Rapid substrate-induced down-regulation in function and surface localization of dopamine transporters: rat dorsal striatum versus nucleus accumbens

The dopamine transporter (DAT) substrates dopamine, d-amphetamine (AMPH), and methamphetamine are known to rapidly and transiently reduce DAT activity and/or surface expression in dorsal striatum and heterologous expression systems. We sought to determine if similar substrate-induced regulation of DATs occurs in rat nucleus accumbens. In dorsal striatum synaptosomes, brief (15-min) in vitro substrate pre-exposure markedly decreased maximal [³H]dopamine uptake velocity whereas identical substrate pre-exposure in nucleus accumbens synaptosomes produced a smaller, non-significant reduction. However, 45 min after systemic AMPH administration, maximal ex vivo [³H]dopamine uptake velocity was significantly reduced in both brain regions. Protein kinase C inhibition blocked AMPH's down-regulation of DAT activity. DAT synaptosomal surface expression was not modified following either the brief in vitro or in vivo AMPH pre-exposure but was reduced after a longer (1-h) in vitro pre-exposure in both brain regions. Together, our findings suggest that relatively brief substrate exposure results in greater down-regulation of DAT activity in dorsal striatum than in nucleus accumbens. Moreover, exposure to AMPH appears to regulate striatal DATs in a biphasic manner, with an initial protein kinase C-dependent decrease in DAT-mediated uptake velocity and then, with longer exposure, a reduction in DAT surface expression.     

Up-regulation of the neuronal serotoninergic phenotype in vitro: BDNF and cAMP share Trk B-dependent mechanisms

The effects of brain-derived neurotrophic factor (BDNF) and cAMP on the neuronal serotoninergic phenotype were studied in primary cultures of E14 rat embryonic rostral raphe. Short treatments (for 18 h) with BDNF or dibutyryl-cAMP induced an almost two-fold increase in the number of serotoninergic neurones and a dramatic extension and ramification of their neurites. These changes were associated with marked increases in the levels of mRNAs encoding the serotonin transporter, the 5-HT1A and 5-HT1B receptors and the BDNF receptor tyrosine kinase B (TrkB). Concomitant blockade of tyrosine kinases by genistein suppressed all the up-regulating effects of BDNF and cAMP on 5-hydroxytryptamine (5-HT) neurones. These findings suggest that an auto-amplifying mechanism underlies the promoting effect of BDNF on the differentiation of serotoninergic neurones through TrkB activation, which is also triggered by cAMP.     

Neurotrophic and neurotoxic effects of nitric oxide on fetal midbrain cultures

There is evidence suggesting that nitric oxide (NO) may play an important role in dopamine (DA) cell death. Thus, the aim of this study was to investigate the effects of NO on apoptosis and functionality of DA neurones and glial cells. The experiments were carried out in neuronal-enriched midbrain cultures treated with the NO donor diethylamine-nitric oxide complexed sodium (DEA-NO). DEA-NO, at doses of 25 and 50 microM, exerted neurotrophic effects on dopamine cells, increasing the number of tyrosine hydroxylase positive (TH(+)) cells, TH(+) neurite processes, DA levels and [(3)H]DA uptake. A dose of 25 microM DEA-NO protected DA cells from apoptosis. In addition, it induced de novo TH synthesis and increased intracellular reduced glutathione (GSH) levels, indicating a possible neuroprotective role for GSH. However, in doses ranging from 200 to 400 microM, DEA-NO decreased TH(+) cells, DA levels, [(3)H]DA uptake and the number of mature oligodendrocytes (O1(+) cells). No changes in either the amount or morphology of astrocytes and glial progenitors were detected. A dose- and time-dependent increase in apoptotic cells in the DEA-NO-treated culture was also observed, with a concomitant increase in the proapoptotic Bax protein levels and a reduction in the ratio between Bcl-xL and Bcl-xS proteins. In addition, DEA-NO induced a dose- and time-dependent increase in necrotic cells. 1H-[1,2,4]oxadiazolo[4, 3a]quinoxaline-1-one (ODQ, 0.5 microM), a selective guanylate cyclase inhibitor, did not revert the NO-induced effect on [(3)H]DA uptake. Glia-conditioned medium, obtained from fetal midbrain astrocyte cultures, totally protected neuronal-enriched midbrain cultures from NO-induced apoptosis and rescued [(3)H]DA uptake and TH(+) cell number. In conclusion, our results show that low NO concentrations have neurotrophic effects on DA cells via a cGMP-independent mechanism that may implicate up-regulation of GSH. On the other hand, higher levels of NO induce cell death in both dopamine neurones and mature oligodendrocytes that is totally reverted by soluble factors released from glia.     

Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.     

The tyrosine phosphatase Shp-2 interacts with the dopamine D(1) receptor and triggers D(1) -mediated Erk signaling in striatal neurons

We report a novel mechanism for dopamine D(1) receptor (D(1) R)-mediated extracellular signal-regulated kinases (Erk) activation in rat striatum. Erk signaling depends on phosphorylation and dephosphorylation events mediated by specific kinases and phosphatases. The tyrosine phosphatase Shp-2, that is required for Erk activation by tyrosine kinase receptors, has been recently shown to regulate signaling downstream of few G protein-coupled receptors. We show that the D(1) R interacts with Shp-2, that D(1) R stimulation results in Shp-2 tyrosine phosphorylation and activation in primary striatal neuronal cultures and that D(1) R/Shp-2 interaction is required for transmitting D(1) R-dependent signaling to Erk1/2 activation. D(1) R-mediated Erk1/2 phosphorylation in cultured striatal neurons is in fact abolished by over-expression of the inactive Shp-2(C/S) mutant and by small interfering RNA-induced Shp-2 silencing. Moreover, by using selective inhibitors we show that both D(1) R-induced Shp-2 activation and Erk1/2 phosphorylation are dependent on the cyclic AMP/protein kinase A pathway and require Src. These results, which were substantiated also in transfected human embryonic kidney 293 cells, provide a novel mechanism by which to converge D(1) R signaling to the Erk pathway and suggest that Shp-2 or the D(1) R/Shp-2 interface could represent a potential drug target for disorders of dopamine transmission involving malfunctioning of D(1) R signaling.     

Insulin-like growth factor-1 increases activity and surface levels of the GLAST subtype of glutamate transporter

Glutamate uptake systems are the primary mechanisms involved in excitatory amino acids clearance, their regulation is extremely important for proper neuronal function. Using cultured chick cerebellar Bergmann glia cells, the involvement of receptor tyrosine kinases in glutamate uptake was studied. Treatment of the cells with insulin-like growth factor-1 but not epidermal growth factor or neuronal growth factor, induces a dose and time dependent increase in [(3)H]-D-aspartate uptake that is sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Saturation experiments show a significant increase in V(max), suggesting that the amount of transporter molecules at the cell membrane under insulin-like growth factor-1 treatment is augmented. This interpretation was strengthen by equilibrium-binding experiments and by the fact that the increase in [(3)H]-D-aspartate uptake was not dependent on protein synthesis. The present studies suggest that insulin-like growth factor-1 signaling is involved in modulation of glutamate transporter cell surface expression.     

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.     

Phosphatidylinositide 3-kinase and protein kinase C zeta mediate retinoic acid induction of DARPP-32 in medium size spiny neurons in vitro

Mature striatal medium size spiny neurons express the dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels, or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include retinoic acid (RA), brain-derived neurotrophic factor, and estrogen (E₂). We now demonstrate that RA regulates DARPP-32 mRNA and protein in primary striatal neuronal cultures. Furthermore, DARPP-32 induction by RA in vitro requires phosphatidylinositide 3-kinase, but is independent of tropomyosin-related kinase B, cyclin-dependent kinase 5, and protein kinase B. Using pharmacologic inhibitors of various isoforms of protein kinase C (PKC), we also demonstrate that DARPP-32 induction by RA in vitro is dependent on PKC zeta (PKCζ). Thus, the signal transduction pathways mediated by RA are very different than those mediating DARPP-32 induction by brain-derived neurotrophic factor. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro , including a novel, important pathway via which phosphatidylinositide 3-kinase regulates the contribution of PKCζ.     

Radicicol Potentiates Neurotrophin-Mediated Neurite Outgrowth and Survival of Cultured Sensory Neurons from Chick Embryo

Radicicol, an antifungal antibiotic with markedly low toxicity, is a potent inhibitor of the Src family of protein tyrosine kinases and causes morphological reversion of v-src-transformed fibroblasts. Recently, this antibiotic was also found to inhibit Raf kinase. In the present study, we found that nanomolar concentrations of radicicol (10 ng/ml) enhanced the survival and neurite outgrowth of neurons from embryonic chick dorsal root ganglia (DRGs) and sympathetic ganglia. It potentiated the trophic effects of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 on the cultured DRG neurons. This concentration of radicicol did not alter the tyrosine phosphorylation of Trk receptors or the activity of mitogen-activated protein (MAP) kinases. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not inhibit radicicol, excluding the involvement of PI3-kinase in the radicicol-dependent trophic actions. These results suggest that radicicol mediates neuronal growth presumably via a mechanism not involving the activation of Trk receptors, MAP kinase, or PI3-kinase.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

Dopamine-dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target-derived neurotrophic signaling (Part 2)

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.     

Qualitative and quantitative re-evaluation of epidermal growth factor-ErbB1 action on developing midbrain dopaminergic neurons in vivo and in vitro: target-derived neurotrophic signaling (Part 1)

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.     

Putative cocaine receptor in striatum is a glycoprotein with active thiol function

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.     

Chronic stimulation of D1 dopamine receptors in human SK-N-MC neuroblastoma cells induces nitric-oxide synthase activation and cytotoxicity

Elevated synaptic levels of dopamine may induce striatal neurodegeneration in l-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that approximately 60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-l-arginine methyl ester and genistein, wortmannin, or NF-kappaB SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-kappaB, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.     

Inhibition of Phosphatidylinositol 3-Kinase Activity Blocks Cellular Differentiation Mediated by Glial Cell Line-Derived Neurotrophic Factor in Dopaminergic Neurons

Abstract: Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µ M 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 n M wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.     

New findings on nuclear gangliosides: overview on metabolism and function

Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ～ 65% in 5 μM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 μM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 μM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 μM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.