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  • 1
    Electronic Resource
    Electronic Resource
    Developmental Expression of Go in Neuronal Cultures from Rat Mesencephalon and Hypothalamus (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 54 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The developmental expression of the α-subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb04903.x
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  • 2
    Electronic Resource
    Electronic Resource
    Tyrosine Hydroxylase Content of Residual Striatal Dopamine Nerve Terminals Following 6-Hydroxydopamine Administration: A Flow Cytometric Study (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 53 (1989), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Fluorescence-activated cell sorting based on im-munolabeling with a monoclonal antibody to tyrosine hydroxylase and a fluorescein-conjugated secondary antibody was used to identify striatal synaptosomes derived from ni-grostriatal dopamine nerve terminals. The amount of tyrosine hydroxylase immunoreactivity in dopaminergic striatal synaptosomes prepared from control rats was compared to the amount in dopaminergic synaptosomes prepared from rats that had received intraventricular injections of 6-hydroxy-dopamine. Although the absolute number of dopaminergic synaptosomes was decreased in lesioned animals, those residual dopamine terminals present contained more tyrosine hydroxylase than did dopamine terminals from control rats. Both the decrease in the absolute number of dopamine terminals and the increase in tyrosine hydroxylase immunoreactivity in residual terminals were proportional to the extent of the lesion, as determined by measurement of striatal dopamine levels. These results suggest that an increase in the amount of tyrosine hydroxylase protein in residual terminals may represent one compensatory mechanism by which residual dopamine neurons maintain normal striatal function after partial destruction of the nigrostriatal dopamine projection.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1989.tb11786.x
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  • 3
    Electronic Resource
    Electronic Resource
    Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus laevis Oocytes (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 54 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 μM) or with the dopamine uptake inhibitors GBR 12909 (1 μM) or cocaine (3 μM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb01929.x
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  • 4
    Electronic Resource
    Electronic Resource
    Regulation of GTP Cyclohydrolase I Gene Expression and Tetrahydrobiopterin Content in Cultured Sympathetic Neurons by Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor (1996)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 66 (1996), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1996.66062541.x
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  • 5
    Electronic Resource
    Electronic Resource
    Tetrahydrobiopterin Cofactor Biosynthesis: GTP Cyclohydrolase I mRNA Expression in Rat Brain and Superior Cervical Ganglia (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridisation. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1,2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2-and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb03614.x
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  • 6
    Electronic Resource
    Electronic Resource
    Tetrahydrobiopterin Turnover in Cultured Rat Sympathetic Neurons: Developmental Profile, Pharmacologic Sensitivity, and Relationship to Norepinephrine Synthesis (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 59 (1992), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We have examined the turnover of 5,6,7,8-tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE-containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half-life of ∼2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4-diamino-6-hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition by N-acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90–95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24-h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels. In contrast, treatment of cultures for 24 h with the direct-acting inhibitor of TH, α-methyl-p-tyrosine, produced an 84% decline in NE content that was maintained over the 4-day treatment period, indicating that the slow decline in NE content following inhibition of BH4 synthesis was not the result of the slow turnover rate of NE. These results demonstrate that despite an almost complete loss of BH4, sympathetic neurons were able to maintain neurotransmitter content, albeit at reduced levels, by retaining a level of TH activity above the value that might have been predicted based on the reduced level of BH4.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1992.tb10093.x
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  • 7
    Electronic Resource
    Electronic Resource
    Quantitation of Rat Dopamine Transporter mRNA: Effects of Cocaine Treatment and Withdrawal (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 59 (1992), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a 〉40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1992.tb08365.x
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  • 8
    Electronic Resource
    Electronic Resource
    Tetrahydrobiopterin Synthesis Rate and Turnover Time in Neuronal Cultures from Embryonic Rat Mesencephalon and Hypothalamus (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: 6-(R)-(L-erythro-1′,2′-Dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N-acetylserotonin (NAS) or 2,4-diamino-6-hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8-h incubation with maximally effective concentrations of NAS (200 μM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady-state levels of BH4 and first-order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first-order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady-state levels of BH4 in HYP cultures (70.3 ± 9.4 pg/culture) were significantly greater than that for MES (46.5 ± 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 ± 0.015/h) and HYP (0.159 ± 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 ± 2.13 pg/culture/h) than for MES (7.11 ± 0.85 pg/culture/h), owing to the greater steady-state concentration of BH4. BH4 turnover time for MES (6.68 ± 0.67 h) and HYP (6.40 ± 0.62 h) and half-life for MES (4.63 ± 0.46 h) and HYP (4.44 ± 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis. These data also indicate that BH4 metabolism may be different between populations of dopamine-containing neurons.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb08830.x
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  • 9
    Electronic Resource
    Electronic Resource
    Immunological Evidence for the Requirement of Sepiapterin Reductase for Tetrahydrobiopterin Biosynthesis in Brain (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 54 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Specific antibodies to sepiapterin reductase were used to investigate its involvement in de novo (6R)-5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis in rat brain. Antisepiapterin reductase (anti-SR) serum totally inhibited NADPH-dependent sepiapterin reductase activity in supernatants from discrete rat brain areas and liver. The anti-SR serum also inhibited the conversion of 7,8-dihydroneopterin triphosphate to BH4 in rat brain extracts. The inhibition was accompanied by a concentration-dependent increase in the formation of 6-lactoyltetrahydropterin (6LPH4), a proposed intermediate in BH4 biosynthesis. In addition, anti-SR serum was used to characterize the distribution and molecular properties of sepiapterin reductase in rat tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting indicated that there was a single polypeptide with the same molecular weight (28,000) as that of the subunit of pure sepiapterin reductase present in all tissues examined except for liver, where an immunoreactive protein of higher molecular weight (30,500) also was detected. Two-dimensional gel electrophoresis of rat striatum and liver demonstrated that the isoelectric point of sepiapterin reductase from both tissues was 6.16 and that the higher molecular weight immunoreactive material in liver had an isoelectric point of 7.06. Our studies with specific anti-SR serum confirmed the results of previous studies using chemical inhibitors of sepiapterin reductase, which suggested that sepiapterin reductase activity was essential for BH4 biosynthesis in the CNS and that 6LPH4 could be a precursor of BH4.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb01951.x
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  • 10
    Electronic Resource
    Electronic Resource
    GTP Cyclohydrolase I Gene Expression in the Brains of Male and Female hph-1 Mice (1999)
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 72 (1999), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract : The hph-1 mouse is characterized by low levelsof GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitativedouble-lable in situ hybridization technique was used to examine CNS GTPCHmRNA expression within serotonin, dopamine, and norepinephrine neurons of maleand female wild-type and hph-1 mice. In wild-type male and femaleanimals the highest levels of GTPCH mRNA expression were observed withinserotonin neurons, followed by norepinephrine and then dopamine neurons.Wild-type female animals were found to express lower levels of GTPCH mRNA ineach cell type when compared with levels seen in wild-type males. GTPCH mRNAabundance in all three cell types was lower in hph-1 male than inwild-type male mice, with the greatest reduction in serotonin neurons. GTPCHmRNA levels were also lower in hph-1 female than in wild-type femalemice, again with the greatest reduction occurring in serotonin neurons.Comparison of hph-1 male and hph-1 female mice revealed thatthe sex-linked difference in GTPCH mRNA expression observed in wild-typeneurons was only present within female dopamine neurons. Overall, theseresults indicate that not only are basal levels of GTPCH mRNA expressionheterogeneous across wild-type murine monoamine cell types but that geneexpression is also modified in a sex-linked and cell-specific fashion by thehph-1 gene locus. The hph-1 mutation does not lie within theGT-PCH mRNA coding region. The 5′ flanking region of the GTPCH gene wascloned and sequenced and shown to be identical for both wild-type andhph-1 genomic DNA. Transient transfection assays performed in PC12cells demonstrated that this 5′ flanking region was sufficient toinitiate transcription of a luciferase reporter gene. Although thehph-1 mutation does not lie within the 5′ flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1999.0720757.x
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