bluuebunny/crossref_metadata_2025_split huggingface dataset:
CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones
Authors: Holm Ninna R. , Christophersen Palle , Hounsgaard Jørn , Gammeltoft Steen , Olesen Søren‐Peter 
Published: March 2003

AbstractNeurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high‐threshold voltage‐activated Ca channels in cultured fetal mouse brain cortical neurones was investigated. Addition of CNTF into serum‐free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18 h. CNTF reduced the current of all pharmacological subtypes of Ca channels as shown by use of selective blockers of L, N, and P/Q type Ca channels (nifedipine, ω‐conotoxin MVIIA, ω‐agatoxin IVA). The Ca channel depression was mediated via the CNTF receptor, because enzymatic cleavage of the α‐subunit glycerophosphatidylinositol anchor of the receptor eliminated the response. The CNTF effect was not elicited through pertussis toxin‐sensitive G proteins. Other neurotrophic factors like neurotrophin‐3 and insulin‐like growth factor‐I had no effect on the Ca2+ currents. These results may have important implications for the possible functions of CNTF in the nervous system, such as altered synaptic activity, neuronal excitability and susceptibility to brain ischaemia.
In vivo monitoring of evoked noradrenaline release in the rat anteroventral thalamic nucleus by continuous amperometry
Authors: Dugast C. , Cespuglio R. , Suaud‐Chagny M. F. 
Published: March 2003

AbstractContinuous amperometry coupled with untreated carbon‐fibre electrodes was used in anaesthetized rats to measure the noradrenaline release evoked in the anteroventral thalamic nucleus by electrical stimulation of the dorsal noradrenergic bundle. As expected, the variations in the oxidation current detected in the anteroventral thalamic nucleus exhibited the characteristics of the in vivo noradrenaline release. They were closely correlated with stimulation and consistent with the anatomy of the noradrenergic system involved. They were abolished by the ejection of tetrodotoxin in the vicinity of the carbon‐fibre electrode, diminished by clonidine, an alpha‐2 agonist, and restored by yohimbine, an alpha‐2 antagonist. Furthermore, the time course of these variations was dramatically increased by desipramine, a specific noradrenaline reuptake blocker. In contrast, neither dopamine nor serotonin reuptake blockers, nor the monoamine oxidase inhibitor pargyline were able to alter them. The main advantage of the present approach is its excellent time resolution. We show here for the first time that after single pulse stimulation, noradrenaline is released and eliminated in 118 milliseconds, this time lapse corresponding to the maximal period beyond which subsequent noradrenaline releases could not add up. These observations are in good agreement with the physiological relationship previously observed between impulse flow and noradrenaline overflow.
Homologous regulation of the heptahelical D1A receptor responsiveness: specific cytoplasmic tail regions mediate dopamine‐induced phosphorylation, desensitization and endocytosis
Authors: Jackson Adele , Iwasiow Rafal M. , Chaar Ziad Y. , Nantel Marie‐France , Tiberi Mario 
Published: March 2003

AbstractIn the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)‐induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild‐type (WT) or truncation forms (Δ425, Δ379 and Δ351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA‐mediated phosphorylation‐dependent desensitization of D1A receptors. However, the longer truncation mutant Δ351 failed to undergo detectable DA‐induced phosphorylation while exhibiting DA‐induced desensitization features similar to the shorter truncation mutant Δ379. These data potentially suggest a novel role for a receptor phosphorylation‐independent process in the DA‐promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA‐mediated receptor endocytosis. Additionally, time‐course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation orkobs, a parameter linked to the receptor‐G protein coupling status. In agreement with the desensitization data, Δ425‐ and Δ379‐expressing cells exhibit an increase ofkobsin comparison with WT‐expressing cells. Nevertheless, Δ351‐expressing cells, which harbor similar desensitization features of Δ379‐expressing cells, display no change inkobswhen compared with WT‐expressing cells. Our results suggest that a defective DA‐induced endocytosis may hamper Δ351 resensitization and concomitant increase inkobs. Thus, our study showing that specific D1A receptor CT sequences regulate DA‐induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.
Low‐density lipoprotein receptor‐related protein levels and endocytic function are reduced by overexpression of the FE65 adaptor protein, FE65L1
Authors: Guénette Suzanne Y. , Chang Yang , Hyman Bradley T. , Tanzi Rudolph E. , Rebeck G. William 
Published: March 2003

AbstractThe FE65 adaptor protein family was identified in two‐hybrid screens as proteins that bind the cytoplasmic domain of the amyloid precursor protein (APP). Studies have shown that FE65 binding to APP modulates APP processing. Increased levels of α‐secretase derived secreted APP (APPsα) and β‐amyloid (Aβ) were recovered from conditioned media upon FE65L1 or FE65 overexpression. These effects were associated with an increase in the ratio of mature/immature APP and increased cell‐surface APP. FE65 has also been reported to bind low‐density lipoprotein receptor‐related protein (LRP). Here we show that FE65L1 overexpression results in decreased LRP steady state levels, LRPs, and LRP endocytic receptor function. These changes in LRP protein levels are not due to decreased transcription of LRP. Furthermore, pulse/chase experiments demonstrate that changes in LRP protein only occurred 12–18 h after translation. We conclude that the decreases in LRP levels likely reflect routing of LRP away from the cell surface into a degradative pathway. Previous studies suggested that LRP plays an important role for Aβ production of Kunitz protease inhibitor forms of APP in the endocytic pathway. These data show that FE65L1 can differentially affect the metabolic fate of APP and LRP. In addition, these data suggest that the LRP decrease observed in FE65L1 overexpressing cells may in part contribute to altered APP processing.
Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson's disease
Authors: Hirrlinger Johannes , Schulz Jörg B. , Dringen Ralf 
Published: March 2003

AbstractTo investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia‐rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 µm DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide‐dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mm DA established almost constant extracellular concentrations of H2O2 of 5 µm and 15 µm, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenalineandnoradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brain's GSH metabolism in Parkinson's disease.
Heterologous sensitization of adenylate cyclase is protein kinase A‐dependent in Cath.a differentiated (CAD)‐D2L cells
Authors: Johnston Christopher A. , Beazely Michael A. , Vancura Amanda F. , Wang James K. T. , Watts Val J. 
Published: March 2003

AbstractPersistent activation of Gαi/o‐coupled receptors results in a paradoxical enhancement of subsequent drug‐stimulated adenylate cyclase activity. The exact mechanism of this up‐regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP‐dependent protein kinase in D2L receptor–mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD‐D2L). Long‐term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two‐fold enhancement of forskolin‐stimulated cyclic AMP accumulation. Similarly, long‐term treatment with the PKA inhibitors, H89 or Rp‐8Br‐cAMP, also enhanced adenylate cyclase activity. In contrast, long‐term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin‐stimulated cyclic AMP accumulation and reduced both quinpirole‐ and H89‐induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT‐PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD‐D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD‐D2L cells.
Analysis of human neuropeptide FF gene expression
Authors: Nystedt Johanna M. , Brandt Annika M. , Mandelin Jami , Vilim Ferdinand S. , Ziff Edward B. , Panula Pertti 
Published: March 2003

AbstractAs an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7‐kb sequence from the promoter region. Primer extension and 5′‐rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U‐251 MG, SK‐N‐SH, SK‐N‐AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real‐time quantitative RT–PCR. The highest promoter activity was measured when a region from − 552 to − 830 bp of the 5′‐flanking region was fused to the constructs, and a potential silencer element waslocalized between nucleotides −220 and −551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between − 61 and − 214 bp of the 5′‐flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.
Functional characterization of alternatively spliced 5‐HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode, Ascaris suum
Authors: Huang Xinyan , Xiao Hong , Rex Elizabeth B. , Hobson Robert J. , Messer William S. , Komuniecki Patricia R. , Komuniecki Richard W. 
Published: March 2003

AbstractSerotonin (5‐HT) receptors play key regulatory roles in nematodes and alternatively spliced 5‐HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5‐HT2As1 and 5‐HT2As2 contain different C‐termini, and 5‐HT2As1Δ4 lacks 42 amino acids at the C‐terminus of the third intracellular loop. 5‐HT2As1 and 5‐HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5‐HT2As isoforms had higher affinity for 5‐HT than their closely related Caenorhabditis elegans homolog (5‐HT2Ce). This increased 5‐HT affinity was not related to the substitution in 5‐HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5‐HT receptors, since a 5‐HT2As1F120Y mutant actually exhibited increased 5‐HT affinity compared with that of 5‐HT2As1. As predicted, cells expressing either 5‐HT2As1 or 5‐HT2As2 exhibited a 5‐HT‐dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5‐HT2As1Δ4 displayed a 10‐fold higher affinity for 5‐HT and 5‐HT agonists than either 5‐HT2As1 or 5‐HT2As2, 5‐HT2As1Δ4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT–PCR, 5‐HT2As1 and 5‐HT2As2 were more highly expressed in pharynx and body wall muscle and 5‐HT2As1Δ4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5‐HT2 receptor isoforms from any system.
Regulation of tyrosine hydroxylase by stress‐activated protein kinases
Authors: Toska Karen , Kleppe Rune , Armstrong Christopher G. , Morrice Nick A. , Cohen Philip , Haavik Jan 
Published: March 2003

AbstractRecombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress‐activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in Vmax and a decrease in Km for 6‐(R)‐5,6,7,8‐tetrahydrobiopterin (BH4), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high‐affinity binding of 14‐3‐3 proteins, but only the interaction of 14‐3‐3 with Ser19 increased the hTH1 activity. The 14‐3‐3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress‐activated protein kinases in the regulation of catecholamine biosynthesis.
Different roles for Gi and Go proteins in modulation of adenylyl cyclase type‐2 activity
Authors: Näsman Johnny , Kukkonen Jyrki P. , Holmqvist Tomas , Åkerman Karl E. O. 
Published: March 2003

AbstractThe effect of Gi/o protein‐coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed α2A‐adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin‐sensitive manner. Activation of Gs proteins switched the response of the α2A‐adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gβγ scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (αo1 + β1γ2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (αi1 + β1γ2) resulted in a potentiation of both the TPA‐ and the Gs‐stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein‐linked signalling.
Functional expression of the norepinephrine transporter in cultured rat astrocytes
Authors: Inazu Masato , Takeda Hiroshi , Matsumiya Teruhiko 
Published: March 2003

AbstractWe assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time‐dependent manner, and this uptake involves temperature‐ and Na+‐sensitive mechanisms. The Na+‐dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 ± 55.4 nm and the Vmax was 1.41 ± 0.03 pmol/mg protein/min. Ouabain, a Na+‐K+ ATPase inhibitor, significantly inhibited Na+‐dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+‐dependent [3H]NE uptake. On the other hand, GBR‐12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT‐PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti‐NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re‐uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.
Mouse brain gene expression changes after acute and chronic amphetamine
Authors: Sokolov Boris P. , Polesskaya Oxana O. , Uhl George R. 
Published: March 2003

AbstractGene expression changes are candidate mechanisms to contribute to long‐term consequences of psychostimulant use. We use microarrays to examine the expression of 6340 genes in brains of mice killed 5 or 20 h following 14 day, twice‐daily treatments with saline (SS), saline followed by a single 7.5 mg/kg amphetamine dose (SA), or repeated 7.5 mg/kg amphetamine doses (AA) that produce sensitization but no clear‐cut neuronal toxicities. Arrays display robust hybridization for about 3600 transcripts. One hundred and seventeen of these expressed transcripts are candidate positives for drug‐related changes, displaying > 1.8‐fold differences from SS control values in whole brains of either SA or AA mice. Five transcripts reveal altered expression in both AA and SA mice. SA mostly enhances expression while AA treatments largely reduce expression. Fourteen SA and four AA changes in whole brain mRNA were replicated by > 1.8‐fold changes in independent microarray assessments of either cerebral cortical or brainstem mRNAs, with more changes identified in frontal than in entorhinal/parietal cortical samples. About one‐quarter of these changes persist in initial studies of mice killed 20 h after the last amphetamine injection. Each of these genes, including transcription factor, cellular regulatory, structural and other gene family members, are candidates to contribute to brain adaptations to psychostimulants.
The ‘glial’ glutamate transporter, EAAT2 (Glt‐1) accounts for high affinity glutamate uptake into adult rodent nerve endings
Authors: Suchak Sachin K. , Baloyianni Nicoletta V. , Perkinton Michael S. , Williams Robert J. , Meldrum Brian S. , Rattray Marcus 
Published: March 2003

AbstractThe excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse‐transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt‐1) in both synaptosomes and GPVs. Uptake of [3H]D‐aspartate or [3H]L‐glutamate into these preparations revealed sodium‐dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine‐o‐sulfate, l‐trans‐2,4‐pyrrolidine dicarboxylate (PDC) (+/–)‐threo‐3‐methylglutamate and (2S,4R )‐4‐methylglutamate. The IC50 values found for these compounds suggested functional expression of the ‘glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 µm dihydrokainate, failed to unmask any functional non‐EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.
Mechanisms by which 2′,3′‐dideoxyinosine (ddI) crosses the guinea‐pig CNS barriers; relevance to HIV therapy
Authors: Gibbs J. E. , Jayabalan P. , Thomas S. A. 
Published: March 2003

AbstractThe influence of transport mechanisms at the blood–brain barrier (BBB) and blood–CSF barrier (choroid plexus) on the CNS distribution of anti‐human immunodeficiency virus (HIV) drugs was examined using guinea‐pig brain perfusion and incubated choroid plexus models. 2′,3′‐dideoxyinosine (ddI) passage across the BBB was demonstrated to be via non‐saturable (Kd = 0.22 ± 0.3 µL/min/g) and saturable (Km = 20.1 ± 15.0 μm, Vmax = 6.5 ± 2.1 pmol/min/g) processes. Cross competition studies implicated an equilibrative nucleoside transporter in this influx. The brain distribution of ddI was unchanged in the presence of additional nucleoside reverse transcriptase inhibitors (NRTIs). ddI transport from blood into choroid plexus was demonstrated to involve an organic anion transporting polypeptide 2‐like transporter. The NRTIs, abacavir, 3′‐azido 3′‐deoxythymidine and (–)‐β‐l‐2′,3′‐dideoxy‐3′‐thiacytidine, competed with ddI for transporter binding sites at the choroid plexus, altering the tissue concentration of ddI. This has clinical implications as the choroid plexus is a site of HIV replication, and suboptimal CNS concentrations of anti‐HIV drugs could result in neurological complications. Furthermore, this may promote the selection of drug resistant variants of HIV within the CNS, which could re‐infect the periphery and lead to HIV therapy failure. This study indicates that understanding drug interactions at the transporter level could prove valuable when selecting drug combinations to treat HIV within the CNS.