Study of a Swiss dopa-responsive dystonia family with a deletion in GCH1: redefining DYT14 as DYT5.

OBJECTIVE: To report the study of a multigenerational Swiss family with dopa-responsive dystonia (DRD). METHODS: Clinical investigation was made of available family members, including historical and chart reviews. Subject examinations were video recorded. Genetic analysis included a genome-wide linkage study with microsatellite markers (STR), GTP cyclohydrolase I (GCH1) gene sequencing, and dosage analysis. RESULTS: We evaluated 32 individuals, of whom 6 were clinically diagnosed with DRD, with childhood-onset progressive foot dystonia, later generalizing, followed by parkinsonism in the two older patients. The response to levodopa was very good. Two additional patients had late onset dopa-responsive parkinsonism. Three other subjects had DRD symptoms on historical grounds. We found suggestive linkage to the previously reported DYT14 locus, which excluded GCH1. However, further study with more stringent criteria for disease status attribution showed linkage to a larger region, which included GCH1. No mutation was found in GCH1 by gene sequencing but dosage methods identified a novel heterozygous deletion of exons 3 to 6 of GCH1. The mutation was found in seven subjects. One of the patients with dystonia represented a phenocopy. CONCLUSIONS: This study rules out the previously reported DYT14 locus as a cause of disease, as a novel multiexonic deletion was identified in GCH1. This work highlights the necessity of an accurate clinical diagnosis in linkage studies as well as the need for appropriate allele frequencies, penetrance, and phenocopy estimates. Comprehensive sequencing and dosage analysis of known genes is recommended prior to genome-wide linkage analysis.

Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN-BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter.

GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine-containing neuroblastoma cell line SK-N-BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br-cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br-cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5'-flanking sequence. Cis-acting elements required for maximal basal and cAMP-dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK-N-BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1-like site and the other a combined CRE-Sp1-CCAAT-box element. EMSA and supershift assays demonstrated that the combined CRE-Sp1-CCAAT-box element recruits ATF-2 and NF-Y but not Sp1-4 or Egr-1-3. NF-Y binding was confirmed using pure recombinant human NF-Y protein. Transcription of the human GTPCH gene in human SK-N-BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF-Y and ATF-2.

Effects of NADH on dopamine release in rat striatum.

Nicotinamide adenine dinucleotide (NADH) may be utilized for the synthesis and regeneration of tetrahydrobiopterin (BH(4)), which in turn is an essential cofactor for tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine (DA). NADH has been reported to relieve some of the symptoms of Parkinson's disease, presumably by altering dopaminergic function. The present study examines the efficacy of NADH in influencing DA activity in the rat striatum. In striatal slices, NADH (350 microM) significantly increased basal DA and DOPAC efflux and caused a 2-fold increase in the DA overflow evoked by high KCl (25 mM). Tissue levels of BH(4), basal BH(4) efflux, and KCl-evoked BH(4) overflow were unaffected by NADH, as was [(3)H]DA uptake into striatal synaptosomes. In contrast to the effects of NADH on DA function in vitro, no effects were observed when NADH was administered systemically. NADH (10 or 100 mg/kg, s.c.) did not influence the tissue content of DA, 5-HT, or their metabolites in the midbrain or striatum, nor did it alter DA extracellular concentrations. These results indicate that NADH can increase DA release from striatal slices, although we are as yet unable to detect this effect in vivo.

Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat GTP cyclohydrolase I gene.

5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5'-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transfection assays of luciferase reporter constructs defined the core promoter as a highly conserved 142-base pair GC-rich sequence upstream from the cap site. DNase I footprint analysis of this region revealed (5' --> 3') a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box. Transcription from the core promoter in PC12 but not C6 or Rat2 cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that both the CRE and CCAAT-box independently contribute to basal and cAMP-dependent activity. The combined CRE and CCAAT-box cassette was also found to enhance basal transcription and confer cAMP sensitivity on a heterologous minimal promoter. The addition of the Sp1/GC box sequence to this minimal promoter construct inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer-binding protein beta, while both PC12 and C6 cell nuclear extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y. Overexpression of ATF-4 in PC12 cells was found to transactivate the GTPCH promoter response to cAMP. These studies suggest that the elements required for cell type-specific cAMP-dependent enhancement of gene transcription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein beta, and nuclear factor Y.

Prenatal ethanol reduces the activity of adult midbrain dopamine neurons.

BACKGROUND: Prenatal ethanol exposure has been demonstrated to reduce dopamine (DA) neurotransmission in the forebrain area, which could be contributed by altered electrical activity in midbrain DA neurons. This hypothesis was tested in the present study. METHODS: The effects of prenatal ethanol exposure on the spontaneous activity of DA neurons in the substantia nigra and ventral tegmental area were investigated with extracellular single-unit recording techniques in adult male rats. Pregnant rats were administered single daily doses of 0, 3, or 5 g/kg ethanol via intragastric intubation from gestation day 8 through 20. An additional control group did not receive the intubation procedure. RESULTS: Prenatal ethanol treatment significantly reduced the number of spontaneously active DA neurons in the substantia nigra and ventral tegmental area in 3- to 5-month-old male offspring. The firing rate and firing pattern of the remaining spontaneously active DA neurons were not altered. There were no differences in the spontaneous activity of DA neurons between the nonintubated and 0 g/kg control groups, indicating possible intubation-induced stress did not influence the activity of DA neurons in adult offspring. Similar prenatal ethanol effects were also determined from older animals (14-16 months old), suggesting that the reduction in the spontaneous activity of DA neurons is a persistent phenomenon in adulthood after prenatal ethanol exposure. Furthermore, the reduction in the number of spontaneously active DA neurons was not the result of a loss in DA neurons per se, as revealed by the results of tyrosine hydroxylase immunohistochemistry. The prenatal ethanol exposure-induced reduction in DA neuronal activity may result from depolarization inactivation, because systemically administered apomorphine (20 microg) increased the spontaneous activity of DA neurons. CONCLUSIONS: Prenatal ethanol exposure induced a long-lasting reduction in the activity of midbrain DA neurons in adult animals. The effect was not the result of cell loss but possible changes in the electrical properties of DA neurons. The decreased electrical activity in midbrain DA neurons could contribute to the prenatal ethanol exposure-induced reduction in DA content and metabolites observed in previous studies and the attention/hyperactivity problems reported in children with fetal alcohol effects/fetal alcohol syndrome.

GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L-phenylalanine-induced increase in tetrahydrobiopterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by L-phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity.

GTP cyclohydrolase I gene expression in the brains of male and female hph-1 mice.

The hph-1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double-label in situ hybridization technique was used to examine CNS GTPCH mRNA expression within serotonin, dopamine, and norepinephrine neurons of male and female wild-type and hph-1 mice. In wild-type male and female animals the highest levels of GTPCH mRNA expression were observed within serotonin neurons, followed by norepinephrine and then dopamine neurons. Wild-type female animals were found to express lower levels of GTPCH mRNA in each cell type when compared with levels seen in wild-type males. GTPCH mRNA abundance in all three cell types was lower in hph-1 male than in wild-type male mice, with the greatest reduction in serotonin neurons. GTPCH mRNA levels were also lower in hph-1 female than in wild-type female mice, again with the greatest reduction occurring in serotonin neurons. Comparison of hph-1 male and hph-1 female mice revealed that the sex-linked difference in GTPCH mRNA expression observed in wild-type neurons was only present within female dopamine neurons. Overall, these results indicate that not only are basal levels of GTPCH mRNA expression heterogeneous across wild-type murine monoamine cell types but that gene expression is also modified in a sex-linked and cell-specific fashion by the hph-1 gene locus. The hph-1 mutation does not lie within the GTPCH mRNA coding region. The 5' flanking region of the GTPCH gene was cloned and sequenced and shown to be identical for both wild-type and hph-1 genomic DNA. Transient transfection assays performed in PC12 cells demonstrated that this 5' flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph-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.

Nigrostriatal dopamine neurons express low levels of GTP cyclohydrolase I protein.

A previous study using the in situ hybridization technique showed that serotonin neurons contain substantially more GTP cyclohydrolase I mRNA than do either dopamine or norepinephrine neurons. The objective of the current study was to determine whether these differences in mRNA abundance are predictive of the amount of GTP cyclohydrolase I protein available for tetrahydrobiopterin biosynthesis. The double-label immunofluorescence technique was used to localize GTP cyclohydrolase I protein to the tyrosine hydroxylase-positive A9 dopamine neurons of the substantia nigra and the A6 norepinephrine neurons of the locus ceruleus or the tryptophan hydroxylase-positive B6/B7 serotonin neurons of the dorsal raphe nucleus. Although GTP cyclohydrolase I immunofluorescence within serotonin and norepinephrine neurons was relatively intense, the fluorescence signal within dopamine neurons was faint to nondetectable. An immunoautoradiographic technique was developed to quantify these apparent differences in GTP cyclohydrolase I protein expression at the cellular level. Significant differences between all three neurochemical subdivisions were found and comparisons showed that on average serotonin neurons contain between 2.3- and 7.3-fold more GTP cyclohydrolase I protein than do either norepinephrine or dopamine neurons, respectively. Nigrostriatal dopamine neurons thus appear to synthesize and maintain tetrahydrobiopterin at low levels. Because dopamine and norepinephrine neurons express essentially equal amounts of GTP cyclohydrolase I mRNA, posttranscriptional events may serve to maintain low levels of GTP cyclohydrolase I protein within dopamine neurons. Phenotypic differences in GTP cyclohydrolase I protein expression across populations of monoamine neurons may be an important control point in neurotransmitter biosynthesis.

Tetrahydrobiopterin biosynthesis in C6 glioma cells: induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment.

The possibility that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 micrograms/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content in cultured sympathetic neurons by leukemia inhibitory factor and ciliary neurotrophic factor.

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 beta-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.

Tetrahydrobiopterin biosynthesis in the rat brain: heterogeneity of GTP cyclohydrolase I mRNA expression in monoamine-containing neurons.

GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. A quantitative in situ hybridization technique was used to study the expression of GTP cyclohydrolase I mRNA in the rat brain at the cellular level. Coronal sections between the diencephalon and myelencephalon were exposed to a 35S-labelled antisense GTP cyclohydrolase I cRNA probe. Sections serial to these were hybridized with a 35S-labelled antisense cRNA probe complementary to tyrosine hydroxylase mRNA. Tyrosine hydroxylase and GTP cyclohydrolase I mRNAs were found to colocalize within catecholamine neurons located throughout the brain. The overall distribution of neurons expressing GTP cyclohydrolase I mRNA was observed to correspond exactly to the known distribution of the dopamine, norepinephrine/epinephrine and serotonin-containing cell groups. Overall, a 30-fold range of GTP cyclohydrolase I mRNA expression was observed, with the transcript being significantly more abundant in serotonin than in dopamine or norepinephrine/epinephrine neurons. Comparisons across serotonin cell groups indicated that neurons of the median raphe nucleus, caudal linear nucleus raphe (B8) and the dorsal raphe (B6/B7) expressed the highest levels of GTP cyclohydrolase I mRNA. Comparisons across dopamine cell groups indicated that the transcript was more abundant in neurons of the ventral tegmental area (A10) than in neurons of the substantia nigra pars compacta (A9) and that both A9 and A10 dopamine neurons exhibited higher levels of expression than the DA neurons of the hypothalamus (A11-A14). Norepinephrine neurons of the locus coeruleus (A6) and subcoeruleus (A6v) exhibited significantly higher levels of GTP cyclohydrolase I mRNA than did neurons in other norepinephrine (A1 and A2) or epinephrine (C1 and C2) cell groups. GTP cyclohydrolase I mRNA could not be detected unequivocally in neurons known to contain nitric oxide synthase. Heterogeneity in the level of expression of GTP cyclohydrolase I mRNA by monoamine-containing neurons may play an important role in determining steady state levels of tetrahydrobiopterin and, ultimately, the regulation of monoamine biosynthesis.

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content by nerve growth factor in cultures of superior cervical ganglia.

Monolayer cultures of superior cervical ganglia free of support cells were maintained in the presence of 100 ng/ml 7S-NGF for 4 days. The concentration of NGF was then changed to between 50 and 400 ng/ml and cultures continued for an additional 7 days. Tetrahydrobiopterin (BH4) content, GTP cyclohydrolase (GTPCH) enzyme activity and mRNA levels were then determined. All three of these measures were found to be elevated between 2- to 4-fold by treatment with increasing concentrations of NGF. Tyrosine hydroxylase (TH) enzyme activity and mRNA levels were increased from 8 to 13-fold by these same treatments. These results indicate that the content of BH4 within sympathetic neurons can be regulated by NGF receptor-mediated changes in GTPCH gene expression. Moreover, concomitant increases in TH enzyme activity and BH4 content demonstrate a coordinated regulation by NGF of this enzyme and its essential cofactor.

Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA.

6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and nuclease protection assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by nuclease protection assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor.

A comparison of the developing dopamine neuron phenotype in cultures of embryonic rat mesencephalon and hypothalamus.

Development of the dopamine (DA) neuron phenotype was monitored in cultures of embryonic rat mesencephalon (MES) and hypothalamus (HYP) maintained for 1 to 21 days in vitro (DIV) in the absence of glial support cells. Cell counts following immunohistochemistry for tyrosine hydroxylase (TH) demonstrated that the number of DA neurons declined by 85% in MES cultures yet increased 5-fold in cultures of HYP, so that by 21 DIV equal numbers of DA neurons were present in these culture systems. After 21 DIV MES DA neurons exhibited a multipolar morphology, with numerous branching processes. HYP DA neurons were primarily fusiform in shape with fewer processes and process branch points. Double-label immunohistochemistry for TH and microtubule-associated protein 2 identified the majority of TH-positive processes in either culture system as dendrites. Individual MES but not HYP DA neurons were also found to generate axons. Western analysis showed that between 1 and 21 DIV the concentration of TH protein increased 2-fold in MES and 4-fold in HYP cultures. After 21 DIV the concentration of TH protein in MES cultures was twice that found in cultures of HYP. In the period between 1 and 21 DIV levels of tetrahydrobiopterin (BH4) increased by 6-fold in MES and 20-fold in HYP cultures. After 21 DIV BH4 content was 3-fold higher in HYP than in MES cultures. The abundance of the mRNA encoding for GTP cyclohydrolase I, the rate-limiting enzyme in BH4 biosynthesis, was similar in MES and HYP cultures despite this difference in BH4 levels. In contrast, TH mRNA was 4-fold more abundant in MES than in HYP cultures. Treatment of MES cultures with the DA neuron toxin 1-methyl-4-phenylpyridinium decreased DA cell numbers, TH protein content and BH4 levels, demonstrating that BH4 is localized primarily to DA neurons. Similar treatment of HYP cultures did not effect any of these parameters. Steady-state levels of DA and the rate of DA synthesis were both 3-fold higher in MES than in HYP cultures. A 95% decline in BH4 content produced by inhibiting BH4 biosynthesis resulted in 64% and 84% declines in the rate of MES and HYP DA synthesis, respectively. Overall, these observations indicate that, with the exception of the capacity to synthesize DA, DA neurons in MES and HYP cultures share few common properties.

Dopamine neuron membrane physiology: characterization of the transient outward current (IA) and demonstration of a common signal transduction pathway for IA and IK.

Dopamine neurons derived from the mesencephalon of embryonic rats were maintained in primary culture, identified and studied with whole-cell patch recording techniques. These neurons demonstrated a rapidly activating and inactivating voltage-dependent outward current which required the presence of K+ ions. This current was termed IA because of its transient nature. It was elicited by step depolarizations from holding potentials more negative than -50 mV and exhibited steady-state inactivation at a membrane potential more positive than -40 mV and half-maximal inactivation observed at -65 mV. This current rapidly achieved peak activation in less than 8 msec and decayed with a time constant (tau) of 58 +/- 5 msec. This current was observed in the presence of tetraethylammonium but was readily blocked by 4-aminopyridine (2-4 mM). This current was also observed to be modulated by stimulation of D2 dopamine receptors (DA autoreceptors) located on the dopamine neurons. Thus, both DA and the D2 receptor agonist quinpirole enhanced the peak IA observed, while the partial D1 receptor agonist SKF 38393 was without effect. The enhancement of IA was confirmed to be due to the activation of D2 receptors as the effects of either DA or quinpirole were blocked by the D2 receptor antagonists eticlopride and sulpiride, but not by the D1 receptor antagonist SCH 23390. Since we have previously demonstrated that the IK present in these cells is also enhanced by D2 receptor stimulation, we investigated the signal transduction pathways involved in coupling DA autoreceptors to both IA and IK. The response of both these potassium currents to DA autoreceptor stimulation was completely abolished by the preincubation of cultures with pertussis toxin, indicating the possible involvement of the G proteins Gi and G(o). In an attempt to further characterize which G protein may be involved, additional experiments were performed. The ability of DA autoreceptor stimulation to augment both currents was also blocked completely when G protein activation was prevented by the intracellular application of GDP beta S (100 microM). In contrast, irreversible activation of G proteins by intracellular application of the nonhydrolyzable GTP analog GTP gamma S (100 microM) mimicked the effects of DA autoreceptor stimulation on both IA and IK. In addition, the intracellular application of a polyclonal antibody that was selective for the alpha-subunit of G(o) completely abolished the DA autoreceptor modulation of both currents while preimmune serum was without effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Dopamine and gamma-aminobutyric acid transporters: differential regulation by agents that promote phosphorylation.

Treatment of striatal synaptosomes with the protein phosphatase inhibitor okadaic acid significantly decreased gamma-aminobutyric acid (GABA) uptake, indicating that the GABA transporter may be regulated by phosphorylation. Forskolin and 8-bromoadenosine-3,5-cyclic monophosphate (8-br-cAMP) inhibited GABA uptake to the same extent as okadaic acid, suggesting the involvement of protein kinase A in GABA transporter regulation. In contrast, the same treatments did not alter dopamine (DA) uptake into striatal synaptosomal preparations. The results suggest that the structurally related GABA and DA transporters may be subject to different post-translational regulation.

Regulation of tetrahydrobiopterin biosynthesis in cultured dopamine neurons by depolarization and cAMP.

Primary cultures containing embryonic rat brain mesencephalic or hypothalamic dopamine neurons were used to examine the effects of membrane depolarization and elevations of cAMP levels on tetrahydrobiopterin cofactor content. Initial studies showed that 24-h incubations with 8-bromo-cAMP or isobutyl methylxanthine increased cofactor levels in either culture system, whereas the stimulatory effects of forskolin or depolarization of membrane potential were only observed in cultures of hypothalamus. 8-Bromo-cAMP was found to increase cofactor content in a concentration-dependent manner, with increases observed up to 5 mM. The time course of the effect of 8-bromo-cAMP was biphasic. Over the short term, an increase of 50% in cofactor content at 2 and 5 h was detected. Over the long term, by 24-48 h, cofactor levels increased by between 100% and 300%. Studies of cofactor turnover indicated that the long-term increase was due to stimulation of tetrahydrobiopterin biosynthesis with no alteration in degradation rate. Inhibitors of gene transcription and translation prevented the long- but not short-term increase in cofactor content. Levels of GTP cyclohydrolase I mRNA were increased 7-10-fold following 5 h of incubation with 8-bromo-cAMP. Tetrahydrobiopterin biosynthesis within cultured dopamine neurons of the hypothalamus and mesencephalon thus appears to be regulated by a cAMP-dependent mechanism involving enhanced gene expression of enzyme(s) involved in cofactor biosynthesis.

Neurotrophic effects of substance P on hippocampal neurons in vitro.

The potential neurotrophic effect of substance P-like immunoreactivity present in culture media was assessed in rat embryonic day 18 hippocampal cultures. The neurokinin-1 (substance P) receptor antagonist CP-96345 induced neurotoxicity that was dose dependent and attenuated by addition of substance P or the neurokinin-1 agonist [Sar9,Met(O2)11]-SP. These studies suggest that under some conditions neurokinin-1 receptor stimulation promotes neuronal survival.

Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.

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 hybridization. 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.

Transfected D2 short dopamine receptors inhibit voltage-dependent potassium current in neuroblastoma x glioma hybrid (NG108-15) cells.

Two isoforms of the D2 dopamine receptor exist, termed D2 short (D2s) and D2 long, which differ by the presence or absence of 29 amino acids. To examine the possible coupling of the D2s isoform to voltage-dependent K+ current, NG108-15 cells that were transfected with and stably express this isoform were studied using whole-cell patch-clamp techniques. In transfected, but not untransfected, cells dopamine and quinpirole (QUIN) reduced the normally observed peak outward K+ current, and this effect was abolished by the D2 antagonist sulpiride but not by the alpha 2-adrenergic receptor antagonist idazoxan or the D1 antagonist (R)-(+)-SCH-23380. The D1 receptor agonist SKF 38393 had no effect. QUIN-induced inhibition of K+ current was prevented by loading the cells with the Ca(2+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting a critical role for intracellular Ca2+ mobilization. In contrast, reduction of the concentration of extracellular Ca2+ and inclusion of the Ca2+ channel blocker cobalt did not modify the reduction of K+ current produced by stimulation of D2s receptors. A critical role for intracellular calcium mobilization in the observed effects was further supported by the observation that increases in cytosolic Ca2+ produced by thapsigargin mimicked the effect of QUIN, whereas intracellular ryanodine, which blocks Ca2+ mobilization, abolished the QUIN responsiveness. Finally, the effect of D2S activation on K+ current was not modified by pretreatment of the cells with pertussis toxin. These results suggest that the D2s dopamine receptor expressed in NG108-15 cells inhibits the activity of native K+ current via a mechanism that is dependent upon the mobilization of intracellular Ca2+ and does not involve a pertussis toxin-sensitive G protein.