bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a System N‐Like Na+‐Dependent Glutamine Transport Activity in Rat Brain Neurons
Authors: Tamarappoo B. K. , Raizada M. K. , Kilberg M. S.
Published: July 2010

Abstract: Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and γ‐aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1‐day‐old rats. The Na+‐dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system Nb. System Nb activity can be monitored by assaying Na+‐dependent [3H]glutamine uptake in the presence of 2 mM concentrations of both 2‐(methylamino)isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system Nb for glutamine, as estimated from Km values, is significantly greater than that observed for the hepatic and muscle Na+‐dependent glutamine transporters, systems N and Nm. In sharp contrast to the hepatic system N transporter, system Nb exhibits a relative insensitivity to pH and does not permit Li+ substitution for Na+ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.
Study of the Interaction of the Myelin Monomeric GTP‐Binding Proteins with Other Brain Proteins
Authors: Rodriguez‐Gabin A. G. , Farooq M. , Norton W. T. , Larocca J. N.
Published: July 2010

Abstract: Although several monomeric GTP‐binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP‐binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP‐binding proteins was explored by incubating those blots with an enriched fraction of 22‐ and 25‐kDa myelin GTP‐binding proteins labeled with radioactive guanine nucleotides. When the GTP‐binding proteins were in the inactive state (GDP‐bound) they interacted with 28‐, 47‐, and 58‐kDa oligodendrocyte polypeptides. Only the 28‐kDa protein was present in myelin. In the active state (GTP‐bound), they interacted only with a 47‐kDa protein in myelin but with 31‐, 38‐, 47‐, 58‐, 60‐, 68‐, and 71‐kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28‐kDa protein did not interact with the GTP‐binding proteins. The fact that the myelin GTP‐binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31‐ and 38‐kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25‐kDa GTP‐binding protein was present in all the complexes. Immunoblot analysis indicated that the 28‐kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP‐binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP‐binding proteins.
Differential Regulation of Fast Axonally Transported Proteins During the Early Response of Rat Retinal Ganglion Cells to Axotomy
Authors: Wodarczyk L. , Merrill V. K. L. , Perry G. W.
Published: July 2010

Abstract: We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two‐dimensional gel electrophoresis. Corresponding changes in retinal GAP‐43 mRNA were measured using northern analysis. Within 2 days of injury, >40% of the transported proteins analyzed, including GAP‐43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP‐43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down‐regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP‐43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.
Potentiation of the Fluoxetine‐Induced Increase in Dialysate Levels of Serotonin (5‐HT) in the Frontal Cortex of Freely Moving Rats by Combined Blockade of 5‐HT1A and 5‐HT1B Receptors with WAY 100,635 and GR 127,935
Authors: Gobert A. , Rivet J.‐M. , Cistarelli L. , Millan M. J.
Published: July 2010

Abstract: In this study, we examined the influence of blockade of serotonin (5‐HT)1A and/or 5‐HT1B autoreceptors on the fluoxetine‐induced increase in dialysate levels of 5‐HT as compared with dopamine (DA) and noradrenaline (NAD) in single samples of the frontal cortex (FCx) of freely moving rats. Fluoxetine (10.0 mg/kg, s.c.) elicited a twofold increase in dialysate levels of 5‐HT relative to baseline values. The selective 5‐HT1A antagonist WAY 100,635 (0.16 mg/kg, s.c.) did not influence 5‐HT release alone but doubled the influence of fluoxetine on basal levels. Similarly, the selective 5‐HT1B/1D antagonist GR 127,935 (2.5 mg/kg, s.c.) did not alter basal 5‐HT levels alone and doubled the fluoxetine‐induced increase in 5‐HT levels. Combined administration of WAY 100,635 and GR 127,935 elicited an (at least) additive rise in the fluoxetine‐induced increase in 5‐HT levels to eightfold basal values, without modifying resting 5‐HT levels. These changes were selective for 5‐HT inasmuch as the parallel (twofold) increase in DA and NAD levels provoked by fluoxetine was not potentiated. The present data demonstrate that combined blockade of 5‐HT1A and 5‐HT1B autoreceptors markedly and selectively potentiates the fluoxetine‐induced increase in dialysate levels of 5‐HT versus DA and NAD in the FCx of freely moving rats. These observations suggest that 5‐HT1A/1B antagonism may represent a novel strategy for the improvement in the therapeutic profile of 5‐HT reuptake inhibitor antidepressant agents and that 5‐HT may be primarily involved in such interactions.
Radical Trapping and Inhibition of Iron‐Dependent CNS Damage by Cyclic Nitrone Spin Traps
Authors: Thomas Craig E. , Ohlweiler David F. , Taylor Vicki L. , Schmidt Christopher J.
Published: July 2010

Abstract: Oxidative damage in the CNS is proposed to play a role in many acute and chronic neurodegenerative disorders. Accordingly, the nitrone spin trap α‐phenyl‐N‐tert‐butylnitrone (PBN), which reacts covalently with free radicals, has shown efficacy in a variety of animal models of CNS injury. We have synthesized a number of cyclic variants of PBN and examined their activity as radical traps and protectants against oxidative damage in CNS tissue. By using electron spin resonance spectroscopy, the cyclic nitrones MDL 101,002 and MDL 102,832 were shown to trap radicals in a manner similar to that of PBN. All cyclic nitrones tested prevented hydroxyl radical‐dependent degradation of 2‐deoxyribose and peroxyl radical‐dependent oxidation of synaptosomes more potently than PBN. The radical scavenging properties of the cyclic nitrones contributed to a three‐ to 25‐fold increase in potency relative to PBN against oxidative damage and cytotoxicity in cerebellar granule cell cultures. Similar to the phenolic antioxidant MDL 74,722, the nitrones minimized seizures and delayed the time to death in mice following central injection of ferrous iron. Although iron‐induced lipid peroxidation was inhibited by MDL 74,722, the nitrones had no effect on this biochemical end point, indicating that iron‐induced mortality does not result solely from lipid peroxidation and suggesting additional neuroprotective properties for the nitrones. These results indicate that cyclic nitrones are more potent radical traps and inhibitors of lipid peroxidation in vitro than PBN, and their ability to delay significantly iron‐induced mortality in vivo suggests they may be useful in the treatment of acute and chronic neurodegeneration. Furthermore, the stability of the spin trap adducts of the cyclic nitrones provides a new tool for the study of oxidative tissue injury.
Malonate‐Induced Degeneration of Basal Forebrain Cholinergic Neurons: Attenuation by Lamotrigine, MK‐801, and 7‐Nitroindazole
Authors: Connop B. P. , Boegman R. J. , Beninger R. J. , Jhamandas K.
Published: July 2010

Abstract: Previously, we have reported that intranigral infusions of malonate, an inhibitor of mitochondrial function, lead to the degeneration of the dopaminergic neurons of the nigrostriatal pathway that is mediated, at least in part, through NMDA receptor activation and nitric oxide formation. In the present study, unilateral focal infusions of malonate into the nucleus basalis magnocellularis (nbM) of male Sprague‐Dawley rats (weighing 250–300 g) resulted in a dose‐related depletion in ipsilateral cortical and amygdaloid choline acetyltransferase (ChAT) activity. Infusion of a 3 µmol dose of malonate into the nbM of vehicle‐treated animals resulted in a 41 and 54% decrease in cortical and amygdaloid ChAT activity, respectively. Systemic pretreatment with lamotrigine (16 mg/kg, i.p.) and MK‐801 (5 mg/kg, i.p.) attenuated the depletions in cortical and amygdaloid ChAT activity that resulted from an infusion of this dose of malonate into the nbM. Acetylcholinesterase (AChE) histochemistry of the nbM following focal infusion of malonate (3 µmol) showed a marked decrease in the number of AChE‐positive neurons that was partially prevented by MK‐801 pretreatment. Before examining the role of nitric oxide formation in malonate‐induced toxicity, the ability of systemic administration of Nω‐nitro‐l‐arginine (l‐NA) to inhibit nitric oxide synthase (NOS) activity in the nbM and cerebellum was investigated. l‐NA (2, 10, and 20 mg/kg, i.p.) produced a dose‐related inhibition of nbM and cerebellar NOS activity that was maximal following a dose of 10 mg/kg l‐NA. This level of NOS inhibition persisted for at least 13 h following l‐NA (10 mg/kg) administration. Subsequently, the effect of l‐NA pretreatment on malonate toxicity was evaluated. Following pretreatment with l‐NA (2 and 10 mg/kg, i.p.), the toxic action of malonate on cortical and amygdaloid ChAT activity was not altered. In addition, infusion of a lower dose of malonate (2 µmol) into the nbM resulted in decreases in cortical and amygdaloid ChAT activity that were not altered by pretreatment with l‐NA (2 and 10 mg/kg, i.p.). In 7‐nitroindazole (7‐NI; 25 and 50 mg/kg, i.p.)‐pretreated animals, malonate (3 µmol) produced decreases in cortical and amygdaloid ChAT activity that were attenuated by both doses of 7‐NI. Thus, malonate‐induced destruction of the basal forebrain cholinergic neurons was attenuated by systemic pretreatment with lamotrigine, MK‐801, and 7‐NI but not by l‐NA.
Relationships Between Phosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin Metabolism in Cultured Oligodendrocytes
Authors: Vos Jan P. , De Haas Catharina G. M. , Van Golde Lambert M. G. , Lopes‐Cardozo Matthijs
Published: July 2010

Abstract: In most cell types the major pathway of sphingomyelin synthesis is the direct transfer of the phosphocholine head group from phosphatidylcholine to ceramide catalyzed by the enzyme l‐acylsphingosine:phosphatidylcholine phosphocholinetransferase (SM synthase; EC 2.7.8.‐). Although this pathway has been demonstrated in brain tissue, its quantitative importance has been questioned. An alternative biosynthetic pathway for sphingomyelin synthesis in brain tissue has been proposed, viz., the direct transfer of phosphoethanolamine from phosphatidylethanolamine to ceramide, followed by methylation of the ethanolamine moiety to a choline group. We have evaluated various possible biosynthetic pathways of sphingomyelin synthesis in rat spinal cord oligodendrocytes, the myelin‐forming cells of the CNS, by labeling cells in culture with radiolabeled choline, ethanolamine, or serine. Our results indicate that, in oligodendrocytes, most of the phosphocholine for the biosynthesis of sphingomyelin is provided by phosphatidylcholine, which is predominantly derived from de novo synthesis. No evidence was found for the operation of the alternative pathway via ceramide‐phosphoethanolamine. Furthermore, our results indicate that a small pool of phosphatidylcholine is provided by methylation of phosphatidylethanolamine, which in turn is formed preferentially by decarboxylation of phosphatidylserine.
Functional Changes of Rat Brain Microsomal Membrane Surface After Learning Task Depending on Dietary Fatty Acids
Authors: Yoshida S. , Miyazaki M. , Takeshita M. , Yuasa S. , Kobayashi T. , Watanabe S. , Okuyama H.
Published: July 2010

Abstract: Biochemical characteristics of brain microsomal membranes were examined before and after the brightness‐discrimination learning tasks in rats that were fed either safflower oil (α‐linolenate‐deficient) or perilla oil (α‐linolenate‐sufficient) diets. We detected small changes in the chain elongation system for polyunsaturated fatty acids in microsomes, whereas no significant difference was detected in the inositol trisphosphate‐induced calcium release and ATP‐induced calcium uptake profiles of microsomes between the two dietary groups. The calcium ion‐induced aggregation rate of microsomes was determined in both groups. We found that the aggregation rate of microsomes in the safflower oil group was significantly greater than that in the perilla oil group. The difference in susceptibility of microsomal membrane phospholipids to phospholipase A2 between the groups was obvious, and the amount of released fatty acids by phospholipase A2 from the perilla oil group microsomes was nearly half of that from the safflower oil group microsomes after the learning task. Susceptibility of sialic acids on the brain microsomal membranes to exogenous sialidase was different only after the learning task in the safflower and perilla oil groups. These results suggest that the biochemical characteristics of membrane surfaces of brain microsomes are affected significantly by the learning task itself in a dietary oil‐dependent manner.
Brain Microvascular and Astrocyte Localization of P‐Glycoprotein
Authors: Pardridge William M. , Golden Pamela L. , Kang Young‐Sook , Bickel Ulrich
Published: July 2010

Abstract: The hypothesis that P‐glycoprotein plays a functional role at the brain capillary endothelium, which makes up the blood‐brain barrier in vivo, is based largely on immunocytochemical studies showing immunoreactive P‐glycoprotein localized to either isolated brain microvessels or microvessels within tissue sections. The present studies use the MRK16 monoclonal antibody to human P‐glycoprotein to demonstrate that the pattern of immunolocalization of P‐glycoprotein in microvessels of human or primate brain is similar to the pattern of immunolocalization of an astrocyte protein, glial fibrillary acidic protein. In contrast, the discontinuous staining pattern of MRK16 is not colocalized with the continuous immunostaining of the brain endothelial GLUT1 glucose transporter. The MRK16 antibody was radiolabeled with [125I]‐iodine, and 125I‐MRK16 avidly bound isolated human brain capillaries via a saturable mechanism. However, the 125I‐MRK16 antibody was not taken up by primate brain capillaries in vivo following intravenous injection. In conclusion, these studies provide evidence that P‐glycoprotein does not play a functional role at the luminal membrane of the brain capillary endothelium in vivo, and that a principal site of immunoreactive P‐glycoprotein in brain microvasculature is localized to astrocyte foot processes.
Expression of Androgen‐Activating Enzymes in Cultured Cells of Developing Rat Brain
Authors: Poletti Angelo , Negri‐Cesi Paola , Melcangi Roberto Cosimo , Colciago Alessandra , Martini Luciano , Celotti Fabio
Published: July 2010

Abstract: Dihydrotestosterone and estradiol, two active metabolites formed locally in the brain from testosterone, modulate several functions of the developing rat CNS; these compounds derive from the 5α‐reduction or the aromatization of the A‐ring of the hormone. Also, progesterone and corticosteroids may be 5α‐reduced and subsequently 3α‐hydroxylated, becoming modulators of specific neuronal functions. Although the aromatase is a single enzyme, two types of 5α‐reductase have been cloned, showing peculiar biochemical properties and probably different functions. Therefore, the isoform(s) of the enzyme 5α‐reductase(s) present in early stage of brain development have been characterized in primary neuronal and glial cell cultures obtained from the fetal or neonatal rat brain, respectively. Aromatase expression was also studied. The results have shown that in all the brain cells examined type 1 5α‐reductase mRNA is expressed. No specific transcript of type 2 5α‐reductase is detectable in any of the cell types examined. Finally, the aromatase gene is expressed only in cultured fetal neurons and especially in those derived from the hypothalamic area of the rat embryos. It is interesting that no aromatase mRNA is detectable in mixed glia or in type 1 astrocytes and oligodendrocytes cultured separately.
Stat3 Is Present in the Developing and Adult Rat Cerebellum and Participates in the Formation of Transcription Complexes Binding DNA at the sis‐Inducible Element
Authors: Planas Anna M. , Berruezo Meritxell , Justicia Carles , Barrón Sonia , Ferrer Isidre
Published: July 2010

Abstract: Stat3 is a member of a recently identified family of proteins named STATs for their ability to act as signal transducers and activators of transcription. Stimulation of epidermal growth factor or cytokine receptors can cause activation of Stat3 and transduction of specific responses to the nucleus. Here the presence of Stat3 has been examined in the rat cerebellum at different times during postnatal development and adulthood by means of immunohistochemistry and western blotting. In addition, DNA binding activity at the sis‐inducible element that is present in the promoter of the c‐fos gene has been studied by electrophoretic mobility shift assay. The results have shown that Stat3 p92 is abundant in the rat cerebellum. Stat3 was found in the external granule cell layer and also within the molecular layer from postnatal day (P) 0 to P7. From P15 to the adult, the internal granule cell layer and Purkinje cells were also stained for Stat3. Nuclear extracts were found to contain DNA binding activity to the sis‐inducible element during development and adulthood. Supershift assays demonstrated that Stat3 mediates the formation of one protein‐DNA complex. The present results suggest that Stat3 participates in intracellular signaling and is involved in maintaining basal c‐fos expression in the cerebellum of the developing and adult rat under physiological conditions.
Overexpression of δ‐Opioid Receptors in Recombinant Baculovirus‐Infected Trichoplusia ni“High 5” Insect Cells
Authors: Wehmeyer Andrea , Schulz Rüdiger
Published: July 2010

Abstract: “High 5” cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ‐opioid receptor. The maximal binding capacity for the narcotic antagonist [3H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [3H][d‐penicillamine2,d‐penicillamine5]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ‐receptors of NG108‐15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100‐fold less effective in competing with [3H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose‐dependent manner by exposure of High 5 membranes to the exogenous G protein Go, which increased the binding capacity for DPDPE. Functional studies revealed a dose‐dependent inhibition (up to 30%) by opioids on forskolin‐stimulated cyclic AMP synthesis, and this effect was potentiated by Go. Quantification of Gαo and Gαi disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ‐opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gαo/mg of membrane protein, and a 20‐fold higher concentration for Gαi. The distinct G‐protein expression in insect cells may be considered an advantage for studying functions of G protein‐coupled receptors.
An mRNA Encoding a Putative GABA‐Gated Chloride Channel Is Expressed in the Human Cardiac Conduction System
Authors: Garret Maurice , Bascles Lionel , Boue‐Grabot Eric , Sartor Pierre , Charron Gisele , Bloch Bertrand , Margolskee Robert F.
Published: July 2010

Abstract: GABA‐gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full‐length cDNA. The predicted protein has many of the features shared by other members of the ligand‐gated ion channel family. This channel subunit has significant amino acid identity (25–40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA χ. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA χ subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.
Retinoic Acid Induces Apoptosis in PC12 Cells Independent of Neurotrophic Factors
Authors: Tong Liqi , Werrbach‐Perez Karin , Perez‐Polo J. Regino
Published: July 2010

Abstract: PC12 cells are known to undergo programmed cell death (apoptosis) when they are deprived of serum. Nerve growth factor (NGF) rescues PC12 cells from serum deprivation‐induced apoptosis. In the present study, we examined the effects of retinoic acid (RA), a classic morphogen, on apoptosis in PC12 cells after serum deprivation and NGF‐mediated rescue. In naive PC12 cells, all trans‐RA treatment induced cell death in the presence of NGF. RA also abolished the protective effects of dibutyryl cyclic AMP or insulin under serum‐free conditions. The death process was accompanied by nuclear condensation and DNA fragmentation, typical of apoptosis. In addition, RA also increased the extent of apoptosis in PC12 cells after serum deprivation. Cycloheximide, an inhibitor of protein synthesis, did not abolish the effects of RA on serum‐deprived PC12 cells. RA also decreased thymidine incorporation and proliferation in NGF‐treated PC12 cells. Furthermore, although the total DNA binding activity of the AP‐1 transcription factor was not changed after RA treatment, RA decreased a specific AP‐1 transcriptional activity. It is surprising that differentiated PC12 cells resisted the toxic effects of RA. These data suggest that RA might function as an endogenous inducer of apoptosis during neural differentiation by a mechanism distinct from that of serum deprivation.
Acute and Chronic Effects of Opioids on δ and μ Receptor Activation of G Proteins in NG108‐15 and SK‐N‐SH Cell Membranes
Authors: Breivogel Christopher S. , Selley Dana E. , Childers Steven R.
Published: July 2010

Abstract: To compare activation of G proteins by opioid receptors, opioid agonist‐stimulated guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTPγS) binding in the presence of excess GDP was assayed in membranes from NG108‐15 (δ) and SK‐N‐SH (primarily μ) cells. Basal [35S]GTPγS binding consisted of a single class of low‐affinity sites (KD 400–500 nM). Addition of agonists produced a high‐affinity site 100–300‐fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10‐fold higher for SK‐N‐SH μ receptors than for NG108‐15 δ receptors. Chronic δ agonist ([d‐Ser2]‐Leu‐enkephalin‐Thr; DSLET) treatment of NG108‐15 cells resulted in an 80% loss of DSLET‐stimulated [35S]GTPγS binding within 1 h. Morphine treatment of SK‐N‐SH cells decreased μ agonist ([d‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin; DAMGO)‐stimulated [35S]GTPγS binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.
Agonist‐Induced Endocytosis of Muscarinic Cholinergic Receptors: Relationship to Stimulated Phosphoinositide Turnover
Authors: Sorensen Scott D. , McEwen Edward L. , Linseman Daniel A. , Fisher Stephen K.
Published: July 2010

Abstract: The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist‐induced internalization has been investigated. Incubation of SH‐SY5Y neuroblastoma cells with oxotremorine‐M resulted in a time‐dependent endocytosis of both muscarinic receptors and α subunits of Gq and G11, but not of isoforms of phosphoinositide‐specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist‐induced increases in diacylglycerol mass and in 32P‐phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist‐induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4‐kinase, none was enriched when compared with cell lysates. Both 32P‐labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5‐bisphosphate, whereas the synthesis of phosphatidylinositol 4‐phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5‐bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.
Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin
Authors: Kuhn Donald M. , Arthur Robert E.
Published: July 2010

Abstract: Tryptophan hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S‐nitroso‐N‐acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro‐) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6S‐isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6R‐isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide‐induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide‐induced inactivation and the combined pterin/nitric oxide‐induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide‐induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.
Presynaptic Nicotinic Modulation of Dopamine Release in the Three Ascending Pathways Studied by In Vivo Microdialysis: Comparison of Naive and Chronic Nicotine‐Treated Rats
Authors: Marshall David L. , Redfern Peter H. , Wonnacott Susan
Published: July 2010

Abstract: The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose‐dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine‐evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine‐sensitive, nicotine‐evoked dopamine release was similar in all three brain regions. However, 10−5M tetrodotoxin totally blocked nicotine‐stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg−1 for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg−1 day−1 increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.
Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures
Authors: Davis Daniel R. , Anderton Brian H. , Brion Jean‐Pierre , Reynolds C. Hugh , Hanger Diane P.
Published: July 2010

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β‐amyloid is mediated via free radicals, and as the deposition of β‐amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF‐τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.
Purification and Characterization of a Human Brain Galectin‐1 Ligand
Authors: Chadli Ahmed , LeCaer Jean‐Pierre , Bladier Dominique , Joubert‐Caron Raymonde , Caron Michel
Published: July 2010

Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin‐1. A soluble ligand of galectin‐1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin‐1‐binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N‐linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix‐assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.
Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum
Authors: Migani Paolo , Fiorini Rosamaria , Ferretti Ersilia , Manini Elena , Chimichi Stefano , Moneti Gloriano
Published: July 2010

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low‐affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration‐dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.
Up‐Regulation of Sodium Channel Subunit mRNAs and Their Cell Surface Expression by Antiepileptic Valproic Acid: Activation of Calcium Channel and Catecholamine Secretion in Adrenal Chromaffin Cells
Authors: Yamamoto Ryuichi , Yanagita Toshihiko , Kobayashi Hideyuki , Yokoo Hiroki , Wada Akihiko
Published: July 2010

Abstract: Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for >24 h caused a time‐dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4‐fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel α‐ and β1‐subunit mRNA levels 1.4‐ and 1.7‐fold at 24 h, and 1.6‐ and 1.8‐fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4–2.1‐fold, even when assayed in the presence of Na+,K+‐ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine‐induced 22Na+ influx by ∼2‐fold in VPA‐treated cells as in nontreated cells. Long‐term treatment with VPA augmented veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage‐dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex 1.2–1.4‐fold with little change in the EC50 value of nicotine, thereby increasing the nicotine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up‐regulates cell surface expression of Na+ channels via the transcription/translation‐dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.
Rapid Anterograde Axonal Transport of the Syntaxin‐SNAP 25‐VAMP Complex
Authors: Shiff Gad , Morel Nicolas
Published: July 2010

Abstract: During the process of docking and fusion of synaptic vesicles to the presynaptic membrane, several presynaptic proteins bind sequentially to a core complex associating two proteins of the presynaptic membrane, syntaxin and SNAP 25, and a protein of synaptic vesicles, VAMP/synaptobrevin. We have immunoprecipitated this core complex after CHAPS solubilization of pure cholinergic synaptosomes of Torpedo electric organ, using anti‐syntaxin or anti‐VAMP immunobeads. In parallel, we studied syntaxin and VAMP, which are transported by the rapid axonal flow to the nerve endings. We found that syntaxin and VAMP accumulating at the proximal end of an electric nerve ligature were already engaged in complexes, as in synaptosomes. In unligated nerves also, significant amounts of VAMP associate with syntaxin. The possibility that these complexes form after solubilization was eliminated because added VAMP was unable to associate with syntaxin in solubilized control nerves and because similar amounts of complex were obtained after sodium dodecyl sulfate or CHAPS solubilization. Hence, syntaxin is already associated with SNAP 25 and VAMP during axonal transport, before reaching nerve endings.
Pituitary Adenylate Cyclase‐Activating Polypeptide Triggers Dual Transduction Signaling in CATH.a Cells and Transcriptionally Activates Tyrosine Hydroxylase and c‐fos Expression
Authors: Muller Andre , Monnier Dominique , Rene Frédérique , Larmet Yves , Koch Bernard , Loeffler Jean‐Philippe
Published: July 2010

Abstract: We used a catecholaminergic neuron‐like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase‐activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase‐PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so‐called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose‐dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 × 10−10 and 2 × 10−9M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was ∼1,000‐fold less potent in stimulating cyclic AMP (with EC50 = 2 × 10−7M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c‐fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c‐fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt‐RAB) of cyclic AMP‐dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c‐fos transcription according to a process that is primarily dependent on the cyclic AMP‐PKA pathway.
Mass Spectrometric Identification and Quantification of Hemorphins Extracted from Human Adrenal and Pheochromocytoma Tissue
Authors: Cerpa‐Poljak Anne , Lahnstein Jelle , Mason Kerryn E. , Smythe George A. , Duncan Mark W.
Published: July 2010

Abstract: The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the β, γ, δ, and ε chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.
Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine
Authors: Delouche Bruno , Pradel Louise‐Anne , Henry Jean‐Pierre
Published: July 2010

Abstract: Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O‐permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of 32P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) 32P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12‐O‐tetradecanoylphorbol 13‐acetate and 1,2‐dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.
The δ‐Opioid Receptor in SK‐N‐BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization
Authors: Namir Noureddine , Polastron Jocelyne , Allouche Stéphane , Hasbi Ahmed , Jauzac Philippe
Published: July 2010

Abstract: The human neuroblastoma cell line SK‐N‐BE expresses δ‐opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 nM etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2‐dopaminergic, α2‐adrenergic, and m2/m4‐muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µM 2‐bromo‐α‐ergocryptine or 1 µM arterenol resulted in a marked desensitization of D2‐dopaminergic and α2‐adrenergic receptors, respectively. Cross‐desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ‐opioid receptor and the D2‐dopaminergic receptor, and pretreatment with 2‐bromo‐α‐ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α2‐adrenergic receptor response, whereas pretreatment with 1 µM arterenol partly desensitized the δ‐opioid receptor response. It is concluded that the δ‐opioid receptor‐mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ‐opioid and D2‐dopaminergic receptors are coupled to adenylyl cyclase via a Gi2 protein, whereas α2‐adrenergic receptor could be coupled to the enzyme via two G proteins, Gi2 and another member of the Gi/Go family.
Calcium‐Activated Neutral Protease Activity Is Decreased in PC12 Cells After Ethanol Exposure
Authors: DePetrillo Paolo B.
Published: July 2010

Abstract: Calcium‐activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence‐based assay with N‐succinyl‐Leu‐Tyr 7‐amido‐4‐methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 µM) or low (140 µM) Ca2+ concentrations in the presence of 20 µM ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: Ft = Ffinal*(1 −e−kobst). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by kobs beginning at 20 mM, to 27.8% of control at 1,400 µM Ca2+ and 29.2% of control at 140 µM Ca2+ in the presence of 20 µM ionomycin. No changes in μ‐calpain or m‐calpain activities were found in cell extracts from cells exposed to 20 mM ethanol, whereas at 40 and 80 mM ethanol, significant decreases in both μ‐calpain and m‐calpain activities were discovered.
Amyloid β‐Peptide Induces Cell Monolayer Albumin Permeability, Impairs Glucose Transport, and Induces Apoptosis in Vascular Endothelial Cells
Authors: Blanc Emmanuelle M. , Toborek Michal , Mark Robert J. , Hennig Bernhard , Mattson Mark P.
Published: July 2010

Abstract: Amyloid β‐peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood‐brain barrier occur in AD. Here we report that Aβ25‐35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25‐35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25‐35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25‐35 were specific because Aβ1‐40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1‐40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25‐35. Damage and death of ECs induced by Aβ25‐35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.
Identification of Candidate Genes Induced by Retinoic Acid in Embryonal Carcinoma Cells
Authors: Cheung William M. W. , Chu Apple H. , Ip Nancy Y.
Published: July 2010

Abstract: Retinoic acid (RA) induced the terminal differentiation of a human embryonal carcinoma cell line (NT2/D1) into several morphologically distinct cell types, including the postmitotic CNS neurons. Although RA has been suggested to play an important role in brain development, little is known about the molecular mechanism by which RA induces neuronal differentiation. In the present study, RNA fingerprinting by arbitrarily primed PCR (RAP‐PCR) was used to identify the transcripts in NT2/D1 cells that were differentially regulated by RA. Northern blot analysis of the differentially amplified PCR fragments revealed 11 genes that were regulated by RA. Of these, seven were up‐regulated and four were down‐regulated along the course of RA treatment. More importantly, four of the RA‐regulated genes that were identified in the present study are novel. Our findings suggested that there are a number of RA‐regulated genes that have yet to be identified. RAP‐PCR provides a useful tool for studying the patterns of transcript expression during the course of RA treatment and allows the cloning of novel genes involved in the process of neuronal differentiation. Furthermore, it provides a basis for the selection of genes that are involved in the RA‐induced signaling pathway in the human CNS.