Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter

Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT amino-terminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery.     

Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains

The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [³H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.     

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.     

Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms.     

Induction of preputium eversion by peptides, serotonin receptor antagonists, and selective serotonin reuptake inhibitors in Biomphalaria glabrata

Abstract. Eversion of the preputium is one of the initial steps in the male copulatory behavior of freshwater pulmonates. Previous experiments have shown that serotonergic mechanisms are involved in eversion in the snail Biomphalaria glabrata because the vertebrate 5-HT₁ receptor antagonist methiothepin caused long-lasting eversion in a dose-dependent manner. In this study, we tested a variety of serotonergic receptor ligands, bioactive peptides, and selective serotonin reuptake inhibitors (SSRIs) for their ability to induce preputium eversion in B . glabrata in order to elucidate the physiological mechanism of eversion. Of 15 compounds tested, five significantly induced preputium eversion: the serotonin receptor antagonists methiothepin (1 and 10 μM; p<0.0001), cyproheptadine (1–10 μM; p<0.007–0.0001), and mianserin (5–50 μM; p<0.01–0.001), the molluscan cardioactive peptide FMRFamide (10 and 50 μM; p<0.0002–0.0001), and the SSRI fluoxetine (=Prozac, 10–100 μM; p<0.0003–0.0001). Serotonin itself neither induced eversion nor blocked methiothepin-induced eversion. This suggests that fluoxetine is not acting as an SSRI, but potentially as a receptor ligand. These preliminary data shed light on the possible physiological mechanism of preputium eversion in B . glabrata and suggest similarity with that of the model freshwater gastropod Lymnaea stagnalis .     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Neurosteroids differentially modulate P2X4 ATP-gated channels through non-genomic interactions

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X₄ receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X₄ receptor. Application of 0.1–10 μM alfaxolone potentiated within 60-s the 1 μM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3–10 μM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the τ_off of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17β-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X₄ receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X₄ receptor more than 10-fold by 10 μM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.     

Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytes

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nM) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10-100 mM). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10-100 mM). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nM) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

Evidence for D2 receptor mediation of amphetamine-induced normalization of locomotion and dopamine transporter function in hypoinsulinemic rats

Dopamine (DA) D2 receptors regulate DA transporter (DAT) activity, and mediate some behavioral effects of amphetamine. DA clearance and amphetamine-stimulated locomotion are reduced in hypoinsulinemic [streptozotocin (STZ)-treated] rats, and these deficits are normalized by repeated treatment with amphetamine. Here, a role for D2 receptors in mediating amphetamine-induced normalization of these parameters was investigated. One week after a saline or STZ injection (50 mg/kg), rats were treated with amphetamine (1.78 mg/kg), raclopride (0.056 mg/kg), saline, or combinations thereof, every-other-day for 8 days with locomotor activity measured following each treatment. Conditioned place preference (CPP) for amphetamine and in vivo chronoamperometry to measure DA clearance were carried out on days 17 and 18, respectively, after STZ or saline. Baseline locomotion and DA clearance were significantly reduced in STZ-treated rats compared with control rats. In STZ-treated rats, amphetamine treatment normalized DA clearance, and restored the locomotor-stimulating effects of amphetamine. Raclopride prevented normalization of these parameters. Amphetamine produced CPP in both STZ-treated and control rats; raclopride significantly attenuated amphetamine-induced CPP in control and not in STZ-treated rats. These results support a role for D2 receptors in regulating DA transporter activity, and further demonstrate that D2 receptors contribute to changes in sensitivity to amphetamine in hypoinsulinemic rats.     

7th Biennial Meeting of the Asian Pacific Society for Neurochemistry, APSN 2006, Singapore, 2-5 July 2006. Abstracts

Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT‐mediated re‐uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein–protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT‐interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.     

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity ( K _m, 2.6 μM) and, although it also binds betaine ( K _m, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX ( K _m, 24 μM) and the betaine-specific SBP BetX ( K _m, 0.6 μM). Unlike most ABC transporter loci, caiX , betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.     

Dopamine neuronal transport kinetics and effects of amphetamine

The dopamine (DA) transporter (DAT) regulates DA neurotransmission by recycling DA back into neurons. Drugs that interfere with DAT function, e.g., cocaine and amphetamine, can have profound behavioral effects. The kinetics of DA transport by DAT in isolated synaptosomal or single cell preparations have been previously studied. To investigate how DA transport is regulated in intact tissue and to examine how amphetamine affects the DAT, the kinetics of DA uptake by the DAT were examined in tissue slices of the mouse caudate-putamen with fast-scan cyclic voltammetry. The data demonstrate that inward DA transport is saturable and sodium-dependent. Elevated levels of cytoplasmic DA resulting from disruption of vesicular storage by incubation with 10 microM Ro 4-1284 did not generate DA efflux or decrease its uptake rate. However, incubation with 10 microM amphetamine reduced the net DA uptake rate and increased extracellular DA levels due to DA efflux through the DAT. In addition, a new, elevated steady-state level of extracellular DA was established after electrically stimulated DA release in the presence of amphetamine, norepinephrine, and exogenous DA. These results from intact tissue are consistent with a kinetic model of the DAT established in more purified preparations in which amphetamine and other transported substances make the inwardly facing DAT available for outward transport of intracellular DA.     

Effect of phenylarsine oxide on fluid phase endocytosis: further evidence for activation of the glucose transporter

We have shown previously that insulin stimulates fluid phase endocytosis in 3T3-L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin-stimulated fluid phase endocytosis, but basal endocytosis as well. The Ki for this process was 6 microM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin-stimulated glucose transport. Although the Ki for insulin-stimulated transport (7 microM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time-dependent reduction to the basal rate (t 1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid-phase endocytosis/exocytosis, which is a vesicle-mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.     

In Situ Regulated Dopamine Transporter Trafficking: There’s No Place Like Home

Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important behaviors, and additionally play a role in DA-related neuropathologies. The presynaptic SLC6 DA transporter (DAT) limits extracellular DA levels by clearing released DA, and is potently inhibited by addictive and therapeutic psychostimulants. Decades of evidence support that the DAT is subject to acute regulation by a number of signaling pathways, and that endocytic trafficking strongly regulates DAT availability and function. DAT trafficking studies have been performed in a variety of model systems, including both in vitro and ex vivo preparations. In this review, we focus on the breadth of DAT trafficking studies, with specific attention to, and comparison of, how context may influence DAT’s response to different stimuli. In particular, this overview highlights that stimulated DAT trafficking not only differs between in vitro and ex vivo environments, but also is influenced by both sex and anatomical subregions.

     

Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms

Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine (METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by (32)PO(4) metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK(1) cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.     

Dopamine transporter relation to levodopa-derived synaptic dopamine in a rat model of Parkinson's: an in vivo imaging study

Studies showed that the dopamine (DA) transporter (DAT) modulates changes in levodopa-derived synaptic dopamine levels (Δ(DA)) in Parkinson's disease (PD). Here we evaluate the relationship between DAT and Δ(DA) in the 6-hydroxydopamine model of Parkinson's disease to investigate these mechanisms as a function of dopaminergic denervation and in relation to other denervation-induced regulatory changes. 27 rats with a unilateral 6-hydroxydopamine lesion (denervation ∼20–97%) were imaged with ¹¹C-dihydrotetrabenazine (VMAT2 marker), ¹¹C-methylphenidate (DAT marker) and ¹¹C-raclopride (D2-type receptor marker). For denervation <75%Δ(DA) was significantly correlated with a combination of relatively preserved terminal density and lower DAT. For denervation <90%, Δ(DA) was significantly negatively correlated with DAT with a weaker dependence on VMAT2. For the entire data set, no dependence on pre-synaptic markers was observed; Δ(DA) was significantly positively correlated with ¹¹C-raclopride binding-derived estimates of DA loss. These findings parallel observations in humans, and show that (i) regulatory changes attempt to normalize synaptic DA levels (ii) a lesion-induced functional dependence of Δ(DA) on DAT occurs up to ∼ 90% denervation (iii) for denervation < 75% relative lower DAT levels may relate to effective compensation; for higher denervation, lower DAT levels likely contribute to oscillations in synaptic DA associated with dyskinesias.     

Fiber development in preanthesis cotton ovules

A tissue culture method was developed to investigate the production of cotton (Gossypium hirsutum L. cv. Texas Marker-1) fibers in vitro. Ovules were excised from 3, 5, 7 and 9 days preanthesis ovaries and placed on an agar-solidified, modified Murashige and Skoog medium containing 2.3 μ M kinetin and 0.45 μ M –2,4–dichlo-rophenoxyacetic acid or 2.3 μ M kinetin and 10.7 μ M naphthaleneacetic acid. Ovules formed fibers and callus tissue. Fibers formed in vitro were up to 10 mm long, 10–22 μ wide and the cell wall was 1–3 μ M thick. Callus tissue cells were subcultured for over 25 weeks and their degree of elongation was monitored. The ability of ovule-derived cells to direct expansion in a longitudinal direction diminished, while lateral expansion increased with time in culture.