bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormality in Translational Regulation of Catalase Expression in Disorders of Peroxisomal Biogenesis
Authors: Singh Inderjit , Kremser Karl , Ghosh Bidyut , Singh Avtar K. , Pai Shashidhar
Published: July 2010

Abstract: Peroxisomal disorders are a newly described group of inherited neurological diseases. In disorders of peroxisomal biogenesis, e.g., Zellweger syndrome, owing to the lack of peroxisomes, catalase, a peroxisomal enzyme, is found to be present in the cytoplasm instead. We observed higher catalase activity (7.59 ± 0.41 mU/mg of protein) in cultured skin fibroblasts from Zellweger patients than in control fibroblasts (4.45 ± 0.29 mU/mg of protein). Moreover, we also found that the majority of the catalase in Zellweger cells was present in the inactive form. The specific activities following reactivation in Zellweger and control cells were 12.1 and 4.9 mU/mg of protein, respectively. To understand the molecular basis of higher levels of catalase in Zellweger than control cells, we examined the rate of synthesis and turnover of catalase and levels of catalase mRNA and protein levels in Zellweger cells as compared with control cells. The initial rates of synthesis of catalase in Zellweger (1.68 ± 0.15 mU/mg of protein) and control (1.51 ± 0.14 mU/mg of protein) cells were similar. The rates of turnover of catalase in Zellweger (t1/2 = 47 ± 8 h) and control (t1/2 = 49 ± 7 h) were also similar. Consistent with the enzyme activity, the levels of catalase protein were higher in Zellweger cells as compared with control cells. On the other hand, there was no difference in the level of catalase mRNA between control and Zellweger cells. Although the rate of synthesis in Zellweger and control cells were initially similar, it was down‐regulated to a lower level at ∼72 h of culture in control fibroblasts as compared with Zellweger cells, which continued to synthesize catalase at the same rate up to 5 days in culture. The presence of similar levels of mRNA in control and Zellweger cells and continued synthesis of catalase in Zellweger cells at a higher level as compared with control cells suggest a loss of regulation at the translational level.
Slow Delivery of the Selective Cholecystokinin Agonist pBC 264 into the Rat Nucleus Accumbens Using Microspheres
Authors: Blanco‐Príeto M. J. , Durieux C. , Daugé V. , Fattal E. , Couvreur P. , Roques B. P.
Published: July 2010

Abstract: Neuropeptides have been shown to play a critical role in adaptational processes, probably by long‐term modulation of neuronal pathways. It could therefore be interesting to study behavioral changes induced by chronic local stimulation of neuropeptide receptors. With this aim poly(lactide‐co‐glycolide) microspheres loaded with a highly potent, peptidase‐resistant, cholecystokinin (CCK)‐B‐selective CCK peptidomimetic agonist (pBC 264) were prepared by a water in oil in water emulsion solvent evaporation method and stereotaxically implanted into the anterior part of the rat nucleus accumbens. Two different kinds of loaded polymeric microspheres differing only by the stabilizing agent [ovalbumin (OVA) or Pluronic F 68] added to the inner emulsion were used. The histological and behavioral studies done 24 h and 8 days after implantation of nonloaded microspheres in the nucleus accumbens indicated that the microspheres were well tolerated. The in vivo release of the selective CCK‐B agonist pBC 264 (associated with a tracer dose of [3H]pBC 264) from microspheres prepared with OVA was very fast (92% after 6 h), whereas only 26% (88 pmol) of pBC 264 was released from the formulation with Pluronic F 68 after 24 h. Eight days after implantation 36% of pBC 264 had diffused from the microspheres, and 8% (∼30 pmol) was still present in the brain concentrated around the site of administration. In all cases the released material was found to correspond to intact pBC 264, thus demonstrating the possibility of obtaining a slow controlled release of peptide in vivo. This method opens up interesting perspectives to study the long‐term effects of neuropeptides.
Developmental Alteration of the Expression and Kinase Activity of Cyclin‐Dependent Kinase 5 (Cdk5)/p35nck5a in the Rat Retina
Authors: Hirooka Kazuyuki , Tomizawa Kazuhito , Matsui Hideki , Tokuda Masaaki , Itano Toshifumi , Hasegawa Eiichi , Wang Jerry H. , Hatase Osamu
Published: July 2010

Abstract: Neuronal Cdc2‐like kinase has been purified from the bovine brain as a proline‐directed serine/threonine kinase. This kinase is a heterodimer of Cdk5 and p35nck5a and influences neuronal maturation or sprouting in the normal brain. In this study, we showed the expression of Cdk5/p35nck5a kinase in the developing rat retina. The expression of Cdk5 and p35nck5a increased between 1 week and 3 weeks after birth. These expression levels were most prominent from 2 weeks to 3 weeks after birth and decreased after 4 weeks. The developmental change of Cdk5/p35nck5a kinase activity coincided with those of the expression of p35nck5a and Cdk5. An immunohistochemical study showed that Cdk5 was expressed in the ganglion cells and in some cells in the inner nuclear layer at an early stage. With retinal development, Cdk5 was expressed in the inner plexiform layer also. In the adult, the expression of Cdk5 was restricted to the inner plexiform layer and to some cells in the inner nuclear layer. These changes of localization in the developing retina were very close to those of B‐50/GAP‐43. On the other hand, the expression of p35nck5a was restricted to the soma of the neuron in the developing retina. This subcellular localization in the developing retina agreed with that in the developing rat brain. The expression levels of Cdk5 and p35nck5a in retina of rats raised from fetus to 3 weeks after birth in darkness were 36% and 40% respectively, of the baseline for control rats. Moreover, the kinase activity in rats raised in darkness was lower than that in control rats. These data suggest that Cdk5/p35nck5a may play a role in neuronal plasticity in the developing rat retina.
Ret 1, a Cis‐Acting Element of the Rat Opsin Promoter, Can Direct Gene Expression in Rod Photoreceptors
Authors: Yu Xiu , Leconte Laurence , Martinez Julian A. , Barnstable Colin J.
Published: July 2010

Abstract: The Ret 1 element, located at −136 to −110 in the rat opsin promoter, binds developmentally regulated retinal nuclear proteins. A similar sequence is found upstream of opsin genes, from humans to Drosophila, as well as many other photoreceptor‐specific genes. The function of the Ret 1 element was tested both in vitro and in two sets of transgenic mice. A mutated Ret 1 element did not bind retinal nuclear proteins in vitro. The same mutations in an otherwise normal 1.9‐kb rat opsin promoter failed to drive expression of a lacZ reporter gene in nine of 12 lines. In the three other lines, expression in photoreceptors was very faint. Four tandem copies of the Ret 1 element maintained the Ret 1 binding specificity in vitro and were able to direct expression of a lacZ transgene in photoreceptors of all nine mouse lines obtained. In two lines, expression was also detected in the ganglion cell layer and the ciliary epithelium. In three lines, a characteristic pattern of expression was found in the nervous system in addition to the normal retinal expression. These results indicate that Ret 1 can and is necessary to drive gene expression in rod photoreceptors. Furthermore, our results suggest that Ret 1‐like elements may also be important in the developing nervous system.
Stimulation of Guanosine 5′‐O‐(3‐[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb
Authors: Olianas Maria C. , Onali Pierluigi
Published: July 2010

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide‐binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 mM NaCl. Exposure to N‐ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [35S]GTPγS binding elicited by pituitary adenylate cyclase‐activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [35S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [35S]GTPγS binding in a concentration‐dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [35S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to Gi/Go, but not to Gs, and support the possibility that activation of Gi/Go mediates the stimulatory effect on adenylyl cyclase activity.
Characterization of Phospholipase A2 Activity Enriched in the Nerve Growth Cone
Authors: Nègre‐Aminou Pascale , Nemenoff Raphael A. , Wood Malcolm R. , De La Houssaye B. A. , Pfenninger K. H.
Published: July 2010

Abstract: Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well‐characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC‐ and PE‐selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at ∼65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI‐selective from a PE‐selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI‐selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane‐associated in the presence of Ca2+. However, its catalytic activity is Ca2+‐independent regardless of whether the enzyme is associated with pure substrate or mixed‐lipid growth cone vesicles. The PE‐selective phospholipase A2 in growth cones was studied in less detail but shares with the PI‐selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane‐associated forms, and Ca2+ independence. Our data indicate growth cones contain two high‐molecular‐weight forms of phospholipase A2 that share many properties with known, Ca2+‐independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI‐selective enzyme may represent a new member of the growing family of cytoplasmic phospholipase A2. The enrichment of the PI‐selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function.
Histaminergic H2 Action Protects Hippocampal CA1 Neurons by Prolonging the Onset of the Anoxic Depolarization in Gerbils
Authors: Fujitani Taro , Adachi Naoto , Nagaro Takumi , Miyazaki Hirofumi , Nakamura Yoichi , Kataoka Kiyoshi , Arai Tatsuru
Published: July 2010

Abstract: The central histaminergic action on ischemia‐induced neuronal damage was examined by evaluating the histological outcome and the direct current (DC) potential shift in the hippocampal CA1 region in gerbils. An intracerebroventricular administration of histamine (10–100 nmol) improved the delayed ischemic damage in hippocampal CA1 pyramidal cells produced by 3 min of transient forebrain ischemia. A high dose (75 nmol) of mepyramine, an H1 antagonist, aggravated ischemia‐induced neuronal damage, but not a low dose (0.75 nmol). Administration of cimetidine (4 nmol) and ranitidine (3 nmol), H2 antagonists, aggravated the neuronal damage. An injection of histamine (100 nmol) prolonged the onset time of the ischemia‐induced sudden shift in the extracellular DC potential (anoxic depolarization; AD) to 133% of that in control animals. Administration of mepyramine (75 nmol) did not markedly change the AD, whereas injections of cimetidine (40 nmol) and ranitidine (3 nmol) reduced the onset latency to 47 and 45%, respectively. These findings suggest that the central H2 action serves to protect neurons by delaying the onset of AD in gerbils.
A Bioluminescent γ‐Aminobutyrate Assay for Monitoring Its Release from Inhibitory Nerve Endings
Authors: Israël M. , Lesbats B.
Published: July 2010

Abstract: A bioluminescent GABA assay is described. The principle of the procedure is based on the action of GABASE (GABA‐aminotransferase plus succinic semialdehyde dehydrogenase), coupled to the detection of succinic semialdehyde and NADH, using Photobacterium luciferase. The method was used for monitoring GABA release from depolarized brain slices.
l‐Deprenyl Protects Mesencephalic Dopamine Neurons from Glutamate Receptor‐Mediated Toxicity In Vitro
Authors: Mytilineou Catherine , Radcliffe Pheona , Leonardi Efthimia Kokotos , Werner Peter , Olanow C. Warren
Published: July 2010

Abstract: l‐Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)‐B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO‐B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µM) and the NMDA receptor blocker MK‐801 (10 µM) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µM) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µM) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.
Regulation of α2A‐Adrenergic Receptor mRNA in Rat Astroglial Cultures: Role of Cyclic AMP and Protein Kinase C
Authors: Reutter Michael A. , Richards Elaine M. , Sumners Colin
Published: July 2010

Abstract: In this study we investigated regulation of α2A‐adrenergic receptor (α2A‐AR) mRNA in rat astroglial cultures by increases in intracellular cyclic AMP levels and by protein kinase C (PKC) activation. Treatment of astroglial cultures with forskolin (FSK), an adenylyl cyclase activator, or with the membrane‐permeable cyclic AMP analogues dibutyryl cyclic AMP or Sp‐adenosine 3′,5′‐cyclic monophosphothioate triethylamine caused time‐ and concentration‐dependent decreases in the levels of a ∼4.0‐kb α2A‐AR mRNA transcript. Levels of α2A‐AR mRNA were reduced to ∼10% of control levels within 4 h of 1 µM FSK treatment. PKC agonists [phorbol 12‐myristate 13‐acetate (PMA), phorbol 12,13‐dibutyrate, and mezerein] also decreased α2A‐AR mRNA levels in a time‐ and concentration‐dependent fashion. A 90% decrease in α2A‐AR mRNA levels was observed with 50 nM PMA in 4 h. The decrease in α2A‐AR mRNA levels caused by FSK and PMA treatment appears to be the result of decreases in transcription of the α2A‐AR gene and is not due to decreases in α2A‐AR mRNA degradation rate. These observations suggest that α2A‐AR mRNA levels are regulated by cyclic AMP and PKC.
Endothelin Stimulates Phospholipase D in Striatal Astrocytes
Authors: Desagher Solange , Cordier Jocelyne , Glowinski Jacques , Tencé Martine
Published: July 2010

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [3H]myristic acid, endothelin (ET)‐1 induced a time‐dependent formation of [3H]phosphatidic acid and [3H]diacylglycerol. In the presence of ethanol, a production of [3H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET‐1 and ET‐3 were equipotent in stimulating PLD activity (EC50 = 2–5 nM). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET‐1, indicating the involvement of a Gi/Go protein. Inhibition of protein kinase C by Ro 31‐8220 or down‐regulation of the kinase by a long‐time treatment with phorbol 12‐myristate 13‐acetate (PMA) totally abolished the ET‐1‐induced stimulation of PLD. In contrast, a cyclic AMP‐dependent process is not involved in the activation of PLD, because the ET‐1‐evoked formation of [3H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8‐bromo‐cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C‐dependent process. However, the ET‐1 and PMA responses were additive. Furthermore, the ET‐1‐evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET‐1, ET‐3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.
Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons
Authors: Brorson James R. , Sulit Reginaldo A. , Zhang He
Published: July 2010

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca2+ concentration ([Ca2+]i). In [Ca2+]i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide‐releasing agent S‐nitrosocysteine produced a delayed rise in [Ca2+]i over a 20‐min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization‐induced [Ca2+]i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca2+]i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP‐regenerating system was supplied via the patch pipette in combined whole‐cell patch‐clamp‐[Ca2+]i fluorimetry experiments, S‐nitrosocysteine had no effect on the resting [Ca2+]i or on the recovery kinetics of [Ca2+]i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca2+]i homeostasis in hippocampal neurons by impairing Ca2+ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca2+]i may contribute to the delayed neurotoxicity of nitric oxide.
Okadaic Acid Induces Hyperphosphorylation of τ Independently of Mitogen‐Activated Protein Kinase Activation
Authors: Ho Duncan T. , Shayan Hossein , Murphy Timothy H.
Published: July 2010

Abstract: Hyperphosphorylation of the microtubule‐associated protein τ is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen‐activated protein kinase (MAPK), a proline‐directed protein kinase, phosphorylates the sites on τ common to Alzheimer's disease. Using an okadaic acid‐induced τ hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor {PD098059 [2‐(2′‐amino‐3′‐methoxyphenyl)oxanaphthalen‐4‐one]} of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose‐dependently blocked basal and okadaic acid‐induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust τ hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12‐myristate 13‐acetate did not result in τ phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce τ hyperphosphorylation.
5‐Hydroxytryptamine‐Facilitated Release of Substance P from Rat Spinal Cord Slices Is Mediated by Nitric Oxide and Cyclic GMP
Authors: Inoue Atsuko , Hashimoto Takashi , Hide Izumi , Nishio Hiroaki , Nakata Yoshihiro
Published: July 2010

Abstract: The role of nitric oxide (NO) in the control of 5‐hydroxytryptamine (5‐HT)‐induced release of substance P was investigated in rat spinal cord in vitro. 5‐HT facilitated the 60 mM K+‐evoked release of substance P‐like immunoreactive materials (SPLI) from the superfused rat dorsal spinal cord slices without affecting spontaneous SPLI release. The facilitatory effect of 5‐HT was significantly inhibited by ICS 205‐930 or granisetron (potent and specific 5‐HT3 receptor antagonists), by NG‐monomethyl‐l‐arginine (NMMA, a NO synthase inhibitor), and by methylene blue or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (MB or ODQ, respectively; both are inhibitors of soluble guanylyl cyclase) and was mimicked by 2‐methylserotonin (2‐m‐5‐HT, a selective 5‐HT3 receptor agonist), l‐arginine (a precursor of NO), or 8‐bromo‐cyclic GMP. NMMA, MB, or ODQ inhibited the 2‐m‐5‐HT‐induced increase of cyclic GMP levels in the rat dorsal spinal cord slices. These data suggest that the facilitatory effect of 5‐HT on the release of SPLI is mediated by the 5‐HT3 receptor and that the intracellular signaling is mediated via NO by an increase in cyclic GMP production.
Down‐Regulation of Norepinephrine Transporters on PC12 Cells by Transporter Inhibitors
Authors: Zhu Meng‐Yang , Ordway Gregory A.
Published: July 2010

Abstract: To investigate the regulation of norepinephrine transporters (NETs) in vitro, we measured the binding of the NET‐selective ligand [3H]nisoxetine in homogenates of PC12 cells after exposure of intact cells to the NET inhibitor desipramine (DMI). A 3‐day exposure of PC12 cells to DMI robustly reduced the Bmax, but not the KD, of [3H]nisoxetine binding to NETs. Reduction of the binding of [3H]nisoxetine was dependent on both the concentration of DMI and the time of exposure to DMI. Reduction of [3H]nisoxetine binding to NETs produced by a 1‐day exposure to DMI reverted to preexposure levels 48 h after cessation of DMI exposure. Similar down‐regulation of NETs was found when PC12 cells were exposed to another NET‐selective drug, nisoxetine, which is structurally unrelated to DMI. In contrast, exposure of cells to the serotonin transporter‐selective drug citalopram, or the NET substrate norepinephrine, had no effects on the binding of [3H]nisoxetine to NETs. The down‐regulation of NETs was paralleled by a DMI‐induced reduction in the uptake of [3H]norepinephrine in intact PC12 cells. It can be inferred from these data that inhibitors of the NET can down‐regulate NETs directly, and do so in the absence of changes in the synaptic concentration of norepinephrine.
Dual Modulation of Dopamine Release from Anterior Nucleus Accumbens Through Cholecystokinin‐B Receptor Subsites
Authors: Léna Isabelle , Roques Bernard P. , Durieux Christiane
Published: July 2010

Abstract: Previous binding studies have suggested the existence of two affinity states for cholecystokinin‐B (CCK‐B) receptor. One study, using BC 197 and BC 264, two highly selective CCK‐B agonists, has shown that BC 197 is selective for one subsite, B1, and that BC 264 has the same affinity for the two subsites, B1 and B2. Therefore, the possible involvement of CCK‐B subsites in the modulation of endogenous dopamine (DA) release from slices of the anterior part of the nucleus accumbens was investigated with these two agonists in order to associate a functional response with activation of each subsite. The selective B1 agonist BC 197 produced a dose‐dependent increase of 35 mM K+‐stimulated DA release. In contrast, at a low concentration (20 nM), BC 264 inhibited the K+‐evoked DA release, whereas at a higher concentration (1 µM), it stimulated the DA release. These two opposing effects were suppressed by the CCK‐B antagonist PD‐134,308, but not by the CCK‐A antagonist L‐364,718 and were not prevented by tetrodotoxin, a Na+‐channel blocker. Moreover, BC 264 at 20 nM, in the presence of PD‐134,308 at a concentration that would block the B2 subsites (0.1 nM), increased the evoked DA release. All together, these results support further the existence of distinct CCK‐B subsites and suggest that, in the anterior nucleus accumbens, their stimulation mediates opposite effects on K+‐stimulated DA release via a presynaptic mechanism.
Gene‐Based Neurotransmitter Modulation in Cerebellar Granule Neurons
Authors: Fritz Jeffery D. , Utz Andrea L. , Hale Nannette M. , Wu Lan , Powers Alvin C. , Verdoorn Todd A. , Robertson David
Published: July 2010

Abstract: The human glutamic acid decarboxylase (GAD) gene was transferred into rat cerebellar granule neurons. Following adenoviral‐mediated gene transfer, nearly 100% of the neurons had transgene expression that persisted for the duration of their survival in culture. GABA levels were elevated both in the growth media and in lysates of GAD‐modified granule neurons. In GAD‐modified neurons, extracellular GABA levels steadily increased with time, whereas intracellular GABA levels peaked 10 days after gene transfer. GAD‐modified neurons released both glutamate and GABA into the surrounding media before and after potassium‐induced stimulation, but only the release of glutamate was sensitive to potassium stimulation. These data suggest that glutamatergic neurons, which initially contained no detectable GABA, can be genetically modified to release GABA constitutively.
Intranasal Administration of the Dopaminergic Agonists l‐DOPA, Amphetamine, and Cocaine Increases Dopamine Activity in the Neostriatum: A Microdialysis Study in the Rat
Authors: De Souza Silva M. A. , Mattern C. , Häcker R. , Nogueira P. J. C. , Huston J. P. , Schwarting R. K. W.
Published: July 2010

Abstract: The effectiveness of intranasal drug administration to stimulate central neuronal systems is well known from drug addiction and has also been considered as an alternative pharmacokinetic approach to treat brain disorders such as Parkinson's disease. In the present study, the possible neurochemical effects of intranasal administration of the psychostimulants cocaine and amphetamine and of the antiparkinsonian drug l‐DOPA were analyzed. By using in vivo microdialysis in the urethane‐anesthetized rat, it was found that unilateral intranasal administration of either of the psychostimulants led to huge and rapid increases of extracellular dopamine levels in the neostriatum followed by decreases of its metabolites dihydroxyphenylacetic acid and homovanillic acid. Furthermore, intranasal administration of l‐DOPA, but not of the saline vehicle, also led to increased extracellular levels of neostriatal dopamine and to increases of its metabolites. Because the effect of intranasal l‐DOPA on neostriatal dopamine was observed only ipsilaterally but not contralaterally to the side of intranasal drug administration, it can be hypothesized that l‐DOPA was not effective via passage through the circulation but may have acted through a neuronal or an extraneuronal route. These data provide neurochemical evidence that the intranasal route may not only be efficient in drug abuse, but may also be useful to target the brain therapeutically, as in the case of neurodegenerative brain disorders.
Altered μ‐Opiate Receptor‐G Protein Signal Transduction Following Chronic Morphine Exposure
Authors: Wang Lin , Gintzler Alan R.
Published: July 2010

Abstract: This laboratory has demonstrated that the longitudinal muscle/myenteric plexus (LMMP) preparation manifests pleiotropic responses to opioid agonists. For example, the μ‐selective opiate receptor agonist sufentanil can produce a naloxone‐reversible increase or decrease in the electrically stimulated formation of cyclic AMP, depending on its concentration. The present study demonstrates that the sufentanil facilitation and inhibition of stimulated cyclic AMP formation are mediated via Gs‐ and Gi‐like G proteins, respectively. Inactivation of Gi (via pertussis toxin) not only abolishes sufentanil inhibition of cyclic AMP formation but also unmasks a facilitory effect. The latter response is eliminated following treatment with cholera toxin. In tolerant/dependent LMMP tissue, previously inhibitory concentrations of sufentanil produce a facilitation of cyclic AMP formation. However, this unmasked facilitory effect is resistant to cholera toxin. Thus, although inactivation of the inhibitory signal transduction pathway (via pertussis toxin) is sufficient to unmask excitatory sufentanil effects in opiate naive preparations, this mechanism cannot explain the reversal of sufentanil inhibition to facilitation that is observed in tolerant/dependent tissue. Instead, the chronic morphine‐induced emergence of a μ‐opiate receptor‐coupled facilitory pathway that is either not expressed or not fully manifest in opiate naive LMMP tissue is suggested.
A Role for 4‐Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β‐Peptide
Authors: Mark Robert J. , Lovell Mark A. , Markesbery William R. , Uchida Koji , Mattson Mark P.
Published: July 2010

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4‐hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β‐peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25‐35‐induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein‐bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time‐ and concentration‐dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+,K+‐ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ‐induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.
Quantification of Local De Novo Synthesis Versus Blood Contributions to Quinolinic Acid Concentrations in Brain and Systemic Tissues
Authors: Heyes Melvyn P. , Morrison Paul F.
Published: July 2010

Abstract: The source of the neurotoxin quinolinic acid (QUIN) in brain and systemic tissues under normal and pathologic circumstances reflects either de novo synthesis from l‐tryptophan and other precursors, or entry of QUIN itself from the blood. To quantify the relative contributions of blood‐ versus tissue‐derived QUIN, [13C7]QUIN was infused subcutaneously via osmotic pumps (0.55 µl/h, 30 mM) in gerbils, and the fraction of QUIN in tissue (Ti; measured in tissue homogenates) derived from blood (BI; measured in serum) was calculated by the formula ([13C7]QUINTi/QUINTi)/([13C7]QUINBl/QUINBl). In controls, blood QUIN contributed 38–49% of QUIN in brain, 70% in CSF, between 40 and 70% in kidney, heart, and skeletal muscle, but <5% in spleen, lung, liver, and intestine. Systemic endotoxin (450 µg/kg) increased blood, brain, CSF, and systemic tissue QUIN levels. Notably, the relative proportion of QUIN derived from blood in brain, spleen, lung, and intestine was unchanged by endotoxin, but increased in kidney, heart, and skeletal muscle. In contrast, cerebral ischemic injury (10 min of bilateral carotid artery occlusion) increased regional brain QUIN concentrations at 4 days post ischemia, with a proportional increase in the amount of QUIN derived from de novo synthesis by brain tissue. In the blood and systemic tissues of postischemic gerbils, there were no changes in systemic tissue or blood QUIN levels, or changes in the relative proportions of blood‐ versus systemic tissue‐derived QUIN. These results establish that the brain normally synthesizes QUIN, that the blood is a significant source of QUIN in controls and during acute systemic immune activation, and that the rate of QUIN formation by brain tissue increases in conditions of brain and systemic immune activation.
Reduced [3H]Cyclic AMP Binding in Postmortem Brain from Subjects with Bipolar Affective Disorder
Authors: Rahman Shafiqur , Li Peter P. , Young L. Trevor , Kofman Ora , Kish Stephen J. , Warsh Jerry J.
Published: July 2010

Abstract: Findings of increased Gsα levels and forskolin‐stimulated adenylyl cyclase activity in selective cerebral cortical postmortem brain regions in bipolar affective disorder (BD) implicate increased cyclic AMP (cAMP)‐mediated signaling in this illness. Accumulating evidence suggests that intracellular levels of cAMP modulate the abundance and disposition of the regulatory subunits of cAMP‐dependent protein kinase (cAMP‐dPK). Thus, in the present study, we tested further whether hyperfunctional Gsα‐linked cAMP signaling occurs in BD by determining [3H]cAMP binding, a measure of the levels of regulatory subunits of cAMP‐dPK, in cytosolic and membrane fractions from discrete brain regions of postmortem BD brain. Specific [3H]cAMP (5 nM) binding was determined in autopsied brain obtained from 10 patients with DSM‐III‐R diagnoses of BD compared with age‐ and postmortem delay‐matched controls. [3H]cAMP binding was significantly reduced across all brain regions in cytosolic fractions of BD frontal (−22%), temporal (−23%), occipital (−22%) and parietal (−15%) cortex, cerebellum (−36%), and thalamus (−13%) compared with controls, but there were no differences in [3H]cAMP binding in the membrane fractions from these same regions. These results suggest that changes occur in the cAMP‐dPK regulatory subunits in BD brain, possibly resulting from increased cAMP signaling. The possibility that antemortem lithium and/or other mood stabilizer treatment may contribute to the above changes, however, cannot be ruled out.
Key Residues Defining the μ‐Opioid Receptor Binding Pocket: A Site‐Directed Mutagenesis Study
Authors: Mansour Alfred , Taylor Larry P. , Fine Jeffrey L. , Thompson Robert C. , Hoversten Mary T. , Mosberg Henry I. , Watson Stanley J. , Akil Huda
Published: July 2010

Abstract: Structural elements of the rat μ‐opioid receptor important in ligand receptor binding and selectivity were examined using a site‐directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for μ/δ selectivity (Ile198 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H‐labeled d‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three‐ to 20‐fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β‐endorphin1–31, JOM‐13, deltorphin II, dynorphin1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor‐binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile198 to Val resulted in a four‐ to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.
Arginine‐Modulated Receptor‐Activated Calcium Influx via a NO/Cyclic GMP Pathway in Human SK‐N‐SH Neuroblastoma Cells
Authors: Liu Pei‐Shan , Shaw Yi‐Hsin
Published: July 2010

Abstract: The effects of arginine on calcium mobilization in human SK‐N‐SH neuroblastoma cells were examined. It was found that arginine potentiated an increase in carbachol‐induced Ca2+ from the external Ca2+ influx as opposed to an internal Ca2+ release from intracellular pools. The potentiation effect of arginine on carbachol‐induced calcium mobilization was mimicked by either 8‐bromo cyclic GMP or sodium nitroprusside. In addition, it was found that arginine induced NO production and an increase in cyclic GMP. Moreover, arginine‐induced potentiation, NO production, and cyclic GMP increases were all suppressed after the preincubation of cells with N‐methyl‐l‐arginine or N‐nitro‐l‐arginine, nitric oxide synthase inhibitor. It is suggested that the NO production and subsequent cyclic GMP elevation induced by arginine are responsible for the potentiation of carbachol‐induced Ca2+ increase. Our results show the existence of a NO/cyclic GMP pathway and an interconnection of NO and Ca2+ signaling pathways in human SK‐N‐SH neuroblastoma cells. We also observed that NO, which is produced by endothelial CPAE cells, has a modulating effect on cyclic GMP elevation in human SK‐N‐SH neuroblastoma cells. The intercellular communication role of NO and its cell‐diffusing character may also affect the regulation of nonneuronal cells in their interactions with neuronal cells.
Expression of Microtubule‐Associated Protein‐2a and Other Novel Microtubule‐Associated Protein‐2 Transcripts in Human Fetal Spinal Cord
Authors: Kalcheva Nellie , Weidenheim Karen M. , Kress Yvonne , Shafit‐Zagardo Bridget
Published: July 2010

Abstract: In human fetal spinal cord (HFSC), six additional microtubule‐associated protein‐2 (MAP‐2) transcripts are generated by alternative splicing of two recently described exons, exon 8 and exon 13. The following three translated proteins are detected by western blot analysis: MAP‐2b expressing exon 8 (MAP‐2b+8; MAP‐2a), MAP‐2b expressing exon 13 (MAP‐2b+13), and MAP‐2c expressing exon 8 and exon 13 (MAP‐2c+8+13). The finding that MAP‐2b+8 is expressed in HFSC demonstrates for the first time the presence of MAP‐2a in human fetal CNS. Immunocytochemical studies show that exon 8‐specific antibody and exon 13‐specific antibody stain independent and overlapping populations of neurons in the lumbar region of the HFSC. Antibody 13‐immunopositive neurons have predominantly cytosolic staining, whereas in the antibody 8‐immunoreactive neurons staining was observed in the cytosol, dendrites, and some synapses. The prenatal expression of MAP‐2a, which has been used as a marker of synaptogenesis, not only demonstrates the presence of a mature MAP‐2 isoform in HFSC, but suggests that MAP‐2a is important during human fetal as well as postnatal synaptogenesis.
Energy Stress‐Induced Dopamine Loss in Glutathione Peroxidase‐Overexpressing Transgenic Mice and in Glutathione‐Depleted Mesencephalic Cultures
Authors: Zeevalk Gail D. , Bernard Laura P. , Albers David S. , Mirochnitchenko Oleg , Nicklas William J. , Sonsalla Patricia K.
Published: July 2010

Abstract: The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 µM buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 µmol) into the left side had significantly less loss of striatal dopamine than their hGPE‐negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.
Neuronal Nicotinic Acetylcholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and Genomic Organization of Receptor Subunits
Authors: Campos‐Caro A. , Smillie F. I. , Domínguez del Toro E. , Rovira J. C. , Vicente‐Agulló F. , Chapuli J. , Juíz J. M. , Sala S. , Sala F. , Ballesta J. J. , Criado M.
Published: July 2010

Abstract: Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the α3 subunit, which we have previously cloned, the presence of α5 and β4 but not of β2 subunits was detected by reverse transcription‐PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that α3, α5, and β4 subunits are coexpressed in all chromaffin cells. The primary structure of α5 and β4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other β4‐containing nicotinic receptors, the ones formed by the bovine β4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of α3 and α5 subunits, which together with the β4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the α3 and α5 genes overlap at their 3′ ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.
Synthesis and Cytotoxic Profile of a Diphtheria Toxin‐Neurotrophin‐4 Chimera
Authors: Negro Alessandro , Skaper Stephen D.
Published: July 2010

Abstract: A diphtheria toxin‐neurotrophin‐4/5 (NT‐4/5) chimera (DAB389‐NT4), in which the native receptor binding domain of diphtheria toxin was replaced with a synthetic gene encoding rat NT‐4/5, was expressed, refolded, and purified. This fusion toxin has a deduced molecular mass of 60,163 and is formed by joining the first 389 amino acids of diphtheria toxin to amino acids 1–130 of mature rat NT‐4/5, using an NH2‐terminal bridge of 33 additional amino acids including six consecutive histidines. Neural cell types expressing only p75LNGFR or p75LNGFR and full‐length or truncated TrkB were used to evaluate the cytotoxic efficacy of DAB389‐NT4. The fusion toxin produced a concentration‐dependent killing of all cell populations, with LC50 values that largely reflected the known NT‐4/5 binding affinities for these receptor proteins. Mean LC50 values ranged from 2,960 pM in p75LNGFR‐expressing neuro‐2a neuroblastoma cells to 1,075 and 70 pM, respectively, in hippocampal astrocytes (p75LNGFR+/truncated TrkB+) and cerebellar granule cells (p75LNGFR+/TrkB+). The LC50 for DAB389‐NT4 in receptor‐negative 3T3 fibroblasts was 20 nM. NT‐4/5 and brain‐derived neurotrophic factor but not ciliary neurotrophic factor added in excess neutralized DAB389‐NT4 cytotoxicity. NT‐4/5, however, did not reduce the cytotoxicity of intact diphtheria toxin.
Regulation of δ‐Opioid Receptor mRNA Levels by Receptor‐Mediated and Direct Activation of the Adenylyl Cyclase‐Protein Kinase A Pathway
Authors: Buzas Beata , Rosenberger John , Cox Brian M.
Published: July 2010

Abstract: The effects of activation of the adenylyl cyclase‐protein kinase A pathway on the expression of δ‐opioid receptor mRNA in the NG108‐15 neuroblastoma × glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in δ‐receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclc AMP (db‐cAMP) mimicked the effect of PGE1. Down‐regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of δ‐opioid receptor transcripts. d‐Ser2‐Leu‐enkephalin‐Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db‐cAMP treatment. Inhibitors of protein kinase A(PKA) partially reversed the PGE1‐and db‐cAMP‐mediated repression of the δ‐opioid receptor mRNA levels. The rate of degradation of δ‐opioid receptor mRNA in the presence of actinomycin D was not altered in response to db‐cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of δ‐opioid receptor mRNA levels by db‐cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.
Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis
Authors: Rocha L. L. , Evans C. J. , Maidment N. T.
Published: July 2010

Abstract: Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation‐induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 ± 2.1 days (mean ± SEM) after the last stage V seizure], in comparison with values obtained from the sham‐kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40–60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin‐A (1–6), dynorphin‐A (1–8), and Leu‐enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.