bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic AMP Regulates the Expression of Neurokinin1 Receptors by Neonatal Rat Spinal Neurons in Culture
Authors: Abrahams L. G. , Reutter M. A. , McCarson K. E. , Seybold V. S.
Published: March 2003

Abstract: Neurokinin1 (NK1) receptors are up‐regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)‐responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl‐cAMP (dbcAMP) resulted in a time‐dependent increase in 125I‐Bolton‐Hunter‐substance P (BHSP) binding in the cultures; treatment with dibutyryl‐cyclic GMP did not. Treatment with forskolin plus 3‐isobutyl‐1‐methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP‐induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP‐induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)‐induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.
α1‐Antichymotrypsin‐Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme “Prohormone Thiol Protease”
Authors: Hook Vivian Y. H. , Tezapsidis Nikolaos , Hwang Shin‐Rong , Sei Catherine , Byrne Michael , Yasothornsrikul Sukkid
Published: March 2003

Abstract: Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary α1‐antichymotrypsin (ACT), referred to as ACT‐like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56‐ and 60‐kDa ACT‐like proteins I and II, respectively, to enkephalin‐containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE‐Sepharose and chromatofocusing resulted in the separation of ACT‐like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56‐kDa ACT‐like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60‐kDa ACT‐like protein II was isolated by butyl‐Sepharose, Sephacryl S200, and concanavalin A‐Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT‐like protein I, with a Ki,app of 35 nM, but ACT‐like protein II was less effective. ACT‐like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT‐like proteins that differentially inhibit PTP. The colocalization of the ACT‐like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT‐like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.
Cyclic AMP‐Dependent Activation of the Proenkephalin Gene Requires Phosphorylation of CREB at Serine‐133 and a Src‐Related Kinase
Authors: Kobierski Linda A. , Wong Allison E. , Srivastava Smita , Borsook David , Hyman Steven E.
Published: March 2003

Abstract: The transcription factor CREB [cyclic AMP response element (CRE)‐binding protein] is activated by several kinase pathways on phosphorylation of serine‐133. Phosphorylation of CREB at serine‐133 is required for the induction of target gene expression. The proenkephalin gene is a target of cyclic AMP‐dependent agonists like forskolin, and its expression is driven by the enhancer element CRE‐2. It has been shown that CREB binds CRE‐2 in extracts from striatum and hypothalamus. However, these studies did not show a functional requirement for CREB serine‐133 phosphorylation in CRE‐2 function. We demonstrate that CREB binds CRE‐2 in primary astrocyte cultures and that transcriptional activation of CRE‐2 requires CREB phosphorylation at serine‐133. In addition, it has recently been shown that, at least in some contexts, CREB phosphorylation is not sufficient to activate target gene expression and that another intracellular signal seems to be required. Therefore, we also sought to determine if another signaling event, in addition to CREB phosphorylation, might be involved in cyclic AMP‐mediated induction of the proenkephalin gene. We have found that the inhibition of src‐related nonreceptor tyrosine kinases blocks forskolin‐induced proenkephalin gene expression without having any effect on serine‐133‐phosphorylated CREB levels and that constitutively activated src kinase can activate the proenkephalin promoter.
ATP‐Stimulated Ca2+ Influx and Phospholipase D Activities of a Rat Brain‐Derived Type‐2 Astrocyte Cell Line, RBA‐2, Are Mediated Through P2X7 Receptors
Authors: Sun Synthia H. , Lin Lian‐Bin , Hung Amos C. , Kuo Jon‐Son
Published: March 2003

Abstract: This study characterizes and examines the P2 receptor‐mediated signal transduction pathway of a rat brain‐derived type 2 astrocyte cell line, RBA‐2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP‐stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), in a concentration‐dependent manner. The agonist 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2‐methylthioadenosine triphosphate tetrasodium, and ATPγS were much less effective, whereas UTP, ADP, α,β‐methylene‐ATP, and β,γ‐methylene‐ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP‐ and BzATP‐stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration‐ and time‐dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration‐dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP‐stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+‐dependent PLD in RBA‐2 astrocytes. Furthermore, protein kinase C regulates this PLD.
Extensive Alternative Splicing in the 5′‐Untranslated Region of the Rat and Human Neuropeptide Y Y5 Receptor Genes Regulates Receptor Expession
Authors: Parker Eric M. , Xia Ling
Published: March 2003

Abstract : Neuropeptide Y (NPY) elicits a plethora of physiological effects by interacting with several distinct G protein‐coupled receptors. Activation of one of these receptors, the NPY Y5 receptor, is thought to result in increased food intake, anticonvulsant effects, attenuation of opiate withdrawal, inhibition of neuronal activity, and alteration of renal function. Several alternatively spliced human and rat NPY Y5 receptor cDNAs have been isolated that use different combinations of exons in the 5′‐untranslated region. The various human NPY Y5 receptor cDNAs appear to be differentially expressed in different brain regions. The level of human NPY Y5 receptor expressed transiently in COS1 cells was significantly influenced by the sequence of the 5′‐untranslated region. These results indicate that alternative splicing in the 5′‐untranslated region of the human and rat NPY Y5 receptor genes occurs in a tissue‐specific manner and is one mechanism by which cells control the level of NPY Y5 receptor expression.
Different Roles for RhoA During Neurite Initiation, Elongation, and Regeneration in PC12 Cells
Authors: Sebök Ágnes , Nusser Nóra , Debreceni Balázs , Guo Zhong , Santos Marinilce F. , Szeberenyi József , Tigyi Gábor
Published: March 2003

Abstract : The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)‐induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid‐regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites ; however, they remained responsive to NGF, as indicated by the activation of extracellular signal‐regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered ; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were ~50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis ; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF‐induced neuronal differentiation and neurite regeneration.
Cholinergic Stimulation of Early Growth Response‐1 DNA Binding Activity Requires Protein Kinase C and Mitogen‐Activated Protein KInase Kinase Activation and Is Inhibited by Sodium Valporate in SH‐SY5y Cells
Authors: Grimes Carol A. , Jope Richard S.
Published: March 2003

Abstract : Activation of muscarinic receptors in human neuroblastoma

SH‐SY5Y cells with carbachol stimulated a rapid and large increase in early

growth response‐1 (Egr‐1, also called zif268 and NGF1‐A) protein levels and

DNA binding activity. Egr‐1 DNA binding activity was stimulated within 15 min

of treatment with carbachol and maintained a maximum 20‐fold increase over

basal between 1 and 2 h after treatment, and the EC50 was ~

μM carbachol. Carbachol‐stimulated Egr‐1 DNA binding activity was

dependent on protein kinase C, as it was potently inhibited by GF109203×

(IC50 ~0.1 mM) and was reduced by 85 ± 5% by down‐regulation of protein kinase C. Inhibitors of increases in intracellular calcium levels reduced carbachol‐induced Egr‐1 DNA binding activity by 25‐35%. Carbachol‐stimulated activation of Egr‐1 was reduced 35% by genistein, a tyrosine kinase inhibitor, and 60% by PD098059, an inhibitor of mitogen‐activated protein kinase kinases 1/2 (MEK1/2) that activates extracellular‐regulated kinases 1/2 (ERK1/2). A novel inhibitory action was caused by chronic (7‐day) administration of sodium valproate but not by two other bipolar disorder therapeutic agents, lithium and carbamazepine. Valproate treatment reduced carbachol‐stimulated Egr‐1 DNA binding activity by 60% but did not alter carbachol‐induced activation of ERK1/2 or p38 or increases in Egr‐1 protein levels. These results reveal that muscarinic receptors activate Egr‐1 through a signaling cascade primarily encompassing protein kinase C, MEK1/2, and ERK1/2 and that valproate substantially inhibits Egr‐1 DNA binding activity stimulated by carbachol or protein kinase C.
Regulation of Phospholipase Cγ in the Mesolimbic Dopamine System by Chronic Morphine Administration
Authors: Wolf Daniel H. , Numan Suzanne , Nestler Eric J. , Russell David S.
Published: March 2003

Abstract: Neurotrophic signaling pathways have been implicated in the maintenance of the mesolimbic dopamine system, as well as in changes in this system induced by chronic morphine exposure. We found that many of these signaling pathway proteins are expressed at appreciable levels within the ventral tegmental area (VTA) and related regions, although with substantial regional variation. Moreover, phospholipase Cγ1 (PLCγ1) was significantly and specifically up‐regulated within the VTA by 30% following chronic exposure to morphine. PLCγ1 mRNA expression is enriched in dopaminergic neurons within the VTA ; however, the up‐regulation of PLCγ1 in this region was not seen at the mRNA level. In contrast to PLCγ1, insulin receptor substrate (IRS)‐2, a protein involved in phosphatidylinositol 3‐kinase signaling, and another putative IRS‐like protein were significantly down‐regulated within the VTA by 49 and 45%, respectively. Levels of several proteins within the Ras‐ERK pathway were not altered. Regulation of neurotrophic factor signaling proteins may play a role in morphine‐induced plasticity within the mesolimbic dopamine system.
Sublytic Terminal Complement Complexes Decrease POGene Expression in Schwann Cells
Authors: Dashiell Suzanne M. , Koski Carol Lee
Published: March 2003

Abstract : Complement cascade activation on peripheral nerve

myelincan can cause myelin destruction. Although terminal complement complexes

(TCCs) are transiently detected on Schwann cells (SchCs) during inflammatory

neuropathy, SchCs appear resistant to complement‐mediated lysis, and little is

known about the functional consequences of sublytic TCC deposition on SchCs.

We studied the effects of sublytic complement in modulating myelin gene

expression at the posttranscriptional and transcriptional levels. Cultured

SchCs, stimulated to express protein zero (P0), were treated with

sensitizing antibody (Ab) and normal human serum (NHS) complement.

P0 mRNA content decreased by 71% during 12 h. In the presence of

actinomycin D, P0 mRNA levels declined 50% following incubation

with Ab plus 10% NHS over 6 h, compared with control levels, suggesting

enhanced P0 mRNA degradation. The decreases, in part, reflected TCC

formation because C7 reconstitution of Ab plus C7‐depleted human serum (C7dHS)

or TCCs assembled from purified components down‐regulated P0 mRNA

53 and 55% over that of Ab plus C7dHS or heat‐activated components,

respectively. Expression of a P0 promoter/luciferase reporter

consruct transiently transfected into SchCs was reduced 70% by sublytic TCCs

at 6 h, demonstrating that P0 gene transcription was also

inhibited. c‐jun mRNA was up‐regulated within 30 min by sublytic

TCCs, before the reduction in P0 mRNA expression. Our data suggest that sublytic complement activation on SchCs may contribute to peripheral nerve demyelination by decreasing expression of genes important in myelin formation and compaction.
Glial and Neuronal Cells Express Functional Chemokine Receptor CXCR4 and Its Natural Ligand Stromal Cell‐Derived Factor 1
Authors: Bajetto Adriana , Bonavia Rudy , Barbero Simone , Piccioli Patrizia , Costa Alfredo , Florio Tullio , Schettini Gennaro
Published: March 2003

Abstract : Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell‐derived factor 1, (SDF1), binds to the seventransmembrane G protein‐coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down‐regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down‐modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1α (hSDF1α) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1α with the HIV‐1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.
Neuregulin‐Increased Expression of Acetylcholine Receptorε‐Subunit Gene Requires ErbB Interaction with Shc
Authors: Won Sandra , Si Jutong , Colledge Marcie , Ravichandran Kodimangalam S. , Froehner Stanley C. , Mei Lin
Published: March 2003

Abstract : Selective transcription of acetylcholine receptor (AChR) subunit genes by neuregulin is one of the mechanisms involved in the synaptic localization of AChRs to the neuromuscular junction. Neuregulin stimulates ErbB receptor tyrosine kinases and subsequently activates the Ras/ERK pathway, which is required for neuregulin‐mediated induction of AChR subunit genes in muscle cells and synapse‐specific expression in vivo. Here we investigated the neuregulin transduction mechanism that leads to ERK activation after ErbB receptor tyrosine phosphorylation. Neuregulin increases the association of the adaptor proteins Grb2 and Shc with both ErbB2 and ErbB3 in C2C12 muscle cells. Dephosphorylation of the tyrosine‐phosphorylated ErbB proteins abolished their association with both Grb2 and Shc, suggesting a tyrosine phosphorylation‐dependent interaction. The interaction of Shc with the ErbB receptors is mediated by Shc's phosphotyrosine‐binding domain. In addition, neuregulin increased tyrosine phosphorylation of Shc. Mutagenesis approaches demonstrated that tyrosine phosphorylation of Shc is required for neuregulin induction of AChR subunit gene expression. Taken together, these data indicate that the interaction of ErbB receptors with Grb2 alone is insufficient for neuregulin‐activated transcription, but that ErbB receptor signaling via Shc is necessary and important.
Effect of Benzodiazepines on the Epithelial and Neuronal High‐Affinity Glutamate Transporter EAAC1
Authors: Palmada Mònica , Böhmer Christoph , Centelles Josep Joan , Kinne Rolf K. H.
Published: March 2003

Abstract : EAAC1‐mediated glutamate transport concentrates glutamate

across plasma membranes of brain neurons and epithelia. In brain, EAAC1

provides a presynaptic uptake mechanism to terminate the excitatory action of

released glutamate and to keep its extracellular concentration below toxic

levels. Here we report the effect of well known anxiolytic compounds,

benzodiazepines, on glutamate transport in EAAC1‐stably transfected Chinese

hamster ovary (CHO) cells and in EAAC1‐expressing Xenopus laevis

oocytes. Functional properties of EAAC1 agreed well with already reported

characteristics of the neuronal high‐affinity glutamate transporter

(Km D‐Asp, CHO cells : 2.23 ± 0.15 μM ;

Km D‐Asp, oocytes : 17.01 ± 3.42 μM).

In both expression systems, low drug concentrations (10‐100 μM)

activated substrate uptake (up to 200% of control), whereas concentrations in

the millimolar range inhibited (up to 50%). Furthermore, the activation was

more pronounced at low substrate concentrations (1 μM), and the

inhibition was attenuated. The activity of other sodium cotransporters such as

the sodium/D‐glucose cotransporter SGLT1, stably transfected in CHO cells, was

not affected by benzodiazepines. In electrophysiological studies, these drugs

also failed to change the membrane potential of EAAC1‐expressing Xenopus

laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.
Structural Requirements of 5‐Hydroxytryptamine‐Moduline Analogues to Interact with the 5‐Hydroxytryptamine1B Receptor
Authors: Plantefol Mathieu , Rousselle Jean Claude , Massot Olivier , Bernardi Elizabeth , Schoofs Alain René , Pourrias Bernard , Ollivier Roland , Fillion Gilles
Published: March 2003

Abstract : 5‐Hydroxytryptamine‐moduline is an endogenous cerebral

tetrapeptide that regulates the activity of 5‐hydroxytryptamine1B

receptors. Direct binding of 5‐[3H]hydroxytryptamine‐moduline on

rat brain homogenate evidenced the existence of two interacting sites for the

peptide, very likely corresponding to different conformations of the

5‐hydroxytryptamine1B receptor : The peptide first binds to a

low‐affinity state of the receptor (pIC50 = 7.68 ± 0.14) and

then induces (or stabilizes) a high‐affinity complex (pIC50 = 11.62

± 0.18). This work focuses on the ability of

5‐hydroxytryptamine‐moduline analogues to recognize the high‐ and low‐affinity

sites for 5‐hydroxytryptamine‐moduline. The results obtained show that the two

conformers of the 5‐hydroxytryptamine1B receptor have similar but not identical binding pockets for 5‐hydroxytryptamine‐moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low‐affinity site than the high‐affinity one, which only needs a short hydrophobic amino acid in position 2.
Activity‐Dependent Phosphorylation of SNAP‐25 in Hippocampal Organotypic Cultures
Authors: Genoud Sylvie , Pralong William , Riederer Beat M. , Eder Lorenza , Catsicas Stefan , Muller Dominique
Published: March 2003

Abstract: Synaptosomal‐associated protein of 25 kDa (SNAP‐25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP‐25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP‐25 phosphorylation. In addition, immunochemical analysis of two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels shows that SNAP‐25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12, 13‐dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP‐25 among these spots. Phorbol 12, 13‐dibutryate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long‐term potentiation, a property involved in learning mechanisms, and production of seizures with a GABAA receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP‐25 can be phosphorylated in situ by PKC in an activity‐dependent manner.
Subunit Assembly and Domain Analysis of Electrically Silent K+ Channel α‐Subunits of the Rat Kv9 Subfamily
Authors: Stocker Martin , Hellwig Michaela , Kerschensteiner Daniel
Published: March 2003

Abstract:α‐Subunits of the voltage‐gated potassium channel (Kv) subfamily Kv9 show no channel activity after homomultimeric expression in heterologous expression systems. This report shows that heteromultimeric expression of rKv9.1 and rKv9.3 specifically suppresses the currents mediated by α‐subunits of the Kv2 and Kv3 subfamilies but does not affect currents mediated by α‐subunits of the Kv1 and Kv4 subfamilies. To understand the molecular basis of the electrical silence of Kv9 homomultimeric channels, crucial functional domains (amino and carboxy terminus, S4 segment, and pore region) were exchanged between Kv9 α‐subunits and rKv1.3. Electrophysiological studies of these chimeras revealed that the pore region is involved in determining the nonconductive behavior of homomultimeric Kv9 channels. This analysis was extended by protein interaction assays, aiming to identify the region of Kv9 subunits responsible for the specific suppression of rKv2.1‐ and rKv3.4‐mediated currents. We could show that the amino‐terminal domain of Kv9 α‐subunits does not support homomultimeric assembly but interacts specifically with the rKv2.1 amino‐terminal region. Conversely, the specific intersubfamily assembly of rKv3.4 with rKv9.1 or rKv9.3 is governed by the hydrophobic core and not the amino‐terminal domain.
Phosphorylation‐Dependent Akt Cleavage in Neural Cell In Vitro Reconstitution of Apoptosis
Authors: François Fleur , Grimes Mark L.
Published: March 2003

Abstract : Neuronal apoptotic execution uses a cytochrome

c‐dependent caspase activation mechanism that is conserved in other

cell types. Phosphatidylinositol 3‐kinase and its downstream effector,

Akt/protein kinase B, appear to control this mechanism and govern the

life/death decision. We have developed a cell‐free system using cytosol from

human neuroblastoma (SY5Y) cells that reconstitutes biochemical features of

neuronal apoptosis. In the presence of cytochrome c and ATP, caspase‐9 and ‐3 were activated, which initiated chromatin condensation and DNA cleavage in rat pheochromocytoma (PC12) nuclei. Akt was cleaved in reactions where caspase‐3 was activated and its cleavage was prevented by the caspase inhibitor DEVD‐aldehyde. The phosphatase inhibitors orthovanadate and okadaic acid prevented catalytic processing and activation of caspase‐3 and digestion of Akt and partially inhibited cleavage of caspase‐9. Caspase‐dependent destruction of Akt irreversibly inactivates this key mediator of survival signaling, ensuring that the execution pathway will prevail.
Changes of Synaptotagmin Interaction with t‐SNARE Proteins In Vitro After Calcium/Calmodulin‐Dependent Phosphorylation
Authors: Verona Marina , Zanotti Simona , Schäfer Theo , Racagni Giorgio , Popoli Maurizio
Published: March 2003

Abstract: The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein‐protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP‐25, and VAMP‐synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage‐activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation‐dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP‐25 in native complexes was not found to be calcium‐dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin‐syntaxin interaction. Stimulation of Ca2+/calmodulin‐dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP‐25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin‐t‐SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation‐dephosphorylation balance in regulation of the synaptotagmin‐t‐SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.
Uptake of Lipoprotein‐Associated α‐Tocopherol by Primary Porcine Brain Capillary Endothelial Cells
Authors: Goti Daniel , Hammer Astrid , Galla Hans‐Joachim , Malle Ernst , Sattler Wolfgang
Published: March 2003

Abstract: From the severe neurological syndromes resulting from
vitamin E deficiency, it is evident that an adequate supply of the brain with
α‐tocopherol (αTocH), the biologically most active member of the
vitamin E family, is of utmost importance. However, uptake mechanisms of
αTocH in cells constituting the blood‐brain barrier are obscure.
Therefore, we studied the interaction of low (LDL) and high (HDL) density
lipoproteins (the major carriers of αTocH in the circulation) with
monolayers of primary porcine brain capillary endothelial cells (pBCECs) and
compared the ability of these two lipoprotein classes to transfer
lipoprotein‐associated αTocH to pBCECs. With regard to potential binding
proteins, we could identify the presence of the LDL receptor and a putative
HDL3 binding protein with an apparent molecular mass of 100 kDa. At
4°C, pBCECs bound LDL with high affinity (KD = 6
nM) and apolipoprotein E‐free HDL3 with low affinity (98
nM). The binding capacity was 20,000 (LDL) and 200,000
(HDL3) lipoprotein particles per cell. αTocH uptake was
approximately threefold higher from HDL3 than from LDL when
[14C]αTocH‐labeled lipoprotein preparations were used. The
majority of HDL3‐associated αTocH was taken up in a
lipoprotein particle‐independent manner, exceeding HDL3
holoparticle uptake 8‐ to 20‐fold. This uptake route is less important for
LDL‐associated αTocH (αTocH uptake ∼1.5‐fold higher than
holoparticle uptake). In line with tracer experiments, mass transfer studies
with unlabeled lipoproteins revealed that αTocH uptake from
HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic αTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with αTocH in vivo.
Nuclear Ca2+/Calmodulin Translocation Activated by μ‐Opioid(OP3) Receptor
Authors: Wang Danxin , Tolbert Lara M. , Carlson Kurt W. , Sadée Wolfgang
Published: March 2003

Abstract: Previous evidence has suggested a role for calmodulin
(CaM) in opioid receptor signaling. We demonstrate here that morphine
stimulation of the μ‐opioid (OP3) receptor causes rapid CaM
translocation to the nucleus in OP3‐transfected human embryonic
kidney (HEK)‐293 cells and in SH‐SY5Y human neuroblastoma cells.
Ca2+ influx into the cells resulting from OP3 receptor
activation was required for nuclear CaM translocation. Moreover, in
HEK‐OP3 and SH‐SY5Y cells, increased nuclear CaM content was
associated with enhanced phosphorylation of the nuclear transcription factor
cyclic AMP‐responsive element‐binding protein. This appeared to be mediated by
Ca2+/CaM kinases and also by a pathway involving protein kinase C.
CaM was previously shown to bind directly to the OP3 receptor and
to be released from the plasma membrane on agonist stimulation. To test
whether OP3‐mediated CaM release contributes to nuclear CaM
signaling, we used a mutant OP3 receptor (K273A) with reduced
affinity for CaM that fails to release CaM from the plasma membrane.
K273A‐OP3 activated Ca2+ influx to a similar extent as
wild‐type OP3; however, CaM translocation to the nucleus was
attenuated. These results indicate that OP3‐stimulated
Ca2+ influx results in nuclear CaM translocation, which appears to
be enhanced by simultaneous CaM release by OP3 wild‐type receptor
from plasma membranes. These results suggest a novel Ca2+/CaM signaling pathway of opioid receptors in the regulation of transcriptional activity.
Astrocyte Differentiation of Fetal Neuroepithelial Cells by Interleukin‐11 via Activation of a Common Cytokine Signal Transducer, gp130, and a Transcription Factor, STAT3
Authors: Yanagisawa Makoto , Nakashima Kinichi , Arakawa Hirokazu , Ikenaka Kazuhiro , Yoshida Kanji , Kishimoto Tadamitsu , Hisatsune Tatsuhiro , Taga Tetsuya
Published: March 2003

Abstract: The interleukin (IL)‐6 family cytokines utilize membrane
glycoprotein gp130 in common as a critical signal‐transducing receptor
component. IL‐11, a cytokine initially identified as a plasmacytoma growth
factor, belongs to this family. We show here that IL‐11 and its cognate
receptor components are expressed in fetal mouse neuroepithelial cells. We
also show that after 4 days of culture with IL‐11, cells with typical
astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a
marker for astrocytes) come out. This differentiation process is totally
dependent on the gp130‐mediated signal‐transduction pathway involving
activation of a latent cytoplasmic transcription factor, STAT3 (for
signal transducer and activator of
transcription 3), because (a) IL‐11‐induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130‐deficient mice were used, (b) stimulation of neuroepithelial cells by IL‐11 rapidly induces tyrosine‐phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant‐negative form of STAT3 inhibits IL‐11‐induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL‐11‐induced GFAP promoter activation. Taken together, it is suggested that IL‐11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3.
Chronic Blockade of N‐Methyl‐D‐Aspartate Receptors Alters γ‐Aminobutyric Acid Type A Receptor Peptide Expression and Function in the Rat
Authors: Matthews Douglas B. , Kralic Jason E. , Devaud Leslie L. , Fritschy Jean‐Marc , Morrow A. Leslie
Published: March 2003

Abstract: Chronic in vivo or in vitro application of
GABAA receptor agonists alters GABAA receptor peptide
expression and function. Furthermore, chronic in vitro application of
N‐methyl‐D‐aspartate (NMDA) agonists and antagonists alters
GABAA receptor function and mRNA expression. However, it is unknown
if chronic in vivo blockade of NMDA receptors alters GABAA receptor
function and peptide expression in brain. Male Sprague‐Dawley rats were
chronically administered the noncompetitive NMDA receptor antagonist MK‐801
(0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors
significantly increased hippocampal GABAA receptor
α4 and γ2 subunit expression while
significantly decreasing hippocampal GABAA receptor
α2 and β2/3 subunit expression. Hippocampal
GABAA receptor α1 subunit peptide expression was
not altered. In contrast, no significant alterations in GABAA
receptor subunit expression were found in cerebral cortex. Chronic MK‐801
administration also significantly decreased GABAA receptor‐mediated
hippocampal Cl‐ uptake, whereas no change was found in
GABAA receptor‐mediated cerebral cortical Cl‐ uptake.
Finally, chronic MK‐801 administration did not alter NMDA receptor NR1, NR2A,
or NR2B subunit peptide expression in either the cerebral cortex or the
hippocampus. These data demonstrate heterogeneous regulation of
GABAA receptors by glutamatergic activity in rat hippocampus but
not cerebral cortex, suggesting a new mechanism of GABAA receptor regulation in brain.
Distinct Internalization of M2 Muscarinic Acetylcholine Receptors Confers Selective and Long‐Lasting Desensitization of Signaling to Phospholipase C
Authors: Krudewig Riccarda , Langer Barbara , Vögler Oliver , Markschies Nicole , Erl Martin , Jakobs Karl H. , Van Koppen Chris J.
Published: March 2003

Abstract: Although M1‐M4 muscarinic
acetylcholine receptors (mAChRs) in HEK‐293 cells internalize on agonist
stimulation, only M1, M3, and M4 but not
M2 mAChRs recycle to the plasma membrane. To investigate the
functional consequences of this phenomenon, we compared desensitization and
resensitization of M2 versus M4 mAChRs. Treatment with 1
mM carbachol for 1 h at 37°C reduced numbers of cell surface
M2 and M4 mAChRs by 40‐50% and M2 and
M4 mAChR‐mediated inhibition of adenylyl cyclase, intracellular
Ca2+ concentration ([Ca2+]i) increases, and
phospholipase C (PLC) activation by 60‐70%. Receptor‐mediated inhibition of
adenylyl cyclase and [Ca2+]i increases significantly
resensitized within 3 h. However, M4 but not M2
mAChR‐mediated PLC activation resensitized. At 16°C, M2
mAChR‐mediated [Ca2+]i increases and PLC stimulation
desensitized to a similar extent as at 37°C. However, at 16°C, where
M2 mAChR internalization is negligible, both M2 mAChR
responses resensitized, demonstrating that M2 mAChR resensitization
proceeds at the plasma membrane. Examination of M2 mAChR responses
following inactivation of cell surface mAChRs by quinuclidinyl benzilate
revealed substantial receptor reserve for coupling to
[Ca2+]i increases but not to PLC. We conclude that
M2 mAChR internalization induces long‐lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve.
A Minimal Promoter for the GABAA Receptor α6‐Subunit Gene Controls Tissue Specificity
Authors: McLean Pamela J. , Shpektor Diana , Bandyopadhyay Sabita , Russek Shelley J. , Farb David H.
Published: March 2003

Abstract: The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A γ‐aminobutyric acid (GABAA) receptor α6‐subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell‐specific gene expression, the proximal 5′ flanking regions for the human, rat, and mouse α6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat α6 gene‐reporter constructs in primary neuronal cultures reveals that a 155‐bp TATA‐less promoter region (‐130 to +25 bp) constitutes a minimal promoter that can drive cerebellar granule cell‐specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60‐bp region (‐130 to ‐70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60‐bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar‐specific activating sequence is located between ‐324 and ‐130 bp, and a downstream negative regulatory region (+158 to +294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell‐specific expression of the GABAA receptor α6 subunit gene.
Taurine‐Induced Attenuation of MPP+ Neurotoxicity In Vitro
Authors: O'Byrne Muriel B. , Tipton Keith F.
Published: March 2003

Abstract: Taurine is a sulphur‐containing β‐amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin‐induced neuronal damage. Here we report a protective effect of taurine against MPP+‐induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABAA receptors.
Protein Binding of NADH on Chemical Preconditioning
Authors: Huber Roman , Büchner Maren , Li Hongge , Schlieter Martin , Speerfeld Anne Dorte , Riepe Matthias W.
Published: March 2003

Abstract : Chemical preconditioning, an emerging neuroprotective

strategy described in recent years, results in preserved energy metabolism

during hypoxia via yet unknown mechanisms. The hypoxic increase of NADH

content is attenuated by preconditioning. The goal of the present study was to

investigate whether attenuation of the hypoxic NADH increase is due to a shift

between free and protein‐bound NADH. NADH in solution has a fluorescence

maximum at 469.2 nm. In untreated mouse hippocampal slices,

λcontrol onset is 456.2 ± 5.3 nm in CA1 (mean

± SD ; p < 0.01 vs. solution) and 454.6 ± 6.1 nm in

CA3 [p < 0.01 vs. solution, not significant (NS) to

λcontrol onset in CA1]. In slices prepared from animals

pretreated in vivo with 20 mg/kg 3‐nitropropionate,

λpreconditioning onset is 439.2 ± 5.0 nm (p

< 0.001 vs. control) in CA1 and 434.2 ± 6.4 nm in CA3 (p

< 0.001 vs. control ; NS to λpreconditioning onset in

CA1). In controls, the fluorescence maximum shifts to λcontrol

hypoxia 458.2 ± 1.3 nm in CA1 (NS vs. onset) and 456.0 ±

3.6 nm in CA3 (NS vs. onset). On preconditioning with 3‐nitropropionate,

λpreconditioning hypoxia shifts to 446.4 ± 4.3 nm in

CA1 (p < 0.03 vs. onset) and 438.6 ± 6.9 nm in CA3

(p < 0.03 vs. onset). Posthypoxic decay of free and protein‐bound NADH is diminished after preconditioning. We conclude that the free NADH level is reduced on an increase of hypoxic tolerance by chemical preconditioning. Reduction of free NADH content is maintained during hypoxia after preconditioning.
Heat Shock Transcription Factors and the hsp70 Induction Response in Brain and Kidney of the Hyperthermic Rat During Postnatal Development
Authors: Morrison Andrew J. , Rush Sheila J. , Brown Ian R.
Published: March 2003

Abstract : Heat shock transcription factor (HSF) 1 levels increase in brain regions and decline in kidney during postnatal rat development. In both neonatal and adult rats, levels of HSF1 protein in brain and kidney are proportional to the levels of HSF DNA‐binding activity and the magnitude of heat shock protein hsp70 induction after thermal stress. There appears to be more HSF1 protein in adult brain than is needed for induction of hsp70 after thermal stress, suggesting that HSF1 may have other functions in addition to its role as a stress‐inducible activator of heat shock genes. HSF2 protein levels decline during postnatal rat development in brain regions and kidney. Gel mobility shift analysis shows that HSF2 is not in a DNA‐binding form in the neonatal brain and kidney, suggesting that HSF2 may not be involved in the constitutive expression of hsps in early postnatal development. There is no apparent relationship between levels of HSF2 protein and basal levels of hsp90, hsp70, heat shock cognate protein hsc70, and hsp60.
Evidence for Expression of Some Microtubule‐Associated Protein 1B in Neurons as a Plasma Membrane Glycoprotein
Authors: Tanner Sandra L , Franzen Rachelle , Jaffe Howard , Quarles Richard H
Published: March 2003

Microtubule‐associated protein (MAP) 1B is a high‐molecular‐weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well‐known vesicular and plasma membrane protein. MAP1B is also in axolemma‐enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane‐associated following high‐salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle‐rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein.
Modulation of Acetylcholinesterase and Voltage‐Gated Na+ Channels in Choline Acetyltransferase‐ Transfected Neuroblastoma Clones
Authors: Jaco A. De , Ajmone‐Cat M. A. , Baldelli P. , Carbone E. , Augusti‐Tocco G. , Biagioni S.
Published: March 2003

Abstract: Neurotransmitters appear early in the developing embryo

and may play a role in the regulation of neuronal differentiation. To study

potential effects of acetylcholine production in neuronal differentiation, we

used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected

with cDNA for choline acetyltransferase. We tested whether the forced

acetylcholine production can modify the expression or the cellular

localization of different neuronal markers. We studied the activity,

localization, and secretion of acetylcholinesterase in view of its possible

role in the modulation of the morphogenetic action of acetylcholine and of its

proposed role of a regulator of neurite outgrowth. FB5 cells are characterized

by a high level of acetylcholinesterase, predominantly released into the

culture medium. Acetylcholinesterase secretion into the medium was lower in

choline acetyltransferase‐transfected clones than in nontransfected and

antisense‐transfected controls. Moreover, sequential extraction of

acetylcholinesterase revealed that detergent‐extracted, i.e.,

membrane‐associated, activity was higher in the transfected clones expressing

choline acetyltransferase activity than in both control groups. These

observations suggest that a shift occurs in the utilization of

acetylcholinesterase in choline acetyltransferase‐transfected clones from a

secretion pathway to a pathway leading to membrane localization. In addition,

the choline acetyltransferase‐positive clones showed higher densities of

voltage‐gated Na+ channels and enhanced high‐affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.