoxycarbonyltryptophanyl‐d‐norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET‐3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP‐elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression via ETB receptors and that cyclic AMP may be involved in this process.
Hydrogen Peroxide‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+‐Dependent Protein Tyrosine Kinase
Authors: Ito Yuzuru , Nakashima Shigeru , Nozawa Yoshinori
Published: July 2010

Abstract: The mechanism for hydrogen peroxide (H2O2)‐induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid‐labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration‐ or time‐dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31‐8220, did not inhibit but rather slightly enhanced H2O2‐induced PLD activity. Thus, H2O2‐induced PLD activation is considered to be independent of the PLC‐PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration‐dependent inhibition of H2O2‐induced PLD activation. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2‐induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2‐induced PLD activation in a concentration‐dependent manner. Taken together, these results suggest that a certain Ca2+‐dependent protein tyrosine kinase(s) somehow participates in H2O2‐induced PLD activation in PC12 cells.
p53‐Knockout Mice Are Protected Against the Long‐Term Effects of Methamphetamine on Dopaminergic Terminals and Cell Bodies
Authors: Hirata Hiroshi , Cadet Jean Lud
Published: July 2010

Abstract: p53‐knockout mice provide a useful model to test the role of p53 in the neurotoxic effects of drugs in vivo. To test the involvement of p53 in methamphetamine (METH)‐induced toxicity, wild‐type mice, as well as heterozygous and homozygous p53‐knockout male mice, were administered four injections of three different doses (2.5, 5.0, and 10.0 mg/kg) of the drug given at 2‐h intervals within the space of 1 day. METH caused a marked dose‐dependent loss of dopamine transporters in both the striatum and the nucleus accumbens of wild‐type mice killed 2 weeks after drug administration. However, this METH‐induced decrease in dopamine transporters was attenuated in both homozygous and heterozygous p53‐knockout mice, with homozygous animals showing significantly greater protection. The possibility for p53 involvement in METH‐induced toxicity was also supported by the observation that METH caused marked increases in p53‐like immunoreactivity in the striata of wild‐type mice and very little change in heterozygous p53‐knockout mice, whereas no p53‐like immunostaining was detected in the homozygous p53‐knockout mice. Further support for p53 involvement was provided by the fact that METH treatment caused significant decreases in dopamine transporter mRNA and the number of tyrosine hydroxylase‐positive cells in the substantia nigra pars compacta and the ventral tegmental area of wild‐type but not homozygous p53‐knockout mice killed 2 weeks after cessation of METH administration. These results provide concordant evidence for a role of the tumor suppressor, p53, in the long‐term deleterious effects of a drug acting on brain dopamine systems.
Two‐Dimensional Characterization of Paired Helical Filament‐Tau from Alzheimer's Disease: Demonstration of an Additional 74‐kDa Component and Age‐Related Biochemical Modifications
Authors: Sergeant N. , David J.‐P. , Goedert M. , Jakes R. , Vermersch P. , Buée L. , Lefranc D. , Wattez A. , Delacourte A.
Published: July 2010

Abstract: PHF‐tau proteins are the major components of the paired helical filament (PHF) from Alzheimer's disease (AD) neurofibrillary lesions. They differ both qualitatively and quantitatively in their degree of phosphorylation when compared with native tau proteins. However, little is known about the extent and heterogeneity of phosphorylated sites or the isoform composition and the isoelectric variants of PHF‐tau. Therefore, we have characterized PHF‐tau proteins from cortical brain tissue homogenates of 13 AD patients using two‐dimensional gel electrophoresis. Whatever the topographical origin of brain tissue homogenates, PHF‐tau proteins shared the same two‐dimensional gel electrophoresis profile made of a tau triplet of 55, 64, and 69 kDa. A 74‐kDa hyperphosphorylated tau component was detected particularly in the youngest and most severely affected AD patients. This additional component of hyperphosphorylated tau was shown to correspond to the longest brain tau isoform. Furthermore, the isoelectric points of PHF‐tau from older AD patients were significantly more basic, indicating a lower degree of phosphorylation. These results show that the severity of neurofibrillary degeneration of AD is modulated by age.
Mechanisms of Inhibition of Calmodulin‐Stimulated Cyclic Nucleotide Phosphodiesterase by Dihydropyridine Calcium Antagonists
Authors: Sharma Rajendra K. , Wang Jerry H. , Wu Z.
Published: July 2010

Abstract: Calmodulin‐dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interaction between the cyclic nucleotide and Ca2+ second‐messenger systems. CaMPDE exists as tissue‐specific isozymes, and initially these isozymes were designated according to their respective subunit molecular mass. A variety of pharmacological agents have been used to inhibit CaMPDE, and this inhibition occurs mostly via Ca2+‐dependent association with the proteins. We have examined the effect of dihydropyridine Ca2+‐channel blockers felodipine and nicardipine on CaMPDE. The results suggest that the 63‐kDa (PDE 1B1) and 60‐kDa (PDE 1A2) CaMPDE isozymes are inhibited by felodipine and nicardipine by partial competitive inhibition and that these two Ca2+ antagonists appear to counteract each other. This study further demonstrates the existence of a specific site, distinct from the active site on CaMPDE, that exhibits high‐affinity binding of these drugs. Felodipine and nicardipine have similar affinities for 60‐kDa CaMPDE isozymes but bring about different levels of enzyme inhibition, suggesting the possibility of designing specific drugs that can protect the enzyme from inhibition by dihydropyridine Ca2+‐channel blockers.
Lipopolysaccharide and Interleukin‐1β Augmented Histidine Decarboxylase Activity in Cultured Cells of the Rat Embryonic Brain
Authors: Niimi Mitsuhiro , Yamamoto Yumiko , Takemori Hiroshi , Uno Atsuhiko , Kondo Yuki , Yamatodani Atsushi
Published: July 2010

Abstract: We investigated the effect of lipopolysaccharide (LPS) and various inflammatory cytokines on the histidine decarboxylase (HDC) activity in cultured cells of the rat embryonic brain. Histaminergic neuronal cell bodies were supposed to exist in cultured cells of the diencephalon but not in those of the cortex. The HDC activity was elevated by adding LPS and interleukin‐1 β (IL‐1β) but not by tumor necrosis factor‐α (TNF‐α) and IL‐6 to the mixed primary cultures of diencephalon. In the adherent cell fraction of the cultured diencephalon cells, HDC activity was also enhanced by LPS and IL‐1β. In a similar manner, LPS augmented HDC activity in the mixed primary culture of cerebral cortical cells and in its adherent cell fraction. The effects of IL‐1β but not LPS in the mixed primary culture of diencephalon were canceled by a prior exposure to cytosine‐β‐d‐arabinofuranoside. The changes in HDC activity after exposure to LPS for 12 h were not accompanied by increased mRNA levels. In these cell cultures, mast cells were not detected by Alcian Blue staining. These results indicated the presence of the third type of HDC‐bearing cell besides neurons and mast cells in the brain. The increase of HDC activity by IL‐1β might be due to cell proliferation.
Effect of Cocaine on the Histaminergic Neuron System in the Rat Brain
Authors: Ito Chihiro , Onodera Kenji , Sakurai Eiko , Sato Mitsumoto , Watanabe Takehiko
Published: July 2010

Abstract: To examine the effect of cocaine on the brain histamine neuron system, histamine levels and histamine N‐methyl‐transferase activity in the rat brain were measured after the administration of cocaine. Moreover, we examined the effect of l‐histidine on cocaine‐induced wheel‐running behavior. The administration of cocaine (20 mg/kg) increased histamine levels and histamine N‐methyltransferase activity in the striatum, nucleus accumbens, and amygdala 1 h later. The pretreatment with l‐histidine (350 and 700 mg/kg) inhibited the cocaine (20 mg/kg)‐induced increase of wheel‐running activity in a dose‐dependent manner. These findings suggest that cocaine activates the brain histamine neuron system, which may play the role of inhibiting the cocaine‐induced wheel‐running behavior.
A Kinase Insert Isoform of Rat TrkA Supports Nerve Growth Factor‐Dependent Cell Survival but Not Neurite Outgrowth
Authors: Meakin Susan O. , Gryz Ela A. , MacDonald James I. S.
Published: July 2010

Abstract: To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14‐amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC(Ki14) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild‐type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Ki14). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)‐mediated SHC tyrosine phosphorylation and a loss of high‐affinity TrkA‐SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF‐dependent tyrosine phosphorylation of the signaling molecules phospholipase Cγ‐1, MAP kinase/ERK‐1, and SNT. TrkAKi receptors internalize NGF comparable to wild‐type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3‐kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF‐dependent cell survival under serum‐free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate‐early genes (NGF1A, NGF1B, and c‐fos), suggesting that these gene products are not required for NGF‐dependent cell survival responses.
Neuronal Nitric Oxide Synthase and Calmodulin‐Dependent Protein Kinase IIα Undergo Neurotoxin‐Induced Proteolysis
Authors: Hajimohammadreza Iradj , Raser Kadee J. , Nath Rathna , Nadimpalli Ravi , Scott Michele , Wang Kevin K. W.
Published: July 2010

Abstract: Calpain (calcium‐activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N‐methyl‐d‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m‐calpain autolytic fragmentation and nonerythroid α‐spectrin breakdown. Under the same conditions, calmodulin‐dependent protein kinase II‐α (CaMPK‐IIα) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl‐Leu‐Leu‐Nle‐CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified μ‐ and m‐calpain produced fragmentation patterns for CaMPK‐IIα and nNOS similar to those produced in situ. Also, several other calpain‐sensitive calmodulin‐binding proteins (plasma membrane calcium pump, microtubule‐associated protein 2, and calcineurin A) and protein kinase C‐α are also degraded in neurotoxin‐treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK‐IIα and nNOS. The degradation of CaMPK‐IIα, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.
A Multisubstrate Kinetic Mechanism of Dopamine Transport in the Nucleus Accumbens and Its Inhibition by Cocaine
Authors: Povlock Sue L. , Schenk James O.
Published: July 2010

Abstract: Kinetic studies of dopamine transport into suspensions of nucleus accumbens (NAcc) and effects of Na+ and Cl− as cosubstrates were performed using rotating disk electrode voltammetry. To mimic chemical neurotransmission, dopamine was added as a rapid pulse, and transporter‐mediated clearance of dopamine was evaluated kinetically. This paradigm was shown to approximate a zero trans entry transport experiment. Dopamine was taken up with apparent Km and Vmax values of 1.3 µM and 375 pmol/s/g wet weight, respectively. Transport exhibited apparent trans acceleration. Substitution of Na+ with choline or Cl− with isethionate reduced dopamine transport with reaction orders of two and unity, respectively, accompanied by reductions in Vmax with no changes in Km. Apparent KNa and KCl values were 70.0 and 92.1 mM, respectively. Dopamine transport in NAcc was found to follow a partially random, sequential mechanism in which dopamine and Na+ bind randomly to the transporter followed by binding of Cl− before transport. Cocaine inhibited dopamine transport and the influences of the other substrates allosterically with an overall Ki of 0.30 µM. Thus, the general kinetic mechanism of the transport of dopamine in the NAcc is identical to that previously reported by this laboratory for dopamine transport in the striatum. However, the dopamine transporter in the NAcc is more tightly regulated by Na+, possesses a higher kinetic turnover rate, is four times more sensitive to cocaine than the striatal transporter, and exhibits cocaine inhibition independent of [substrate]. These findings suggest that cocaine modulates chemical signaling in NAcc differently than in striatum, providing down‐regulation of function irrespective of [substrate], thereby enhancing dopaminergic signaling more robustly in the NAcc than in the striatum.
Regulation of Actin and Its mRNA by Thyroid Hormones in Cultures of Fetal Human Brain During Second Trimester of Gestation
Authors: Pal Utpal , Biswas Subhas C. , Sarkar Pranab K.
Published: July 2010

Abstract: The effect of thyroid hormones (THs) on the expression of actin gene during fetal human brain development and the period of sensitivity to the hormones have been investigated. Developmental profile of actin in the cytoskeletal (CSK) and noncytoskeletal (non‐CSK) fractions in the fetal cerebra showed a pronounced rise in the level of CSK actin at weeks 17–19. Northern blot analysis also revealed a sharp rise in the level of actin mRNA at weeks 16–18, temporally coinciding with the period of rise of THs and peak expression of TH receptors in the fetal brain. In organ cultures of weeks 13–23 fetal cerebra, THs elicited a general stimulation of CSK proteins at all ages studied with a preferential effect on actin at weeks 17–19. During this period, THs also stimulated the rate of synthesis of actin. Kinetics of induction of actin by TH in the non‐CSK and CSK fractions in organ cultures of week 17 fetal cerebra showed an increased level of actin in both fractions within 1 h. Subsequently (at 5 and 18 h), induction was evident only in the insoluble CSK fraction, suggesting an effect of the hormone on the intracellular distribution of actin between the soluble non‐CSK fraction and the insoluble CSK fraction. Correspondingly, in cultures of week 17 fetal cerebra, THs elicited an increase in actin mRNA level within 30 min of hormonal exposure. The overall results suggest that THs regulate the expression of actin gene by stimulating the rate of synthesis as well as intracellular distribution of actin during the mid phase of the second trimester of gestation.
Selective Aggregation of Endogenous β‐Amyloid Peptide and Soluble Amyloid Precursor Protein in Cerebrospinal Fluid by Zinc
Authors: Brown Abraham M. , Tummolo Donna M. , Rhodes Kenneth J. , Hofmann John R. , Jacobsen J. Steven , Sonnenberg‐Reines June
Published: July 2010

Abstract: Zinc added to buffered solutions of synthetic β‐amyloid peptide (Aβ) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain Aβ, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of Aβ in freshly drawn canine CSF, which contains the same sequence as human Aβ. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous Aβ in CSF, with an EC50 of 120–140 µM. The reaction was specific, because most (≥95%) CSF protein remained soluble under conditions where most Aβ was insoluble, as assayed by scanning densitometry of Coomassie‐stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid‐related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for Aβ (EC50 80 µM). The possibility that zinc‐induced aggregation of APP may precede the deposition of Aβ into plaques is discussed. Investigation of aggregation of Aβ in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.
Adenosine Triphosphate Degradation Products After Oxidative Stress and Metabolic Dysfunction in Cultured Retinal Cells
Authors: Rego Ana Cristina , Santos Maria S. , Oliveira Catarina R.
Published: July 2010

Abstract: The alteration in energy metabolic products was analyzed in cultured retinal cells submitted to oxidative stress, hypoxia, glucopenia, or ischemia‐like conditions. Ischemia highly reduced cellular ATP and increased AMP formation, without significant changes in ADP. Ischemia induced a significant increase in extracellular adenosine (ADO) and hypoxanthine (HYP), and to a lesser extent inosine (INO). Glucopenia reduced cellular ATP by about two‐ to threefold, which was not compensated for by AMP formation. Under glucopenia, extracellular ADO and HYP were significantly increased, although a major increase in extracellular INO was observed. 5‐(4‐Nitrobenzyl)‐6‐thioinosine (10 µM) reduced extracellular ADO during glucopenia or ischemia by ∼80%, indicating that ADO accumulation occurs mainly via the transporter. Intracellular ATP, ADP, or AMP and extracellular ADO, INO, or HYP were not apparently changed after oxidative stress or hypoxia. Nevertheless, in the presence of 10 µM erythro‐9‐(2‐hydroxy‐3‐nonyl)adenosine, oxidative stress was shown to increase significantly the accumulation of ADO, which was reduced in the presence of 200 µMα,β‐methyleneadenosine 5′‐diphosphate, suggesting that ADO accumulation after oxidative stress may result from extracellular degradation of adenine nucleotides. The increase in ADO accumulation resulting from the depletion of cellular ATP was directly related to the release of endogenous glutamate occurring through a Ca2+‐independent pathway after ischemia. Increased metabolic products derived from ATP are suggested to exert a modulating effect against excitotoxic neuronal death.
Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum
Authors: Ikarashi Yasushi , Takahashi Akira , Ishimaru Hirohisa , Arai Tadashi , Maruyama Yuji
Published: July 2010

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D2 antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D2 agonist (±)‐2‐(N‐phenylethyl‐N‐propyl)amino‐5‐hydroxytetralin (N‐434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µM hemicholinium‐3 (HC‐3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug‐treated groups. The degree of relative increase in the level of Ch induced by HC‐3 differed among the drug‐pretreated groups; compared with the control group, the relative increase was larger in the sulpiride‐ and atropine‐treated groups and smaller in the N‐434 and pentobarbital‐treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high‐affinity choline uptake.
Functional Coupling of Endogenous Serotonin (5‐HT1B) and Calcitonin (C1a) Receptors in CHO Cells to a Cyclic AMP‐Responsive Luciferase Reporter Gene
Authors: George S. E. , Bungay P. J. , Naylor L. H.
Published: July 2010

Abstract: A cyclic AMP‐responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose‐dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein‐coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5‐HT) receptor agonist ligands caused an inhibition of forskolin‐stimulated luciferase expression in the rank order 5‐carboxamidotryptamine > 5‐HT > sumatriptan > 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin. The response to 5‐HT was reversed by the 5‐HT1 receptor antagonists cyanopindolol and pindolol, but not the 5‐HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene‐related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8–37). These results were consistent with the presence of 5‐HT1B‐like and calcitonin (C1a‐like) receptors in CHO cells, with the responses to 5‐HT and CGRP being pertussis and cholera toxin‐sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein‐coupled receptors linked to adenylyl cyclase.
Studies of Inositol Phosphate Export from Neuronal Tissue In Vitro
Authors: Roberts Richard E. , Marsden Charles A. , Kendall David A.
Published: July 2010

Abstract: Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] that can be increased in a concentration‐dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P3 could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P3 levels after stimulation with 1 mM carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH‐SY5Y cells. Suprafusion of myo‐[3H]inositol‐prelabelled hippocampal slices with 1 mM carbachol caused an increase in 3H‐inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 ± 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 mM K+. Analysis of the individual 3H‐inositol phosphates in the perfusate revealed that levels of [3H]inositol monophosphate, [3H]inositol bisphosphate, [3H]inositol trisphosphate, and [3H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular 3H‐inositol phosphates was demonstrated in SH‐SY5Y cells incubated with 1 mM carbachol for 30 min. This response was again enhanced by 30 mM K+, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.
Dopamine Transporter Is Required for In Vivo MPTP Neurotoxicity: Evidence from Mice Lacking the Transporter
Authors: Gainetdinov Raul R. , Fumagalli Fabio , Jones Sara R. , Caron Marc G.
Published: July 2010

Abstract: The neurotoxic effect of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29‐fold increase in the level of GFAP mRNA in the striatum of wild‐type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1‐methyl‐4‐phenylpyridinium (MPP+; 10 mM), via a microdialysis probe produced a massive efflux of DA in wild‐type mice (>320‐fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.