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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Acknowledgements
  5. References

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [3H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Abbreviations used:
AMPH

amphetamine

DA

dopamine

DAT

dopamine transporter

hDAT

human DAT

HEK

human embryonic kidney

IGF

insulin-like growth factor

NE

norepinephrine

PKC

protein kinase C

PI

phosphatidylinositol

YFP

yellow fluorescent protein.

Dopamine (DA) is involved in many physiological functions in the CNS, including cognition, emotion and motor activity (Giros and Caron 1993; Hyman 1996; Fumagalli et al. 1998). Dysfunction of dopaminergic neurotransmission plays a role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia and stimulant abuse (Giros and Caron 1993).

Signaling by extracellular DA is regulated by several mechanisms such as diffusion from the synapse, enzymatic degradation and reuptake of DA by the DA transporter (DAT). DAT function is crucial in regulating extracellular DA levels, and ablation of the DAT, either by gene knock out (Jones et al. 1998) or pharmacological blockade (Grace 1995), results in an increase in extracellular DA levels. The DAT is a member of a gene family that includes transporters for norepinephrine (NE), serotonin, gamma-aminobutyric acid, glycine, proline and taurine. The cDNA for the human DAT (hDAT) encodes a primary sequence of 620 amino acids. Hydrophobicity analysis predicted the presence of 12 membrane-spanning segments that are presumed to be α helices (Giros et al. 1992; Ferrer and Javitch 1998).

A number of studies have implicated trafficking of the DAT in the modulation of transporter activity (Daniels and Amara 1999; Melikian and Buckley 1999; Saunders et al. 2000). Several investigators have identified signal transduction pathways that control cell surface expression and activity of the DAT and other neurotransmitter transporters (Batchelor and Schenk 1998; Beckman and Quick 1998; Liu et al. 1999; Blakely and Bauman 2000; Mayfield and Zahniser 2001). Doolen and Zahniser demonstrated that protein tyrosine kinase inhibitors acutely regulate DAT function by altering cell surface expression of the transporter (Doolen and Zahniser 2001). Tyrosine phosphorylation of GAT1 GABA transporter by tyrosine kinases has also been reported to have a role in the trafficking of this membrane protein (Law et al. 2000). Finally, Apparsundaram et al. provided evidence supporting a role of the insulin signaling cascade in the trafficking and function of the NE transporter (Apparsundaram et al. 2001).

Little is known about the role of humoral or hormonal factors in DAT regulation (Figlewicz 1999). Curiously, insulin receptors have been reported to be densely concentrated in limbic areas of the brain (Manzanares et al. 1988; Schulingkamp et al. 2000). In particular, a high density of insulin and insulin-like growth factor (IGF) I receptors has been found in the striatum (Bergstedt and Wieloch 1993; Schulingkamp et al. 2000), a brain region in which DAT is richly expressed (Pilotte et al. 1994; Ciliax et al. 1995; Ciliax et al. 1999). Insulin has been shown to regulate the reuptake of NE both in primary cultures of neonatal rat neurons and in whole brain synaptosomes (Boyd et al. 1986; Raizada et al. 1988), and it is possible that insulin regulates DAT function as well. Indeed, biochemical changes in the dopaminergic system have been identified in streptozotocin-induced diabetic mice (Saitoh et al. 1998). Moreover, resistance to the locomotor and behavioral effects of amphetamine (AMPH), which requires the DAT for its actions, has also been observed in experimentally induced diabetic rodents (Marshall 1978; Saitoh et al. 1998).

Here we explore whether insulin regulates DAT activity in rat brain striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably transfected with the hDAT. Our findings are discussed in the context of how PI 3-kinase, an enzyme activated by insulin through both insulin and IGF1 receptors, regulates hDAT membrane trafficking and transport capacity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Acknowledgements
  5. References

Preparation of synaptosomes

Male Sprague–Dawley rats (250–300 g; Charles River, Wilmington, MA, USA) were housed in a colony room maintained at constant temperature (37°C) and humidity for one week prior to the experiments. Rats had free access to food and water, and a 12 : 12 h light/dark cycle was employed. Experiments were reviewed and approved by the NIDA Animal Care and Use Committee. Rats were killed by decapitation, and their brains were removed and placed in an ice-cooled dish for dissection. The striatum was dissected and homogenized in ice-cold Krebs-Ringer buffer (125 mm NaCl, 1.2 mm KCl, 1.2 mm MgSO4, 1.2 mm CaCl2, 22 mm NaHCO3, 1 mm NaH2PO4, 10 mm glucose, pH 7.4) containing 0.32 m sucrose using a glass homogenizing tube and a Teflon pestle. After centrifugation at 1000 g for 10 min at 4°C, the pellet was discarded and the supernatant centrifuged at 16 000 g for 15 min. The resulting P2 pellet was placed on ice until resuspension.

[3H]DA Uptake in synaptosomes

Synaptosomes were pre-incubated with the indicated concentrations of LY294002 (Calbiochem, San Diego, CA, USA) at 37°C for 30 min (unless otherwise noted) prior to the uptake assays. After pre-incubation, samples were placed on ice and 3H[DA] was added to initiate transport. The assay was performed in Krebs-Ringer buffer containing 0.64 mm ascorbic acid and 0.8 mm pargyline to inhibit substrate degradation. The final concentration of [3H]DA (50 Ci/mmol; Amersham, Piscataway, NJ, USA) was 0.1 μm, except in the kinetic analyses where [3H]DA concentrations were 0.025–1.5 μm. Tubes were incubated for 4 min at 37°C. Non-specific accumulation of [3H]DA was determined in samples incubated at 4°C. The assay was terminated by placing the tubes on ice and adding 5 mL of ice-cold Krebs-Ringer buffer. The solutions were then filtered through Whatman glass microfiber filters (GF/C) presoaked in 0.1% polyethyleneimine to reduce non-specific binding. After filtration, the filters were washed with 2 × 5 mL of ice-cold Krebs-Ringer buffer. They were placed in scintillation vials and 3 mL of Bio-Safe II scintillation fluid (Research Products Int., Moun Prospect, IL, USA) was added to each tube. Radioactivity was determined by liquid scintillation spectrometry. Kinetic parameters were determined by non-linear regression fitting using Prism software (Graph Pad, San Diego, CA, USA).

Plasmid construction, transfection and cell culture

The synthetic hDAT gene tagged at the 5′ end with a FLAG epitope was subcloned into a bicistronic expression vector (Rees et al. 1996) modified to express the synthetic hDAT from a cytomegalovirus promoter and the hygromycin resistance gene from an internal ribosomal entry site as described previously (pciHyg) (Saunders et al. 2000). EM4 cells, an HEK 293 cell line stably transfected with macrophage scavenger (R. Horlick, Pharmacopeia, Cranberry, NJ, USA), were transfected with the FLAG-hDAT using Lipofectamine (Gibco/BRL, Rockville, MD, USA), and a stably transfected pool was selected in 250 μg/mL hygromycin as described (Ferrer and Javitch 1998). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Previous studies have shown that addition of the N-terminal FLAG tag does not alter [3H]DA or [3H]tyramine uptake by the transporter. Similarly, the ability of the transporter to produce substrate-induced currents is unaltered (Saunders et al. 2000).

A fluorescently tagged hDAT was constructed by fusing the C-terminus of the coding region of enhanced yellow fluorescent protein (YFP) from pEYFP-N1 (Clontech, Palo Alto, CA, USA) to the N-terminus of the human synthetic DAT cDNA, thereby creating the fusion construct YFP-hDAT. This construct was subcloned into pciHyg, and stable pools of EM4 cells expressing YFP-hDAT were obtained as described above.

Transient expression of dominant negative dynamin and constitutively active PI 3-kinase

EM4 cells stably expressing FLAG-hDAT were transiently transfected with human wild-type dynamin I or the dominant negative K44A mutant in pCB1 using the PolyFect transfection method (Qiagen, Valencia, CA, USA). Cells were incubated overnight in the presence of 2 μg of DNA per 35 mm dish (immunoflourescence assays) or 0.4 μg per well of a 24-well plate (uptake assays). A constitutively active murine PI 3-kinase p110 α catalytic subunit containing the avian src myristoylation sequence at the amino terminus (Upstate Biotechnology) was subcloned into pcDNA3.1 using HindIII and XbaI and transfected into FLAG-hDAT cells using PolyFect as described above. Immunoblot analysis demonstrated that transient expression of the PI 3-kinase p110 α catalytic subunit did not alter protein levels of DAT with respect to cells transfected with the vector alone (data not shown). The cells were treated 48 h after transfection as indicated for the uptake and confocal microscopy experiments.

Uptake of [3H]DA in FLAG- and YFP-DAT Cells

EM4 cells expressing FLAG-hDAT or YFP-hDAT were seeded into 24-well plates ∼24 h prior to the experiments and grown to confluence (approximately 100 000 cells per well). After 5 h of serum starvation, the cells were incubated with insulin (Sigma, St Louis, MO, USA) or LY294002 in uptake buffer at 37°C. The cells were then placed into an 18°C incubator in uptake buffer (130 mm NaCl, 1.3 mm KCl, 10 mm Hepes, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2.2 mm CaCl2, 10 mm glucose, pH 7.4) containing 100 μm ascorbic acid and 100 mm pargyline and [3H]DA (NEN) uptake was performed immediately. In triplicate or quadruplicate wells, 50 nm[3H]DA together with 10 μm DA was added in a final volume of 250 μL. In the kinetic analyses, the cells were treated in quadruplicate wells with 100 nm[3H]DA (NEN) in the continued presence or absence of LY294002 in addition to various concentrations of unlabeled DA ranging from 0.1 µm to 10 µm. All reagents were diluted in uptake buffer prewarmed to 18°C. The cells were incubated at 18°C for 2 min and then the solutions were aspirated to terminate uptake. After three washes with ice-cold buffer, cells were lysed with 300 μL 1% sodium dodecyl sulfate. Radioactivity was measured in a Beckman scintillation counter with UniverSol cocktail. Specific uptake was defined as total uptake minus non-specific in the presence of 10 μm mazindol. Kinetic parameters were determined by non-linear regression fitting using Prism software (Graph Pad).

Immunofluorescence and confocal microscopy

After 5 h of serum starvation, cells were treated as stated in the text in serum-free medium at 37°C. The steps for immunostaining were conducted at room temperature. The coverslips containing the cells (50–70% confluent) were washed twice with phosphate-buffered saline (PBS; 154 mm NaCl, 11 mm Na2HPO4, 2.7 mm KCl, pH 7.4) and fixed for 25 min with 4% paraformaldehyde. The cells were rinsed twice with PBS and blocked with 5% normal goat serum diluted in 0.05% Triton X-100/PBS (PBST) for 1 h. The blocking solution was aspirated and the cells were rinsed once with PBST. The coverslips were incubated with primary antibody (anti-FLAG M-2 mAb, Sigma) at a 1 : 1200 dilution in PBST for 1 h. Primary antibody was aspirated and the cells were washed 2 times with PBST. The cells were incubated with secondary antibody (goat antimouse IgG (H & L) TriTC, Kirkegarrd & Perry) at a 1 : 200 dilution in PBST for 1 h. The secondary antibody was removed and the cells were washed 2 times with PBST and once with PBS. Coverslips were then mounted onto slides with Crystal Mount (Biomedia) and allowed to dry.

Confocal microscopy was performed on a Nikon Diaphot inverted microscope (Melville, NY) using a Bio-Rad MRC1024 confocal imaging system (Hercules, CA) equipped with a krypton-argon laser and operated with Lasersharp software (Bio-Rad Laboratories, Hercules, CA). The (tetramethyl rhodamine [TMR]) image was obtained using only the 568 nm excitation wavelength with a HQ598DF40 emission filter. Images were obtained with a 100X oil immersion objective with a z-axis resolution of 1 μm unless otherwise stated.

YFP-hDAT real time confocal microscopy was performed on an Olympus IX-70 inverted microscope using a confocal imaging system (Olympus Fluoview 500) equipped with an argon laser. YFP-hDAT images were acquired using a 488-nm excitation wavelength with a 505 long pass filter. The temperature of the bath was held at 37°C by a Bioptechs Focht Chamber System 2 (FCS2).

Cell surface biotinylation and immunoblotting

EM4 cells stably expressing FLAG-synDAT were incubated with DMEM overnight in the absence of serum. The cells were then treated for 30 min at 37°C in DMEM in the absence or in the presence of LY294002 (100 µm) or insulin (1 µm). Cell surface biotinylation with cleavable sulfo-NHS-S-S-biotin (0.3 mg/mL in PBS, pH 9.0) (Pierce Chemical Co., Rockford, IL, USA) to label surface-localized transporter was performed as described (Saunders et al. 2000). After biotinylation cells were incubated for 20 min at 4°C in quenching buffer (20 mm Tris, 10 mm glycine, 140 mm NaCl, pH 7.4) followed by incubation with 10 mm NEM in PBS for 20 min at room temperature. Cells were scraped into PBS-PI buffer (PBS supplemented with 1 µg/mL leupeptin, 1 µg/mL pepstatin, 2 µg/mL aprotinin, 2 µg/mL pefablock, and 10 mm N-ethylmaleimide). Cells were pelleted at 4°C and incubated in PBS-PI supplemented with 0.2% digitonin at 4°C for 20 min Cells were pelleted and then incubated in lysis buffer (PBS-PI containing 1% Triton X-100) at 4°C for 1 h with constant mixing. The extract was centrifuged at 4°C (14 000 × g) for 30 min. An aliquot of the extract was removed for determination of total DAT. The remaining extract was incubated with 15 µL neutrAvidin Plus beads (Pierce Chemical Co.) for 1 h at room temperature (for a 35-mm culture dish). The beads were washed twice with lysis buffer and then eluted with Laemmli sample buffer containing 50 mm dithiothreitol. The crude extracts and the eluted proteins were resolved by SDS–PAGE, transferred to PVDF membranes (Millipore) and blocked for 1 h in 5% dry milk, 1% BSA, 0.1% Tween-20 in TBS. FLAG-DAT was detected by anti-FLAG M2 primary antibody and antimouse-HRP secondary antibody (Santa Cruz Biotechnology, Inc.), with ECL-Plus (Amersham) and fluorescence detection and quantitation on a Fluoro-Chem imager using Alpha EaseTM FC software (Alpha Innotech Corporation, San Leandro, CA, USA).

Results

Insulin regulates [3H]DA uptake

The insulin signaling cascade, which activates PI 3-kinase, modulates vesicular trafficking in general, and the cell surface expression of the insulin-responsive glucose transporter in particular (Katagiri et al. 1996; Katagiri et al. 1997). In order to explore whether insulin regulates hDAT function, we first tested whether this hormone alters [3H]DA uptake in EM4 cells stably expressing FLAG-hDAT. Addition of the N-terminal FLAG tag does not significantly alter [3H]DA uptake by the transporter (Saunders et al. 2000), and the presence of the FLAG tag allows us to use confocal microscopy to analyze the cell distribution of the transporter. Incubation of FLAG-hDAT cells with insulin increased [3H]DA uptake; in the presence of 1 µm insulin the stimulation was maximal at 5 min (128 ± 9% of control conditions) (Fig. 1a). Insulin stimulation of [3H]DA uptake was also concentration-dependent (Fig. 1b). Incubation of FLAG-hDAT cells with 100 nm or lower concentrations of insulin for 10 min did not significantly increase [3H]DA uptake. After a longer incubation time (30 min) 100 nm insulin did significantly increase [3H]DA uptake (140 ± 4% of control conditions, n = 3). Because high concentrations of insulin activate the IGF-I receptor (Baserga et al. 1997) it is possible that in addition to the insulin receptor, the IGF-I receptor may also play a role in regulating [3H]DA uptake.

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Figure 1. Time and concentration dependence of the insulin-stimulated increase in [3H]DA uptake in FLAG-hDAT cells. (a) Treatment of cells with 1 µm insulin increases DA uptake in a time-dependent fashion. The uptake values were normalized with respect to control conditions (no insulin) and are expressed as the mean ± SEM. Data are representative of three experiments conducted in quadruplicate. (b) Increasing concentrations of insulin increase [3H]DA uptake in FLAG-hDAT cells. Cells were incubated with the indicated concentrations of insulin for 10 min, and [3H]DA uptake was determined. Data are normalized to control conditions (no insulin) and are expressed as the mean ± SEM of three different experiments conducted in quadruplicate. For both panels, asterisks indicate significant changes in DA uptake compared with vehicle controls (anova followed by Tukey's test; level of significance p < 0.05).

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Incubation of striatal synaptosomes for 30 min with 100 nm insulin increased [3H]DA uptake (131 ± 10% of control conditions, n = 2). In parallel experiments, 1 μm insulin did not alter glycine transport (99 ± 6% of control conditions, n = 3), which is mediated by the endogenous Na+/Cl- dependent glycine transporter. Therefore, the effect of insulin on [3H]DA uptake is not likely due to global changes in membrane potential or ion gradients.

Inhibition of PI 3-kinase down-regulates DA uptake in FLAG-hDAT cells and in striatal synaptosomes

Although the glucose and glutamate transporters belong to gene families that are unrelated to the Na+/Cl-dependent neurotransmitter transporter family, of which DAT is a member, recent studies have implicated PI 3-kinase in the regulation of their activity and trafficking (Clarke et al. 1994; James and Piper 1994; Davis et al. 1998). Therefore, we examined the effect of an inhibitor of PI 3-kinase, LY294002, on the regulation of [3H]DA uptake. LY294002 binds competitively to the ATP binding site of the PI 3-kinase catalytic subunit, thereby inhibiting its activity. Incubation of FLAG-hDAT cells with LY294002 for 30 min resulted in a concentration-dependent decrease in [3H]DA uptake (Fig. 2a). Treatment with 100 nm wortmannin, another inhibitor of PI 3-kinase, for 1 h also decreased [3H]DA uptake to 76 ± 8 of control conditions (p < 0.01 by paired Student's t-test, n = 3). In contrast, LY294002 (50 μm) did not significantly decrease glycine uptake (data not shown) suggesting that PI 3-kinase selectively regulates DAT activity.

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Figure 2. LY294002 inhibition of [3H]DA uptake in FLAG-hDAT cells and striatal synaptosomes. (a) A 30-min treatment of FLAG-hDAT cells with LY294002 decreases [3H]DA uptake in a concentration- dependent manner. Data are normalized to control conditions (no LY294002, dotted line) and are expressed as the mean ± SEM of three different experiments conducted in quadruplicate. (b) Striatal synaptosomes were treated with increasing concentrations of LY294002 for 30 min before the determination of [3H]DA uptake. (c) [3H]DA uptake in striatal synaptosomes treated for the indicated times with 30 µm LY294002. In all panels, asterisks indicate significant changes in DA uptake compared with vehicle controls (anova followed by Tukey's test; level of significance equal to *p < 0.05 and **p < 0.01).

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The LY294002-induced decrease in [3H]DA uptake was also observed in striatal synaptosomes (Fig. 2b); increasing concentrations of LY294002 progressively decreased [3H]DA uptake both in FLAG-hDAT cells and in striatal synaptosomes (45 ± 1% and 10 ± 4% of control conditions, respectively, at 100 μm LY294002). In striatal synaptosomes, the reduction of [3H]DA uptake was maximal after 30 min with 30 μm LY294002 (Fig. 2c). These results suggest that in synaptosomes and in serum-starved FLAG-hDAT cells the basal level of PI 3-kinase activity tonically regulates hDAT function.

Acute changes in uptake could be produced by altering the number of functional transporters on the cell surface, the rate of turnover, and/or the affinity of the transporter for its substrate. To further characterize the LY294002-induced inhibition of [3H]DA uptake, kinetics studies were performed both in striatal synaptosomes and FLAG-hDAT cells. In striatal synaptosomes, the Vmax of [3H]DA uptake was significantly decreased by treatment with 30 μm LY294002 for 30 min (54% ± 1.2 of control) (Table 1). In contrast, Km values were not significantly affected by LY294002 treatment. LY294002-induced alterations in DAT transporter capacity were also observed in FLAG-hDAT cells. The decrease in [3H]DA uptake in LY294002-treated cells was due to a significant decrease in Vmax (50% ± 6 of control) without any significant effect on the Km (Table 1). These alterations in Vmax in both striatal synaptosomes and FLAG-hDAT cells are consistent with changes in the number of functional transporters at the cell surface, and/or a change in the rate of turnover of the transporter. Similar results were obtained in FLAG-hDAT cells using tyramine, another DAT substrate (data not shown).

Table 1.  Effect of LY294002 on kinetic parameters of DAT activity in rat striatal synaptosomes and FLAG-hDAT cells
 TreatmentVmaxK
  1. Striatal synaptosomes were treated for 30 min at 37°C with vehicle or 30 μm LY294002 before [3H]DA uptake determination. Results are mean ± SEM from three experiments. *p < 0.05 by paired student's t-test. FLAG-hDAT cells were treated for 30 min with vehicle (dimethyl sulfoxide) or 50 μm LY294002. DAT activity was determined with [3H]DA as described under Materials and methods. Vmax and Km values are the mean ± SEM from three experiments. **p < 0.05 by paired Student's t-test.

Synaptosomes pmol/min/mgµm
Control6.74 ± 0.55 0.5 ± 0.006
LY2940023.64 ± 0.08*0.63 ± 0.03
Cells fmol/2 min/105cellsµm
Control337.5 ± 13.740.92 ± 0.34
LY294002163.2 ± 21.17**0.87 ± 0.5
Acute LY294002 treatment blocks the insulin-dependent increase in [3H]DA uptake and causes FLAG-hDAT cell surface redistribution

To evaluate whether the insulin-induced increase in DAT activity in FLAG-hDAT cells (Fig. 1a) is mediated by PI 3-kinase, we treated cells with 1 µm insulin in the presence or absence of 30 µm LY294002 for 10 min. The insulin-mediated stimulation of [3H]DA uptake was inhibited by coincubation with LY294002, with the resulting uptake similar to that produced by LY294002 alone (Fig. 3a). These data suggest that the insulin-induced effect on DAT function is mediated by PI 3-kinase.

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Figure 3. LY294002-induced loss of FLAG-hDAT cell surface expression. (a) Incubation of FLAG-hDAT cells with 1 μm insulin for 10 min increases [3H]DA uptake (125 ± 3.4 relative to control; open bar) (n = 3; **p < 0.001 by paired Student's t-test). This increase was blocked when 30 μm LY294002 was added 20 min before the insulin application (83 ± 4.7% relative to control condition) (n = 3; **p < 0.001 by paired Student's t-test, closed bar). The striped bar shows the effect of 30 μm LY294002 by itself (77 ± 4.7% relative to control conditions) (n = 3; **p < 0.001 by paired Student's t-test). Data are normalized to control conditions (DMSO vehicle for LY294002) (dotted line) and are expressed as mean ± SEM of three different experiments conducted in quadruplicate. Confocal microscopy images of FLAG-hDAT cells incubated in the absence (b) or presence (c) of 50 µm LY294002 for 30 min. The gallery (c) shows z-sections from the top to the bottom of FLAG-hDAT cells treated with LY294002; the six confocal planes 1 µm in thickness show extensive intracellular immunoflourescence. The confocal microscopy images shown are representative of six different experiments.

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We next investigated whether the LY294002-induced decrease in Vmax (Table 1) is due to changes in the subcellular localization of transporter. Figure 3(c) shows that LY294002 treatment caused a significant intracellular accumulation of FLAG-hDAT relative to control cells, in which most of the transporter was detected at the plasma membrane (Fig. 3b; Saunders et al. 2000). Confocal microscopy images of 1 µm steps in depth (Fig. 3c, gallery) illustrate that the fluorescence shifted substantially from the cell surface to the intracellular compartment upon LY294002 treatment. These results are consistent with the Vmax changes (Table 1) and further suggest that the number of DATs on the cell surface decreases after exposure to LY294002.

The LY294002-induced loss of cell surface-associated hDAT is a dynamin-mediated mechanism

Cells expressing FLAG-hDAT were pretreated with 250 µg/mL of concanavalin A to prevent hDAT internalization by stabilizing cell surface integrity due to tetravalent lectin contacts (Toews et al. 1984). As seen in Fig. 4, pretreatment with concanavalin A largely prevented the LY294002-mediated internalization of FLAG-hDAT (compare Figs 4b and c). Pre-incubation with concanavalin A in the absence of LY294002 (data not shown) resulted in an immunofluorescence profile similar to that of control cells (Fig. 4a).

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Figure 4. LY294002-induced FLAG-hDAT internalization is blocked by concanavalin A. Confocal microscopy images of control FLAG-hDAT cells (A) or cells incubated in the presence of 50 μM LY294002 (b) or 50 μM LY294002 plus 250 µg/mL concanavalin A (ConA) (c) for 30 min.

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We evaluated whether the LY294002-induced decrease in DA uptake is a result of the dynamin-mediated cell surface redistribution of FLAG-hDAT by transiently transfecting FLAG-hDAT cells with either wild-type or dominant negative K44A dynamin I. Dynamin is a GTPase that catalyzes the pinching off of clathrin-coated pits from the cell surface (Damke et al. 1994; Zhang et al. 1996). [3H]DA uptake was significantly decreased in response to a 30-min incubation with 50 µm LY294002 in FLAG-hDAT cells expressing wild-type dynamin I (Fig. 5a; 75 ± 3.9% relative to control cells transfected with dynamin I but not treated with LY294002). In contrast, [3H]DA uptake in cells transiently transfected with K44A dynamin I was almost unaffected by treatment with LY294002 (95 ± 3.2% relative to control cells transfected with K44A). Transient expression of K44A dynamin I in FLAG-hDAT cells also blocked the LY294002-induced trafficking of hDAT (Fig. 5b, main panel), whereas expression of wild-type dynamin I did not (Fig. 5b, inset). These data support the hypothesis that the dynamin-dependent cell surface redistribution of hDAT is in large part responsible for the decrease in transporter activity observed with acute LY294002 treatment.

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Figure 5. The LY294002-induced reduction in DA uptake is dynamin-dependent. (a) FLAG-hDAT cells transiently expressing wild-type or K44A dynamin I were treated for 30 min with or without 50 µm LY294002. Uptake of [3H]DA is expressed as a percent of the control (transfected cells not treated with LY294002). The data are the mean ± SEM of two different experiments performed in quadruplicate (**p < 0.01 by paired Student's t-test). (b) Confocal microscopy images of FLAG-hDAT cells transiently transfected with either the K44A dynamin I mutant (main panel) or wild-type dynamin I (inset). The cells were exposed to 50 μm LY294002 for 30 min. The confocal microscopy images shown are representative of four different experiments.

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Consistent with estimates of changes in [3H]DA uptake (Fig. 2A), incubation of FLAG-hDAT cells with 100 µm LY294002 for 30 min produced a significant decrease in the level of hDAT protein recovered in the biotinylated (cell surface) fractions (Fig. 6a; 68% ± 2% of control, n = 3, p < 0.01 by two-tailed t-test). Similar results were observed with 50 µm LY294002 (data not shown). In parallel experiments, incubation with 1 µm insulin for 30 min produced an increase in the level of surface biotinylated hDAT (128% ± 21% of control, n = 3). Although this increase was not statistically significant due to experimental variability, the trend was consistent with the increase in [3H]DA uptake stimulated by insulin (Fig. 1b). Together, these results suggest that the PI 3-kinase regulation of DAT activity likely results from an hDAT trafficking process.

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Figure 6. Effect of LY294002 and insulin on cell surface expression of FLAG-DAT. Cells were treated and then surface biotinylated as described in Experimental Procedures. (a) A representative immunoblot showing duplicate sample of total cell extracts (lanes 1,2,3) and biotinylated extracts (lanes 4,5,6) after treatment with vehicle (lanes 1 and 5), 100 µm LY294002 (lanes 2 and 4), or 1 µm insulin (lanes 3 and 6). (b) Quantification using the Alpha Innotec Fluorochem system of three immunoblots each performed in duplicate. The density of the biotinylated samples eluted from NeutraAvidin beads was normalized by the density of the parallel total extract from which it came to account for small differences in plating density or DAT expression. The normalized values were compared by two-tailed paired t-tests between control and LY treatment and control and insulin treatment. *p < 0.01.

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Insulin attenuates the AMPH-induced cell surface redistribution of hDAT

The insulin regulation of DAT transporter activity was further investigated by following real time changes in hDAT cell surface expression by using EM4 cells stably transfected with YFP-hDAT. The characteristics of the insulin-induced increase in YFP-hDAT activity in this experimental system are similar to those observed with FLAG-hDAT; thus, the YFP moiety does not adversely affect transporter function, and 1 μm insulin increases and 50 μm LY294002 decreases YFP-hDAT transport activity (data not shown). To further determine whether insulin regulates hDAT trafficking, we examined insulin-mediated redistribution of YFP-hDAT in live cells at 37°C. The YFP-DAT was first internalized by treating the cells with 10 μm AMPH (Saunders et al. 2000). After AMPH was added, confocal images were collected every two min for a duration of 52 min. At the time of AMPH administration, YFP fluorescence was seen primarily at the cell surface (Fig. 7a1). A gradual increase in intracellular fluorescence, concurrent with a decrease in plasma membrane fluorescence, was observed over 30 min of exposure to AMPH (Fig. 7a2). At this time, 5 μm insulin was added to the bath solution with AMPH still present. As a result, a gradual recovery of the plasma membrane fluorescence was observed. After 20 min of exposure to AMPH plus insulin, the intracellular fluorescence was reduced and the plasma membrane fluorescence increased to a level similar to that seen in control conditions (Fig. 7a3). In contrast, the internalization in response to 10 μm AMPH in the absence of subsequent insulin persisted for over 50 min (data not shown).

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Figure 7. Insulin regulates AMPH-induced hDAT cell surface redistribution in YFP-DAT cells. Confocal images collected from YFP-hDAT cells every two minutes for a duration of 52 minutes. Panel a shows that a 30-min exposure to 10 μm AMPH induces an increase of intracellular YFP-hDAT (a2) with respect to untreated control cells (a1). Addition of 5 μm insulin for 20 min in the presence of AMPH (a3) reduces the intracellular signal and restores plasma membrane fluorescence to levels similar to those seen in untreated control cells. Treatment of YFP-DAT cells with cycloheximide (40 μg/mL) alone for 4 h prior to and during the confocal microscopy experiment did not affect the cell membrane distribution of YFP-DAT (b1). In cycloheximide treated cells, application of 10 μm AMPH for 30 min still induces YFP-DAT cell surface redistribution (b2). In addition, cycloheximide treatment did not affect the ability of insulin to regulate AMPH-induced YFP-DAT cell surface redistribution (b3). Panel c shows that the fluorescence profile of YFP-DAT remained the same for the duration of the experiment in the absence of either AMPH or insulin. The confocal microscopy images shown are representative of three different experiments for each experimental condition.

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The increase in transporter protein at the cell surface upon insulin coapplication could result from either recycling of internalized hDAT to the plasma membrane or delivery of nascent transporters to the plasma membrane. To prevent the de novo synthesis of new YFP-hDAT, protein synthesis was inhibited by incubating YFP-DAT cells in cycloheximide for 4 h prior to and during collection of the confocal images. In cycloheximide-treated cells (Figs 7b1), 10 μm AMPH still induced an increase in intracellular fluorescence (Figs 7b2), and 5 μm insulin in the presence of AMPH was able to reduce the level of intracellular fluorescence to a level similar to that seen in control conditions (Fig. 7b3). These data suggest that YFP-DAT can recycle to the plasma membrane after being internalized. Figure 7(c) shows that without application of AMPH or insulin, the transporter is localized in the plasma membrane for the duration of the experiment.

Exogenous PI 3-kinase activates [3H]DA uptake

Multiple lines of evidence have documented the involvement of PI 3-kinase in the insulin stimulation of glucose transport (Elmendorf and Pessin 1999). In particular, it has been shown that expression of a constitutively active form of PI 3-kinase is sufficient to induce the translocation of the GLUT4 transporter to the cell surface (Elmendorf and Pessin 1999). In order to determine whether exogenous PI 3-kinase also modulates [3H]DA uptake, we transiently transfected FLAG-hDAT cells with constitutively active PI 3-kinase. [3H]DA uptake was 28 ± 5% higher in these cells than in control FLAG-hDAT cells transfected with the empty vector (n = 3, experiments conducted in quadruplicate; p < 0.005 by paired Student's t-test) (Fig. 8a).

image

Figure 8. Exogenous PI 3-kinase increases [3H]DA uptake and regulates AMPH-induced FLAG-hDAT trafficking. (a) FLAG-hDAT cells were transiently transfected with empty vector (control, open bar) or with constitutively active PI 3-kinase (closed bar). Uptake of [3H]DA is expressed as a percent of control. (b) Confocal microscopy images of FLAG-hDAT after incubation with 10 µm AMPH for 1 h in cells transfected with either constitutively active PI 3-kinase (inset) or the empty vector (main panel). The confocal microscopy images shown are representative of four different experiments.

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We then explored whether constitutively active PI 3-kinase affects AMPH-induced cell surface redistribution of FLAG-hDAT. We exposed FLAG-DAT cells transfected with constitutively active PI 3-kinase (Fig. 8b, inset) or vector (Fig. 8b, main panel) to 10 μm AMPH for 1 h. Expression of constitutively active PI 3-kinase antagonized the AMPH-induced trafficking of FLAG-hDAT. These data suggest that PI 3-kinase activity regulates AMPH induced cell surface redistribution of hDAT.

Discussion

In non-brain tissue such as skeletal muscle, the binding of insulin to its receptor stimulates transporter proteins that mediate the influx of glucose. Insulin crosses the blood–brain barrier (Banks and Kastin 1998), but neurons utilize insulin-independent mechanisms to obtain glucose. Therefore, insulin in the CNS could modulate cellular processes distinct from those related to the supply and metabolism of glucose.

Insulin has been shown to regulate dopaminergic neurotransmission, but the underlying mechanism has been under considerable debate (Patterson et al. 1998). Regulation of hDAT cell surface expression may represent an important mechanism by which insulin signaling modulates the dopaminergic system. DA uptake in striatal preparations from fasted hypoinsulinemic rats was significantly decreased as compared to control rats (Patterson et al. 1998). This reduction in DA uptake resulted from a decrease in transporter Vmax without a change in the Km for DA. Incubation of striatal suspensions from fasted animals with 1 nm insulin for 30 min restored the Vmax of DA uptake to control levels (Patterson et al. 1998). These data suggested that modifications in insulin levels affect DA uptake in the striatum. Interestingly, no changes in DA uptake were observed in the nucleus accumbens of food-deprived rats, suggesting that insulin levels could selectively affect different brain regions.

Here, we demonstrate that insulin causes an increase in DA uptake in FLAG-hDAT cells (Fig. 1) and in striatal synaptosomes. Several observations suggest that PI 3-kinase is a key player in the insulin regulation of DAT activity in these preparations. First, a pharmacological inhibitor of PI 3-kinase, LY294002, reverses the insulin-evoked increase in DA uptake (Fig. 3a). LY294002 also reduced transporter activity in striatal synaptosomes and FLAG-hDAT cells in the absence of insulin (Fig. 2, Table 1), suggesting that basal levels of PI 3-kinase activity in these systems modulate hDAT function. Moreover, wortmannin, a structurally distinct inhibitor of PI 3-kinase, also reduced DA uptake. Finally, expression of a constitutively active mutant of PI 3-kinase in FLAG-hDAT cells increased DA uptake (Fig. 8A). These results, for the first time, suggest a direct correlation between increased DA uptake and PI 3-kinase activity.

As transporter efficiency depends on the maximal rate of the transport process as well as on the number of active transporters on the cell surface, the PI 3-kinase regulation of DA uptake in FLAG-hDAT cells and striatal synaptosomes could indicate an increase in DAT cell surface expression. Indeed, a role for PI 3-kinase in regulating the trafficking of the DAT is evident from our data. Using confocal microscopy, we found that exposure of cells to LY294002 caused a large increase in the amount of intracellular FLAG-hDAT (Fig. 3). Our biotinylation studies confirmed that LY294002 caused a reduced density of DATs in the plasma membrane (Fig. 6). These data suggest a direct correlation between the trafficking of hDAT and PI 3-kinase activity.

A number of studies have implicated internalization of neurotransmitter transporters as a mechanism of transporter regulation (Qian et al. 1997; Zhang et al. 1997; Zhu et al. 1997; Apparsundaram et al. 1998; Ramamoorthy and Blakely 1999; Blakely and Bauman 2000). For example, PKC activation decreases hDAT cell surface binding, hDAT-associated currents, and the DAT Vmax with no change in Km (Vaughan et al. 1997; Zhu et al. 1997; Pristupa et al. 1998; Daniels and Amara 1999; Melikian and Buckley 1999). This inhibition of DA transport was associated with a rapid internalization of carriers from the plasma membrane (Daniels and Amara 1999; Melikian and Buckley 1999) mediated by the GTPase dynamin I (Daniels and Amara 1999). Similarly, we demonstrate here that inhibition of PI 3-kinase with LY294002 reduces DAT activity (Table 1) and decreases hDAT cell surface expression (Figs 3 and 6). The LY294002-induced hDAT cell surface redistribution was blocked by pre-incubation with concanavalin A (Fig. 4) and by expression of a dominant negative mutant of dynamin I (Fig. 5), suggesting that this inhibition in DA uptake may also occur via clathrin-mediated endocytosis. The possibility that the LY294002-induced inhibition of DAT activity only involves a modification of the transporter or an associated protein without a resulting alteration in cell surface expression of DAT seems unlikely, as the dominant negative mutant of dynamin I (Fig. 5) almost completely reversed both the LY294002-induced internalization and decreased uptake.

Our data suggest that altered trafficking of hDAT underlies the effects of wortmannin and LY294002 on basal DAT activity. It remains to be determined whether elevated PI 3-kinase activity stimulates hDAT cell surface expression and DA uptake by increasing the rate of DAT exocytosis and/or by diminishing its rate of endocytosis. It is known that insulin in adipocytes and muscles induces the translocation of intracellular GLUT4 glucose transporter containing vesicle to the plasma membrane (Cheatham and Kahn 1995). Our biotinylation analyses detected a non-significant increase in hDAT cell surface expression upon insulin stimulation (Fig. 6). Thus, our data do not eliminate the possibility that insulin stimulates DA transport by pathways that are independent of protein trafficking, as recently reported for the norepinephrine transporter (Apparsundaram et al. 2001).

Increasing PI 3-kinase activity by expression of the constitutively active mutant (Fig. 8b) reduced AMPH- induced hDAT cell surface redistribution. In addition, real time confocal microscopy of YFP-hDAT suggested that insulin reverses the cell surface redistribution of the hDAT induced by AMPH (Fig. 7). Blocking protein synthesis with cycloheximide did not alter this insulin effect. Therefore, it is likely that internalized hDAT returns to the cell surface upon stimulation with insulin. Melikian and Buckley have proposed a similar trafficking mechanism for protein kinase C (PKC)-mediated DAT cell surface redistribution. They suggested that internalized DAT traffics through the recycling endosome compartment back to the cell membrane (Melikian and Buckley 1999). Curiously, in MDCK cells a GFP-DAT was not recycled, suggesting that trafficking pathways are not identical in every cell (Daniels and Amara 1999).

These data suggest that PI 3-kinase activity and insulin stimulation regulate AMPH-induced trafficking of the hDAT. This novel hormonal regulation of DAT trafficking could explain why anorexia, stereotyped behavior, and locomotor activity induced by AMPH are markedly attenuated in diabetic (i.e. alloxan-treated) rats. This attenuation was reversed by subsequent administration of insulin (Marshall 1978). Similar results were obtained when rats received streptozotocin (Rowland et al. 1985; Carr et al. 2000), an anticancer drug that induces a diabetic condition. Therefore, our results could provide a mechanism for hormonal regulation of drug abuse, and suggest that the insulin signaling pathway may represent a new cellular target for substance abuse therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Acknowledgements
  5. References

We are grateful to Dr Mark Caron for the gift of wild-type dynamin and dynamin (K44A) cDNA. This work was supported by a NARSAD Young Investigator Award (AG), and by grants from the National Institute of Health (NIH) (DA13975, DA14684) (AG), and DA11495 and MH57324 (JAJ).

References

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