bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine Hydroxylase in Bovine Adrenal Chromaffin Cells: Angiotensin II‐Stimulated Activity and Phosphorylation of Ser19, Ser31, and Ser40
Authors: Bobrovskaya Larisa , Cheah Tat B. , Bunn Stephen J. , Dunkley Peter R.
Published: July 2010

Abstract: The aim of this study was to determine the effect of angiotensin II (AII) on tyrosine hydroxylase (TOH) activity and phosphorylation in bovine adrenal chromaffin cells (BACCs). We report here that stimulation of BACCs with AII (100 nM) produced a significant increase in both TOH activity and phosphorylation over a period of 10 min. The increase in TOH activity was receptor‐mediated. Tryptic phosphopeptide analysis by HPLC revealed that AII stimulated an increase in phosphorylation of three sites on TOH, Ser19, Ser31, and Ser40, with the largest increase being observed for Ser31 phosphorylation. Pretreatment of the cells with the protein kinase C inhibitor Ro 31‐8220 (10 µM, 15 min) did not affect TOH activity or phosphorylation produced by AII. The inhibitor also did not affect the TOH activity or Ser40 phosphorylation produced by forskolin (10 µM, 10 min). In contrast, Ro 31‐8220 fully inhibited the TOH activation as well as Ser31 and Ser40 phosphorylation of TOH produced by phorbol 12, 13‐dibutyrate (500 nM, 10 min). Removal of extracellular Ca2+ from the incubation medium inhibited the AII‐induced TOH activity by 50% and significantly blocked Ser19 and Ser31 phosphorylation but did not affect Ser40 phosphorylation in response to AII. These results indicate that AII activates a complex and perhaps novel signaling pathway leading to the phosphorylation and activation of TOH. The TOH activation by AII appears to be partially independent of Ser40 phosphorylation, suggesting a potentially important role for Ser31 phosphorylation.
Functional Modulation of P2×2 Receptors by Cyclic AMP‐Dependent Protein Kinase
Authors: Chow Yu‐Wen , Wang Hung‐Li
Published: July 2010

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage‐ and ligand‐gated channels are modulated. The intracellular carboxyl terminus of P2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)‐dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2×2 purinoceptor could be regulated by the protein phosphorylation. Whole‐cell voltage‐clamp recording was used to record ATP‐evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2×2 receptor. Dialyzing HEK 293 cells with phorbol 12‐myristate 13‐acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP‐induced cationic current. The role of PKA phosphorylation in modulating the function of the P2×2 receptor was investigated by internally perfusing HEK 293 cells with 8‐bromo‐cAMP or the purified catalytic subunit of PKA. Both 8‐bromo‐cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP‐activated current without affecting the inactivation kinetics and the value of reversal potential. Site‐directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2×2 receptors, intracellular perfusion of 8‐bromo‐cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP‐evoked current. These results suggest that as with other ligand‐gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2×2 receptor and ATP‐mediated physiological effects in the nervous system.
Cloning of Zebrafish Neurofilament cDNAs for Plasticin and Gefiltin: Increased mRNA Expression in Ganglion Cells After Optic Nerve Injury
Authors: Asch William S. , Leake Devin , Canger Anthony K. , Passini Marco A. , Argenton Francesco , Schechter Nisson
Published: July 2010

Abstract: During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.
Retrovirus‐Mediated Double Transduction of the GTPCH and PTPS Genes Allows 6‐Pyruvoyltetrahydropterin Synthase‐Deficient Human Fibroblasts to Synthesize and Release Tetrahydrobiopterin
Authors: Laufs Stephanie , Blau Nenad , Thöny Beat
Published: July 2010

Abstract: The tetrahydrobiopterin (BH4) cofactor is essential for the aromatic amino acid hydroxylases that are involved in phenylalanine degradation and catecholamine and serotonin biosynthesis. Furthermore, BH4 is an essential and limiting cofactor for all types of nitric oxide synthases. BH4 deficiency results in hyperphenylalaninemia and monoamine neurotransmitter depletion associated with progressive mental retardation and is most commonly due to autosomal recessive mutations in 6‐pyruvoyltetrahydropterin synthase (PTPS), the second enzyme for cofactor biosynthesis. Due to the relatively poor blood‐brain barrier penetration of the cofactor, conventional therapy requires, besides oral doses of synthetic BH4, administration of neurotransmitter precursors and an aromatic amino acid decarboxylase inhibitor. The outcome of this therapy is not always beneficial. In this study we transduced into primary patient fibroblasts the human cDNAs for the BH4 biosynthetic enzymes GTP cyclohydrolase I and PTPS, expressed from different retroviral vectors. This allowed BH4 biosynthesis in originally PTPS‐deficient cells. Moreover, the double‐transduced fibroblasts released between 200 and 800 pmol of BH4/106 cells/day. Such engineered fibroblasts may be grafted into the central nervous system and used as depository cells for constitutive delivery of BH4.
Differentiation of a Catecholaminergic CNS Cell Line Modifies Tyrosine Hydroxylase Transcriptional Regulation
Authors: Lazaroff Meredith , Qi Yanping , Chikaraishi Dona M.
Published: July 2010

Abstract: Recently, a tyrosine hydroxylase (TH)‐expressing CNS‐derived cell line, CAD, was obtained that is capable of undergoing reversible morphological differentiation. The isolation of the CAD line allowed us to ask whether different DNA regulatory elements direct TH transcription when cells are growing and undifferentiated versus postmitotic and differentiated. To this end, we compared expression of a transiently transfected bacterial chloramphenicol acetyltransferase reporter gene under the transcriptional control of TH 5′ flanking DNA in CAD cells grown in the presence and absence of serum. Mutational analysis indicates that CAD cells differently regulate TH transcription depending on their state of differentiation. In both states, the cyclic AMP response element and AP1 site each activate transcription. However, in undifferentiated cells, the dyad/E‐box element represses expression by ∼2.7‐fold, whereas it modestly activates transcription in differentiated cells. The role of the dyad/E‐box as a repressor correlates well with the two‐ to threefold lower amount of endogenous TH protein present in the undifferentiated CAD cells. This study demonstrates the differential use of TH DNA regulatory elements in proliferating, undifferentiated and nonproliferating, differentiated immortalized neuronal cells.
Regional Expression and Regulation of Alternative Forms of mRNAs Derived from Two Distinct Transcription Initiation Sites of the Rat mGluR5 Gene
Authors: Yamaguchi Shun , Nakanishi Shigetada
Published: July 2010

Abstract: Metabotropic glutamate receptor (mGluR) subtype 5 is expressed in both neuronal and glial cells and is thought to play an important role in neuronal plasticity. This expression is up‐regulated during the early postnatal period and is induced in cultured astrocytes by specific growth factors. To investigate the mechanism underlying the regulation of mGluR5 expression, we isolated and characterized genomic clones containing the 5′‐upstream exons and their flanking regions of the mGluR5 gene. On the basis of the mGluR5 genomic structure, cDNA recloning of the 5′‐extreme region of mGluR5 as well as primer extension analysis indicated that mGluR5 mRNA is generated from two alternative first exons, termed exon 1A and exon 1B, which are separated by 1,949 bp and then connected to the common exon 2. Northern blot and in situ hybridization analyses indicated that two distinct transcription initiation sites are commonly used in the expression of mGluR5 mRNA in various, but specialized, brain regions and that these two alternative forms of mGluR5 mRNA are similarly up‐regulated or down‐regulated during the early postnatal period, depending on the brain regions. The two mRNAs are also expressed in cultured astrocytes but respond differently to growth factor‐mediated induction. This study provides the genetic basis indicating the diverse mechanisms involved in the regulation of mGluR5 expression.
Activation of Endogenous Antioxidant Defenses in Neuronal Cells Prevents Free Radical‐Mediated Damage
Authors: Duffy Steven , So Austin , Murphy Timothy H.
Published: July 2010

Abstract: Dopamine (DA) is oxidized to the neurotoxic prooxidant species H2O2, OH•, and DA quinones. We tested whether dimethyl fumarate (DMF), an electrophile shown to induce a pleiotropic antioxidant response in nonneuronal cells, could reduce the toxicity of DA metabolites in neural cells. Treatment of the N18‐RE‐105 neuroblastoma‐retina hybridoma cell line with 30–150 µM dopamine led to cell death within 24 h, which increased steeply with dose, decreased with higher plating density, and was blocked by the H2O2‐metabolizing enzyme catalase. Pretreatment with DMF (30 µM, 24 h) significantly attenuated DA and H2O2 toxicity (40–60%) but not that caused by the calcium ionophore ionomycin. DMF treatment also elevated total intracellular GSH and increased activities of the antioxidant enzymes quinone reductase (QR), glutathione S‐transferase (GST), glutathione reductase, and the pentose phosphate enzyme glucose‐6‐phosphate dehydrogenase. To assess the protective efficacy of QR and GST, a stable cell line was constructed in which these enzymes were overexpressed. Cell death in the overexpressing line was not significantly different from that in a cell line expressing normal QR and GST activities, indicating that these two enzymes alone are insufficient for protection against DA toxicity. Although the relative importance of a single antioxidant enzyme such as QR or GST may be small, antioxidant inducers such as DMF may prove valuable as agents that elicit a broad‐spectrum neuroprotective response.
Oxidative Stress Induces a Form of Programmed Cell Death with Characteristics of Both Apoptosis and Necrosis in Neuronal Cells
Authors: Tan Shirlee , Wood Malcolm , Maher Pamela
Published: July 2010

Abstract: Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinson's disease, and Alzheimer's disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT‐22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate‐treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.
AMPA Receptor‐Mediated Excitotoxicity in Human NT2‐N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+
Authors: Itoh Takayuki , Itoh Aki , Horiuchi Kazumi , Pleasure David
Published: July 2010

Abstract: Human NT2‐N neurons express Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid glutamate receptors (AMPA‐GluRs) and become vulnerable to excitotoxicity when AMPA‐GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca2+ levels ([Ca2+]i) was 1.9‐fold greater in the presence than in the absence of cyclothiazide, Ca2+ entry via AMPA‐GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40‐fold rise in [Na+]i, which induced a delayed [Ca2+]i rise. Transfer of the neurons to a 5 mM Na+ medium after AMPA‐GluR activation accelerated the delayed [Ca2+]i rise and intensified excitotoxicity. Low‐Na+ medium‐enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK‐801), and completely blocked by removal of extracellular Ca2+, suggesting that Ca2+ entry by reverse operation of Na+/Ca2+ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na+ loading. Our results serve to emphasize the central role of neuronal Na+ loading in AMPA‐GluR‐mediated excitotoxicity in human neurons.
Increase of Adenylate Kinase Isozyme 1 Protein During Neuronal Differentiation in Mouse Embryonal Carcinoma P19 Cells and in Rat Brain Primary Cultured Cells
Authors: Inouye Sachiye , Seo Misuzu , Yamada Yasue , Nakazawa Atsushi
Published: July 2010

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double‐immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron‐like cells, but not in glia‐like cells. The amount of brain‐type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.
The Calcitonin Gene‐Related Peptide‐Induced Acetylcholinesterase Synthesis in Cultured Chick Myotubes Is Mediated by Cyclic AMP
Authors: Choi Roy C. Y. , Yung Lisa Y. , Dong Tina T. X. , Wan David C. C. , Wong Yung H. , Tsim Karl W. K.
Published: July 2010

Abstract: In vertebrate neuromuscular junctions, post‐synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene‐related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N6,O2′‐dibutyryladenosine 3′,5′‐cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Gαs, the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Gαi cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen‐tailed AChE was up‐regulated by the cAMP pathway. These findings indicated that CGRP‐induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron‐derived factor.
Age‐Dependent Differential Regulation of Sensory Neuropeptides by Glial Cell Line‐Derived Neurotrophic Factor
Authors: Adler Joshua E.
Published: July 2010

Abstract: Glial cell line‐derived neurotrophic factor markedly enhances survival of neonatal dorsal root sensory neurons in vitro, an effect seen even in the presence of anti‐nerve growth factor. Furthermore, it increases levels of substance P, inducing more than a sixfold rise that is maximal at 10 ng/ml. At the same dose, it potentiates the action of nerve growth factor on substance P but not on survival. Neither factor increases somatostatin content in neonatal neurons. Although its effect on substance P diminishes with age, glial cell line‐derived neurotrophic factor dramatically increases somatostatin levels in neurons from adult rats. Glial cell line‐derived neurotrophic factor is therefore the second trophic factor found to promote survival and regulate substance P in neonatal sensory neurons. More significant is that it is the first and sole neurotrophic factor reported to regulate somatostatin in sensory neurons at any age, with its effect restricted to the adult. These results suggest mechanisms for differential regulation of somatostatin versus substance P in nociceptive pathways.
Inhibition of Phosphorylation of TrkB and TrkC and Their Signal Transduction by α2‐Macroglobulin
Authors: Hu Yi‐qun , Koo Peter H.
Published: July 2010

Abstract: Monoamine‐activated α2‐macroglobulin (α2M) was shown to reduce the dopamine concentration in corpus striatum of adult rat brains and inhibit other neuronal functions in vivo and in vitro. As brain‐derived neurotrophic factor, neurotrophin‐4, and neurotrophin‐3 are important neurotrophic factors for dopaminergic neurons, the effect of monoamine‐activated α2M on signal transduction by trkB and trkC was investigated. The results show that monoamine‐activated α2M binds to trkB and inhibits brain‐derived neurotrophic factor/neurotrophin‐4‐promoted autophosphorylation of trkB in a dose‐dependent manner in both trkB‐expressing NIH3T3 (NIH3T3‐trkB) and human neuroblastoma SH‐SY5Y cells. Monoamine‐activated α2M also blocks tyrosine phosphorylation of phospholipase C‐γ1 and extracellular signal‐regulated protein kinase (ERK)‐1, which are key intracellular proteins involved in trkB signal transduction. Similarly, monoamine‐activated α2M inhibits tyrosine phosphorylation of neurotrophin‐3‐induced trkC and its signal transduction in a dose‐dependent manner in NIH3T3 cells expressing trkC (NIH3T3‐trkC). In contrast to monoamine‐activated α2M, normal α2M has little or no significant inhibitory effect on the phosphorylation of trkB and trkC. In addition, the retinoic acid‐promoted tyrosine phosphorylation of phospholipase C‐γ1, ERK‐1, and/or ERK‐2 in SH‐SY5Y cells was unaffected by monoamine‐activated α2M; this suggests that the inhibitory effect of activated α2M on the neurotrophin‐stimulated phosphorylation of intracellular signalling proteins may be specific. Taken together, the data indicate that activated α2M is a pan‐trk inhibitor, which by virtue of its binding to trk receptors may block trk‐mediated signal transduction in dopaminergic neurons and lead to reduction of dopamine concentration in corpus striatum.
Nitric Oxide‐Induced Release of Acetylcholine in the Nucleus Accumbens: Role of Cyclic GMP, Glutamate, and GABA
Authors: Prast H. , Tran M. H. , Fischer H. , Philippu A.
Published: July 2010

Abstract: We have previously shown that the basal acetylcholine release in the ventral striatum is under the enhancing influence of endogenous nitric oxide (NO) and that NO donors cause pronounced increases in the acetylcholine release rate. To investigate the role of cyclic GMP, glutamate, and GABA in the NO‐induced acetylcholine release, we superfused the nucleus accumbens, (Nac) of the anesthetized rat with various compounds through a push‐pull cannula and determined the neurotransmitter released in the perfusate. Superfusion of the Nac with the NO donors diethylamine/NO (DEANO; 100 µmol/L), S‐nitroso‐N‐acetylpenicillamine (SNAP; 200 µmol/L), or 3‐morpholinosydnonimine (SIN‐1; 200 µmol/L) enhanced the acetylcholine release rate. The guanylyl cyclase inhibitor 1H‐(1,2,4)‐oxodiazolo(4,3‐a)quinoxalin‐1‐one (ODQ; 10 µmol/L) abolished the effects of DEANO and SIN‐1. 6‐(Phenylamino)‐5,8‐quinolinedione (LY‐83583; 100 µmol/L), which also inhibits cyclic GMP synthesis, inhibited the releasing effects of DEANO and of SNAP, whereas the effect of SIN‐1 on acetylcholine release was not influenced. The DEANO‐induced release of acetylcholine was also abolished in the presence of 20 µmol/L 6,6‐dinitroquinoxaline‐2,3‐dione (DNQX) and 10 µmol/L (±)‐2‐amino‐5‐phosphonopentanoic acid (AP‐5). Simultaneous superfusion with 50 µmol/L quinpirole and 10 µmol/L 7‐bromo‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine (SKF 83566) was ineffective. Superfusion with 500 µmol/L DEANO decreased the release of acetylcholine. The inhibitory effect of 500 µmol/L DEANO was reversed to an enhanced release on superfusion with 20 µmol/L bicuculline. Bicuculline also enhanced the basal release rate. These findings indicate that cyclic GMP mediates the NO‐induced release of acetylcholine by enhancing the outflow of glutamate. Dopamine is not involved in this process. Only high concentrations of NO increase the output of GABA, which in turn decreases acetylcholine release. Our results suggest that cells that are able to release glutamate, such as glutamatergic neurons, are the main target of NO in the Nac.
Enzymatic and Molecular Biological Analysis of Palmitoyl Protein Thioesterase Deficiency in Infantile Neuronal Ceroid Lipofuscinosis
Authors: Cho Seongeun , Dawson Glyn
Published: July 2010

Abstract: Infantile neuronal ceroid lipofuscinosis (INCL) is a neurodegenerative lysosomal storage disease that results from a deficiency of palmitoyl protein thioesterase (PPT), a deacylating enzyme that removes cysteine‐bound palmitate from proteins. We developed an in vitro PPT enzyme assay that can be readily used for the clinical diagnosis of both INCL patients and carriers. The substrate is a palmitoylated peptide (IRY[14C]palmitoyl‐CWLRR) synthesized by reacting [14C]palmitoyl‐CoA with a synthetic octapeptide from the PNS P0 glycoprotein. The PPT assay performed in immortalized lymphoblastoid B‐cells or the postmortem brain homogenate showed the optimal enzyme activity at pH 5.0, consistent with the findings that PPT is a lysosomal enzyme. PPT activity in lymphoblasts from INCL patients was <4% of that of control lymphoblasts. In addition, obligatory carriers showed 74% of the control activity. Other pathological controls, including the juvenile form of NCL, showed PPT activities that were not different from normal. Furthermore, one brain sample from an INCL patient contained only 7% PPT activity when compared with an unaffected brain. Thus, the enzyme activity assay gives a confident diagnosis of INCL. In contrast, when the total RNA extract from lymphoblasts was probed with 32P‐labeled PPT by northern blot analysis, the level of transcript varied among independent INCL families and was not related to PPT activity. In conclusion, whereas variant genetic modifications result in PPT deficiency, all giving similar INCL phenotype, both affected patients and heterozygote carriers can now be screened with a reliable in vitro PPT assay.
Lactate Is Released and Taken Up by Isolated Rabbit Vagus Nerve During Aerobic Metabolism
Authors: Véga Céline , Poitry‐Yamate Carol L. , Jirounek Petr , Tsacopoulos Marcos , Coles Jonathan A.
Published: July 2010

Abstract: To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing d‐[U‐14C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6‐phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (µg dry wt)−1 from the exogenous glucose and 14.2 pmol (µg dry wt)−1 from endogenous substrates. Lactate release was not increased when bath Po2 was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U‐14C]lactate at a total concentration of 2.13 mM and 1 mM glucose, 14C was incorporated in CO2 and glutamate. The initial rate of formation of CO2 from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.
Synaptic Vesicle Protein SV2B, but Not SV2A, Is Predominantly Expressed and Associated with Microvesicles in Rat Pinealocytes
Authors: Hayashi Mitsuko , Yamamoto Akitsugu , Yatsushiro Shouki , Yamada Hiroshi , Futai Masamitsu , Yamaguchi Akihito , Moriyama Yoshinori
Published: July 2010

Abstract: Microvesicles are endocrine counterparts of neuronal synaptic vesicles, and accumulate and secrete classic neurotransmitters. In mammalian pinealocytes, microvesicles accumulate l‐glutamate through a vesicular glutamate transporter and secrete it through exocytosis. To characterize the molecular organization of microvesicles in more detail, we investigated in this study the expression and localization of synaptic vesicle protein 2 (SV2) in rat pinealocytes. RT‐PCR analysis indicated that transcripts specific for two isoforms, SV2A, a ubiquitous form present in neuronal and endocrine cells, and SV2B, a neuron‐specific form, are amplified in pineal RNAs. Northern blotting with specific transcripts indicated that the mRNA for SV2B is predominantly expressed, whereas that for SV2A is below the detection limit. Site‐specific antibodies against SV2B recognized a single 72‐kDa polypeptide in the pineal membrane fraction, whereas anti‐SV2A antibodies did not recognize any polypeptides. Immunohistochemical analysis of cultured cells indicated that SV2B is expressed in pinealocytes but not in other types of cells. SV2B is present in somata and is especially rich in processes, which are filled with microvesicles. SV2B is colocalized with synaptophysin and synaptotagmin, markers for microvesicles. Immunoelectron microscopy indicated that SV2B is associated with microvesicles. These results indicated that SV2B, but not SV2A, is expressed in rat pinealocytes and associated with microvesicles. As SV2B is also expressed in cultured αTC6 clonal pancreatic α cells, SV2B is not a protein specific for neurons.
Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells
Authors: Furuya Shigeki , Mitoma Junya , Makino Asami , Hirabayashi Yoshio
Published: July 2010

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP‐1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell‐permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP‐1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell‐permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.
Phosphatidic Acid‐Dependent Phosphorylation of a 29‐kDa Protein by Protein Kinase Cα in Bovine Brain Cytosol
Authors: Yokozeki T. , Homma K. , Kuroda S. , Kikkawa U. , Ohno S. , Takahashi M. , Imahori K. , Kanaho Y.
Published: July 2010

Abstract: Activation of phospholipase D (PLD) is involved in receptor‐mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PA‐regulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain‐specific 25‐kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti‐p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an anti‐protein kinase C (PKC)α antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA‐ and PS‐stimulated PARK activity was extremely augmented by the presence of 1 µM free Ca2+. In the presence of 1 mM EGTA, phorbol 12‐myristate 13‐acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCα. From these results, it is suggested that the PARK activity purified might be attributed to PKCα. In p25‐depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti‐p25 antibody, PA‐dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCα in bovine brain cytosol was depleted by its precipitation with the anti‐PKCα antibody, neither p29 nor p32 in this PKCα‐depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCα, which, in turn, phosphorylates p29, which may be identical with p25.
Binding Characteristics of a Potent AMPA Receptor Antagonist [3H]Ro 48‐8587 in Rat Brain
Authors: Mutel Vincent , Trube Gerhard , Klingelschmidt Agnès , Messer Jürg , Bleuel Zaiga , Humbel Urs , Clifford Merwyn M. , Ellis Gareth J. , Richards J. Grayson
Published: July 2010

Abstract: A new AMPA receptor antagonist, Ro 48‐8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H]kainate, and [3H]MK‐801 binding to rat brain membranes and on AMPA‐ or NMDA‐induced depolarization in rat cortical wedges. [3H]Ro 48‐8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48‐8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48‐8587 > 6‐nitro‐7‐sulphamoylbenzo[f]quinoxaline‐2,3‐dione (NBQX) > YM 90K > 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (∼95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3–6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (∼2.6 and 0.4 pmol/mg of protein, respectively). Ro 48‐8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.
Tyrosine Hydroxylase in the European Eel (Anguilla anguilla): cDNA Cloning, Brain Distribution, and Phylogenetic Analysis
Authors: Boularand Sylviane , Biguet Nicole Faucon , Vidal Bernadette , Veron Michèle , Mallet Jacques , Vincent Jean‐Didier , Dufour Sylvie , Vernier Philippe
Published: July 2010

Abstract: We report the isolation of a full‐length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3′ untranslated region and by a diversity restricted to the 3′ end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU‐rich 3′ noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N‐terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH‐expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.
M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools
Authors: Wotta Diane R. , Parsons Ann M. , Hu Jingru , Grande Andrew W. , El‐Fakahany Esam E.
Published: July 2010

Abstract: This study shows that activation of M1 muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)‐K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca2+ concentration and nNOS activity. We examined the relationship between receptor‐mediated increases in intracellular Ca2+ concentration and activation of nNOS over both short and long intervals using guanosine 3′,5′‐cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca2+ from intracellular stores in both the CHO M1/nNOS transfected cells and in neuroblastoma (N1E‐115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M1 muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l‐[3H]arginine to l‐[3H]citrulline as well as cGMP formation following stimulation of M1 muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca2+ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca2+.
Transforming Growth Factor β1 Regulates the Expression of Cyclooxygenase in Cultured Cortical Astrocytes and Neurons
Authors: Luo Jia , Lang Julie A. , Miller Michael W.
Published: July 2010

Abstract: The hypothesis that transforming growth factor β1 (TGFβ1) regulates the synthesis of prostaglandins by CNS tissue was tested by using purified cultures of cortical astrocytes or neurons that were obtained from rat pups on postnatal day 4 or 5 or fetuses on gestational day 16, respectively. The cells were exposed to TGFβ1 for 2 days. The synthesis of prostaglandins depends upon the production and conversion of arachidonic acid, steps that are catalyzed by phospholipase A2 (PLA2) and cyclooxygenase (COX), respectively. Prostaglandin E2 (PGE2) concentration was determined by radioimmunoassay. The expression of cytosolic PLA2 and COX (the constitutive COX1 and the inducible COX2) was assessed by using immunohistochemical and quantitative immunoblotting procedures. Astrocytes produced much more PGE2 than neurons, suggesting that glial cells are an important source of PGE2 in the CNS. TGFβ1 increased the production of PGE2 by astrocytes and neurons in a concentration‐dependent manner. Furthermore, TGFβ1 enhanced COX activity; the inhibitor indomethacin completely blocked TGFβ1‐mediated PGE2 synthesis. Cultured astrocytes and neurons expressed the three enzymes: cytosolic PLA2, COX1, and COX2. Cytosolic PLA2 expression was unaffected by TGFβ1 treatment. In contrast, COX expression was altered by TGFβ1 treatment in a concentration‐dependent fashion. COX1 was increased by TGFβ1, but only in astrocytes. TGFβ1 increased COX2 expression in astrocytes and neurons. Thus, TGFβ1‐induced increases in PGE2 concentration are regulated by COX. This study suggests that TGFβ1 is an important regulator of immune and inflammatory processes in the CNS.
Activity and Cyclic AMP‐Dependent Regulation of Nociceptin/Orphanin FQ Gene Expression in Primary Neuronal and Astrocyte Cultures
Authors: Buzas Beata , Rosenberger John , Cox Brian M.
Published: July 2010

Abstract: The regulation of nociceptin/orphanin FQ (N/OFQ) gene expression by neuronal activity and by activation of the cyclic AMP signaling pathway in primary neuronal and astroglial cultures is described. Neuronal activity mimicked by veratridine‐mediated depolarization profoundly increased N/OFQ gene expression in primary striatal neurons. Calcium entry through L‐type, but not N‐type, voltage‐sensitive calcium channels activated by depolarization appears to be involved, because nitrendipine and nifedipine, but not ω‐conotoxin, reduced the induction of N/OFQ expression by veratridine. A selective inhibitor of calcium/calmodulin kinases (KN‐62) also antagonized the depolarization‐induced increase in N/OFQ mRNA levels, suggesting a role for these enzymes in the activity‐dependent induction of N/OFQ gene expression. Constitutively expressed transcription factors may mediate N/OFQ gene expression levels, because veratridine induction of N/OFQ transcription was insensitive to the protein synthesis inhibitor cycloheximide. Regulation of N/OFQ gene expression by depolarization and cyclic AMP is not restricted to striatal neurons, because similar regulation was also observed in neuronal cultures derived from the cerebral cortex. Veratridine did not increase N/OFQ mRNA levels in primary astrocyte cultures; however, elevated intracellular cyclic AMP levels lead to a dramatic, 30‐fold induction of N/OFQ mRNA levels in these cells.
Membrane Transport of Neuronal Nitric Oxide Synthase Substrate l‐Arginine Is Constitutively Expressed with CAT1 and 4F2hc, but Not CAT2 or rBAT
Authors: Stevens Bruce R. , Vo Chi B.
Published: July 2010

Abstract: The potential induction of cationic and zwitter‐ionic amino acid transport systems and mRNA transcripts was investigated in primary neuronal cultures from rat hypothalamus/brainstem. Cultures exposed to bacterial lipopolysaccharide (LPS) plus interferon‐γ (IFNγ) were assessed with respect to northern blot analyses, l‐leucine/l‐arginine cross‐inhibition uptake profiles in the presence and absence of Na+, and initial rate sodium‐independent l‐arginine transport kinetics. l‐Arginine uptake activity was constitutively expressed along with uninduced steady‐state levels of CAT1 and 4F2hc transcripts. However, neither the high‐affinity nor the low‐affinity alternatively spliced inducible isoforms of CAT2 or CAT2a transcripts (encoding system y+ in control astrocytes, lymphocytes, or liver) nor the rBAT transcripts (encoding system bo,+ in control intestinal epithelial cells) were detected by northern analysis of neuronal mRNA. Cross‐inhibition profiles were consistent with physiologic system y+ activity, but not system bo,+ or system y+L. Transport kinetics gave a single component with Vmax = 113 ± 7 pmol/min/mg of protein and Km = 47 ± 8 µMl‐arginine; these kinetic parameters were not influenced by addition of LPS/IFNγ at concentrations that up‐regulated CAT2 mRNA and system y+ activity in control astroglia from the same area of the brain. The data are consistent with l‐arginine membrane uptake occurring via only system y+ encoded by constitutive CAT1, with possible physiologic contribution by constitutive 4F2hc transcripts in primary neuronal cultures.
Up‐Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation
Authors: Cuadra Adolfo E. , El‐Fakahany Esam E.
Published: July 2010

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M1 receptors and nNOS, with 3.3 µM or 1 mM carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor‐stimulated nNOS activity as determined by l‐[3H]citrulline and cyclic [3H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist‐pretreated cells, as determined by specific [N‐methyl‐3H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor‐stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist‐pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin‐stimulated nNOS activity was greater in CCh‐pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E‐115 neuroblastoma cells, which endogenously express nNOS and muscarinic M1 receptors. Thus, the enhancement of maximal receptor‐stimulated nNOS activity following agonist pretreatment can be attributed to up‐regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down‐regulation and desensitization of multiple steps involved in nNOS activation.
Corticotropin‐Releasing Factor Administered Centrally, but Not Peripherally, Stimulates Hippocampal Acetylcholine Release
Authors: Day Jamie C. , Koehl Muriel , Le Moal Michel , Maccari Stefania
Published: July 2010

Abstract: In addition to corticotropin‐releasing factor's well‐known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin‐releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin‐releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin‐releasing factor (0.5–5.0 µg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose‐dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin‐releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin‐releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin‐releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress.