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Keywords:

  • TrkB;
  • biotinylation;
  • brain-derived neurotrophic factor;
  • dopamine transporter;
  • dopamine uptake;
  • tyrosine kinases

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Termination of dopamine neurotransmission is primarily controlled by the plasma membrane-localized dopamine transporter. In this study, we investigated how this transporter is regulated by tyrosine kinases in neuronal preparations. In rat dorsal striatal synaptosomes, inhibition of tyrosine kinases by genistein or tyrphostin 23 resulted in a rapid (5–15 min), concentration-dependent decrease in [3H]dopamine uptake because of a reduction in maximal [3H]dopamine uptake velocity and dopamine transporter cell surface expression. The reduced transporter activity was associated with a decrease in phosphorylated p44/p42 mitogen-activated protein kinases. In primary rat mesencephalic neuronal cultures, the tyrosine kinase inhibitors similarly reduced [3H]dopamine uptake. When cultures were serum-deprived, acute activation of tyrosine kinase-coupled TrkB receptors by 100 ng/mL brain-derived neurotrophic factor significantly increased [3H]dopamine uptake; the effects were complex with increased maximal velocity but reduced affinity. The facilitatory effect of brain-derived neurotrophic factor on dopamine transporter activity depended on both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Taken together, our results suggest that striatal dopamine transporter function and cell surface expression is constitutively up-regulated by tyrosine kinase activation and that brain-derived neurotrophic factor can mediate this type of rapid regulation.

Abbreviations used:
BDNF

brain-derived neurotrophic factor

BSA

bovine serum albumin

DA

dopamine

DAT

dopamine transporter

DIV

days in vitro

E15

embryonic day 15

GAT

GABA transporter

HBSS

Hank’s balanced saline solution

KRH

Krebs-Ringer HEPES buffer

MAPK

mitogen-activated protein kinase

MEK

mitogen-activated protein kinase kinase

NET

norepinephrine transporter

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PBS/Mg/Ca

PBS/1 mmol/L MgCl2/0.1 mmol/L CaCl2

PI3K

phosphatidylinositol 3-kinase

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

PP2A

protein phosphatase 2A-A

SDS

sodium dodecyl sulfate

SERT

serotonin transporter

SLC

solute carrier

STR

dorsal striatal

TH

tyrosine hydroxylase

TK

tyrosine kinase

TrkB

tropomysin-related kinase

TTBS

0.1% Tween-20/Tris-buffered saline

The neurotransmitter dopamine (DA) is essential for normal CNS functions, including motor activity, cognition, affect, and reward (Zahniser and Sorkin 2004; Bannon 2005). The DA transporter (DAT) provides a primary means for controlling the duration and magnitude of DA neurotransmission (Torres et al. 2003). The DAT is a member of the solute carrier (SLC)6 gene family of neurotransmitter transporters, which also includes the norepinephrine (NET), serotonin (SERT), and GABA (GAT) transporters (Gether et al. 2006). Functional DATs are localized on the plasma membrane of axons, pre-synaptic axon terminals, and dendrites of DA neurons (Nirenberg et al. 1996).

DAT activity and cell surface expression are regulated by exposure to DAT substrates, DAT inhibitors, and a variety of receptor signaling cascades, including protein kinases and phosphatases (Zahniser and Doolen 2001; Gulley and Zahniser 2003; Melikian 2004). Protein kinase C (PKC) is the most well-studied of the kinases, with PKC activation decreasing DAT activity through clathrin-mediated endocytosis (Daniels and Amara 1999; Sorkina et al. 2005), an event associated with an increase in DAT phosphorylation (Foster et al. 2006). Activation of PKC also triggers ubiquitination of the DAT, which leads to transporter internalization through an endocytotic mechanism mediated by the E3 ubiquitin ligase NEDD4-2 (Miranda et al. 2005; Sorkina et al. 2006).

Tyrosine kinases (TKs) are also well-situated to regulate the DAT. DA neurons express multiple receptor TKs, including tropomysin-related kinase (TrkB) and insulin receptors (Numan and Seroogy 1999; Figlewicz et al. 2003). TrkB receptors are activated through binding of brain-derived neurotrophic factor (BDNF; Huang and Reichardt 2003). As DA neurons also express BDNF, TrkB receptors can be activated in an autocrine or paracrine manner (Seroogy et al. 1994). Indeed, BDNF is a potent growth factor promoting DA neuronal survival and activity following chronic administration (Hyman et al. 1994; Shen et al. 1994). In addition, BDNF has a critical role in activity-dependent neuronal plasticity (Poo 2001). Recent studies (Horger et al. 1999; Grimm et al. 2003; Hall et al. 2003; Pu et al. 2006) suggest that BDNF’s effects on DA neurons and synaptic plasticity may converge to augment not only normal DA neurotransmission, but also cocaine-induced increases in DA-mediated synaptic and behavioral plasticity.

The report that TK inhibitors decrease DAT activity in dorsal striatal (STR) homogenates (Simon et al. 1997) suggests that TrkB and insulin receptors on DA neurons receive a basal tone, through which DAT activity is up-regulated. Moreover, TK inhibitor-induced reduction in DA transport is associated with a loss of cell surface DATs in DAT-expressing oocytes (Doolen and Zahniser 2001). However, the full extent to which TKs regulate DAT function, kinetics, and cell surface expression in brain preparations is not well understood. Recently, it was reported that acute insulin treatment, acting through phosphatidylinositol 3-kinase (PI3K) and Akt, up-regulate DAT activity through an increase in DAT cell surface expression (Carvelli et al. 2002; Garcia et al. 2005). Furthermore, using findings with other SLC6 transporters as a guide, TKs could regulate DAT activity via several mechanisms. Prasad et al. (1997) found that TK activation up-regulates SERT activity and cell surface expression. Furthermore, BDNF or insulin activation of receptor TKs increases the activity of GAT1, SERT, and NET, whereas TK inhibitors exert the opposite effect (Law et al. 2000; Apparsundaram et al. 2001; Gil et al. 2003; but see Mossner et al. 2000). Interestingly, however, the insulin-mediated increase in NET activity is not because of enhanced transporter insertion into the plasma membrane (Apparsundaram et al. 2001), which is the mechanism by which insulin up-regulates DAT activity (Carvelli et al. 2002; Garcia et al. 2005).

TK agonists such as BDNF primarily activate two signal transduction pathways in neurons: (i) mitogen-activated protein kinases (MAPKs) and (ii) PI3K (Huang and Reichardt 2003). MAPKs are serine-threonine kinases activated by MAPK kinase (MEK) and play key roles in neuron proliferation and differentiation, as well as synaptic and behavioral plasticity (Sweatt 2001; Licata and Pierce 2003). PI3K also plays a significant role in neuron survival and differentiation, especially through activation of its downstream serine–threonine kinase Akt (Huang and Reichardt 2003). Recent reports have demonstrated that DAT activity and cell surface expression are positively regulated by constitutive MAPK and PI3K/Akt activity (Carvelli et al. 2002; Lin et al. 2003; Moron et al. 2003; Garcia et al. 2005).

The purpose of the present study was to understand whether TK inhibition and acute BDNF-mediated activation rapidly regulates DAT kinetics, uptake activity, and/or cell surface expression in brain preparations (STR synaptosomes and primary mesencephalic neurons). In addition, the involvement of the MAPK and PI3K signaling pathways in this regulation was examined.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Male (150–200 g; 6–8 weeks old) and timed pregnant female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA), housed on a 12-h light/dark cycle with ad libitum food and water, were used in these experiments. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center.

Materials

Chemicals were purchased from either RBI/Sigma (St. Louis, MO, USA) or Pierce Chemical (Rockford, IL, USA), with the following exceptions: (−)-cocaine HCl was obtained from the National Institute on Drug Abuse (Research Triangle Institute International, Research Triangle Park, NC, USA), BDNF and LY294002 were obtained from EMD Biosciences (San Diego, CA, USA), K252a was obtained from Tocris Bioscience (Ellisville, MO, USA), U0126 was obtained from Cell Signaling Technology (Beverly, MA, USA), Ham’s F12 and fetal bovine serum were obtained from Hyclone (Logan, UT, USA) or Gibco/Invitrogen (Invitrogen Corp., Carlsbad, CA, USA), and [3H]DA (specific activity of 39–60 Ci/mmol) and [3H]alanine (specific activity of 85 Ci/mmol) were purchased from Perkin-Elmer Life Sciences (Boston, MA, USA). Primary antibodies used included: mouse monoclonal DAT (generous gift from Dr Roxanne Vaughan, University of North Dakota, Grand Forks, ND, USA), rabbit polyclonal TrkB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal tyrosine hydroxylase (TH; Immunostar Inc., Hudson, WI, USA), mouse monoclonal protein phosphatase 2A-A (PP2A; Santa Cruz Biotechnology), rabbit polyclonal phospho Akt (Ser473) and Akt, and mouse monoclonal phospho p42/44 MAPK (Thr202/Tyr204) and rabbit polyclonal p44/42 MAPK (Cell Signaling Technology, Beverly, MA, USA).

Preparation of dorsal striatal synaptosomes

Following decapitation, brains from male rats were rapidly removed, and both STR hemispheres were dissected. STR tissue was homogenized in 2 mL of ice-cold phosphate buffer (3.3 mmol/L NaH2PO4 + 12.7 mmol/L Na2HPO4) containing 0.32 mol/L sucrose (pH 7.4) with a glass/Teflon homogenizer. The sucrose buffer used to homogenize synaptosomes for DAT biotinylation experiments also contained 1 mmol/L EDTA, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 0.1 mmol/L aminoethylbenzenesulfonyl fluoride hydrochloride. Homogenization was followed by centrifugation at 1000 g for 12 min at 4°C. Next, the supernatant was centrifuged at 12 500 g to isolate the P2 pellet that was resuspended at either 15 mg/mL (wet weight of tissue for [3H]ligand uptake and/or MAPK westerns) or 100 mg/mL (wet weight of tissue for DAT biotinylation and concomitant [3H]DA uptake) and pre-incubated with an appropriate concentration of drug(s) for 5–30 min at 37°C. For determination of the drugs’ concentration responses, time courses, and kinetic analyses, uptake of [3H]DA into the synaptosomes was then measured.

For the MAPK western blot and DAT biotinylation experiments, the STR synaptosomes were further processed. Synaptosomes used to measure phospho and total p44/p42 MAPK levels after drug pre-treatment were split. One aliquot was used to measure drug-induced changes in [3H]DA uptake and the other aliquot was mixed with 1X protease inhibitor cocktail (RBI/Sigma) supplemented with the phosphatase inhibitors NaF and Na3VO4 (1 mmol/L each), centrifuged at 12 500 g for 15 min at 4°C, and then resuspended in STE buffer (1% sodium dodecyl sulfate [SDS], 1 mmol/L EDTA, 10 mmol/L Tris, pH 8.0). Samples were briefly sonicated and stored at −20°C until western blot analysis. The protein concentration was determined with the BCA protein assay kit (Pierce Chemical) using bovine serum albumin (BSA) as the standard. The STR synaptosomes used to measure total and cell surface-localized DAT were also split after drug pre-treatment. One sample was used to measure drug-induced changes in [3H]DA uptake and the second was further processed for cell surface biotinylation of DAT.

[3H]ligand uptake into dorsal striatal synaptosomes

Following drug pre-treatment, STR synaptosomes were incubated with assay buffer (in mmol/L: 134 NaCl, 4.8 KCl, 1.3 CaCl2, 1.4 MgSO4, 3.3 NaH2PO4, 12.7 Na2HPO4, 11 glucose, and 1 ascorbic acid, pH 7.4) containing 1 μmol/L pargyline for 10 min at 37°C before addition of [3H]DA or [3H]alanine at a final concentration of 0.5 or 10 nmol/L, respectively. For kinetic analysis, unlabeled DA (5, 10, 50, 100, 200, and 500 nmol/L) was added concomitantly with the [3H]DA. The uptake assay was performed for 3 min at 37°C. Non-specific uptake of [3H]DA was determined with 1 mmol/L cocaine. Non-specific uptake of [3H]alanine was determined in samples incubated at 4°C. The assay was stopped by placing the samples on ice, adding 3 mL of ice-cold 0.32 mol/L sucrose solution and filtering through Whatman GF/C glass microfiber filters with a cell harvester. The filters were washed twice more with the sucrose solution before radioactivity was determined by liquid scintillation spectrometry. Synaptosomal protein concentrations were determined according to the method of Bradford (1976), using BSA as the standard.

Cell surface biotinylation of dopamine transporter in dorsal striatal synaptosomes

Levels of total and cell surface-localized DAT were determined in STR synaptosomes using the membrane impermeable biotinylation reagent sulfo-NHS-biotin as described previously (Zhu et al. 2005b) with the following exceptions. Synaptosomes were incubated with 2 mg/mL EZ-Link sulfo-NHS-biotin (Pierce Chemical) for 45–60 min at 4°C with constant shaking. The biotin was inactivated with a 10-min incubation in 1.0 mol/L glycine in phosphate-buffered saline (PBS)/Ca/Mg buffer (in mmol/L: 138 NaCl, 2.7 KCl, 1.5 KH2PO4, 9.6 Na2HPO4, 0.1 CaCl2, 1 MgCl2, pH 7.3) at 4°C. Synaptosomes were lysed with 1% Triton X-100 buffer (10 mmol/L Trizma base, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mL Triton X-100, 0.1% SDS, 1% Na deoxycholate, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.1 mmol/L aminoethylbenzenesulfonyl fluoride hydrochloride, pH 7.4) for 30 min at 4°C before centrifugation at 20 000 g for 30 min at 4°C. Lysate was incubated overnight at 4°C with freshly prepared monomeric avidin beads as per the manufacturer’s instructions (Pierce Chemical). Biotinylated proteins were eluted with Lammeli buffer (62.5 mmol/L Trizma base, pH 6.8, 20% glycerol, 2% SDS, 5%β-mercaptoethanol, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.1 mmol/L aminoethylbenzenesulfonyl fluoride hydrochloride) for 1 h at 22°C–25°C. Samples were stored at −20°C until western blot analysis.

Western blot analysis of dorsal striatal synaptosomes

Mitogen-activated protein kinase

Dorsal striatal synaptosomes were thawed and 5 μg aliquots were mixed with loading buffer (62.5 mmol/L Trizma base, pH 6.8, 2% SDS, 10% glycerol, 5%β-mercaptoethanol, trace of bromophenol blue). Before loading, samples were heated to 100°C for 5 min and then separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes (Perkin-Elmer Life Sciences) using the Genie transfer apparatus (Idea Scientific, Minneapolis, MN, USA). Membrane blots were blocked for 1 h at 22°C–25°C in 5% non-fat dry milk in 0.1% Tween 20/Tris-buffered saline (TTBS; 140 mmol/L NaCl, 20 mmol/L Trizma base, pH 7.6). After blocking, membranes were incubated with an antibody directed against phospho or total p44/p42 MAPK overnight at 4°C. The next day, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at 22°C–25°C. Immunoreactive bands were visualized by enhanced chemiluminescence (Pierce Chemical) on X-ray film.

Dopamine transporter

Total and biotinylated DAT samples prepared from STR synaptosomes were thawed and subjected to western blot analysis as described above, with the following exceptions. Equal volumes of each sample were heated to 74°C for 5 min before loading, and following SDS-PAGE and transfer, blots were blocked at 22°C–25°C in 3% BSA in TTBS for 1.5 h, followed by 5% non-fat dry milk in TTBS for 30 min. To confirm that the sulfo-NHS-biotin did not label intracellular proteins in the STR synaptosomes, DAT blots were stripped for 1 h at 60°C with Restore western blot stripping buffer (Pierce Chemical), re-probed with an antibody directed against PP2A, and then processed as described above.

Primary neuronal co-cultures

Timed pregnant dams were deeply anesthesized with an intramuscular injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) on embryonic day (E)15, and fetuses were rapidly removed from the uterus and placed in ice-cold Hank’s balanced saline solution (HBSS). Following the protocols of Dunnett and Bjorklund (1992), the striatum and mesencephalon were removed under aseptic conditions, washed in HBSS, triturated, centrifuged at 300 g for 5 min at 10°C, and resuspended in Ham’s F12 supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin. Following filtration through a 70 μm cell strainer, mesencephalic and striatal neurons were counted by trypan blue exclusion and plated together at a density of 120 000 or 240 000 cells/cm2 and 30 000 or 60 000 cells/cm2, respectively, onto 24-well plates (uptake) or 12-well plates containing glass coverslips (immunocytochemistry). For western blot analyses, mesencephalic and striatal neurons were plated together at a density of 240,000 cells/cm2 and 60,000 cells/cm2, respectively, onto 12-well plates. Plates had previously been coated with 0.2% polyethylenimine in 0.2 M borate buffer (300 mL 0.2 M boric acid + 200 mL 0.05 M sodium tetraborate, pH 8.3). Co-culturing dissociated mesencephalic and striatal neurons has been reported to positively influence DA neuron survival, growth, and uptake activity (Prochiantz et al. 1981; Schinelli et al. 1993). Neuronal co-cultures were maintained in supplemented Ham’s F12 with 5% CO2 at 37°C. Medium was changed every 2–3 days. At 5-9 days in vitro (DIV), neurons were used either for measurement of [3H]DA uptake, fluorescent immunocytochemical detection of TH (marker for DA neurons) and TrkB receptors, or for western blot detection of phospho and total p44/p42 MAPK and Akt.

[3H]DA uptake into dissociated mesencephalic neurons

Uptake assays involving BDNF were preceded by a 2-h serum deprivation during which the neurons continued to be incubated at 37°C, but with 10% pre-conditioned medium supplemented with Ham’s F12, 2 mmol/L glutamine, and streptomycin/penicillin. All other uptake assays with the neuronal co-cultures remained in serum-containing media. Subsequently, neurons were treated with drug(s) for 5–30 min at 37°C. The neurons were rinsed and then assayed in Krebs-Ringer HEPES buffer (KRH; in mmol/L: 120 NaCl, 4.7 KCl, 2.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 glucose, 10 HEPES, pH 7.4) supplemented with 10 μmol/L pargyline, 10 μmol/L ascorbic acid, and 10 μmol/L catechol. Assays (0.5 mL) included 50 or 100 nmol/L [3H]DA. For kinetic analysis, unlabeled DA (200, 500, 1000, 2000, and 4000 nmol/L) was added concomitantly with the [3H]DA. Non-specific [3H]DA accumulation was determined in the presence of 1 mmol/L cocaine. After 10 min of incubation at 37°C, uptake was terminated by quickly washing the neurons three times with 1 mL of ice-cold KRH. Neurons were then solubilized in 0.5 mL of 3% trichloroacetic acid for 60 min with gentle shaking. Radioactivity was determined by liquid scintillation counting.

Western blot analysis of primary neuronal co-cultures

At six DIV, neuronal cultures were treated with drug for 30 min at 37°C. Neurons were then treated with KRH lysis buffer (in mmol/L: 1 EDTA, 1 EGTA, 1 NaF, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 10% glycerol, 0.1 Na3VO4, 20 disodium p-nitrophenylphosphate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mmol/L aminoethylbenzenesulfonyl fluoride hydrochloride). Neurons were harvested with a cell scraper and incubated on ice for 15 min. Cellular debris was removed by centrifugation for 10 min at 12 000 g. Protein concentrations were determined with the BCA protein assay kit (Pierce Chemical) using BSA as the standard. Equal amounts of protein (5 μg per lane) were resolved by SDS-PAGE and membrane blots were then probed for phospho and total MAPK as described above or for phospho and total Akt. Membrane blots probed for Akt were processed similarly to MAPK with the following exception: the primary antibody was directed against phospho or total Akt.

Fluorescence immunocytochemistry and microscopy

At 7–9 DIV, the mesencephalic-striatal neuronal co-cultures grown on glass coverslips were fixed with 4% paraformaldehyde for 30 min at 22°C–25°C, followed by three rinses in PBS supplemented with 1 mmol/L MgCl2 and 0.1 mmol/L CaCl2 (PBS/Mg/Ca). Cultures were then pre-incubated in PBS/Mg/Ca containing 1% BSA and 0.05% saponin for 30 min at 22°C–25°C, followed by incubation in PBS/Mg/Ca containing 1% BSA, 0.01% saponin and primary antibodies directed against TH (1:500) and TrkB (1:100) for 1 h at 22°C–25°C. Neurons were then rinsed and incubated with either a CY3-labeled anti-mouse secondary antibody for TH or FITC-labeled anti-rabbit secondary antibody for TrkB (Jackson Laboratories, West Grove, PA, USA) for 30 min at 22°C–25°C. Following three rinses in PBS/Mg/Ca, neurons were mounted onto slides with Mowiol (EMD Biosciences) and stored at 4°C until microscopic analysis.

Images were acquired through CY3 and FITC filter channels using a MarianasTM Imaging workstation and SlideBook 4.1 software (Intelligent Imaging Innovation, Inc., Denver, CO, USA). Final arrangement of all images was performed using Adobe Photoshop.

Data analysis

Data are presented as mean ± SEM, unless otherwise noted. Significant differences were defined as those with p < 0.05. Drug-induced alterations in [3H]DA uptake were analyzed by one-way anova followed by Holm-Sidak post hoc tests. Substrate affinity (Km) and maximal uptake velocity (Vmax) values were estimated from the [3H]DA uptake kinetic experiments by non-linear regression analysis using Prism software (GraphPad, San Diego, CA, USA). Drug-induced differences in Km and Vmax were analyzed by paired t-tests. Densitometric analyses of X-ray films of the western blots were performed using AlphaEase software (version 5.5; Alpha Innotech Corp., San Leandro, CA, USA) to determine the linear range of the chemiluminescence signals. For western blot analyses of biotinylated DAT, densitometry values were obtained for the total and biotinylated DAT signals for each condition. The densitometry value for each biotinylated DAT band was normalized to its respective total band to account for differences in amount of loaded protein. Data are expressed as biotinylated/total DAT signal within each condition. These values were analyzed by one-way anova followed by Holm-Sidak post hoc tests. For the MAPK and Akt western blot analyses, densitometry values were obtained for the total and phospho p44/p42 MAPK or Akt signals for each condition. The densitometry value for each phospho p44/p42 MAPK or Akt band was normalized to its respective total p44/p42 MAPK or Akt band to account for differences in amount of loaded protein. Data are expressed as a percent of control for phospho/total MAPK or phospho/total Akt. These values were analyzed by one-way anova followed by Holm-Sidak post hoc tests.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tyrosine kinase inhibitors and [3H]DA uptake into dorsal striatal synaptosomes

To examine the role of TKs in the rapid functional regulation of the DAT in rat STR synaptosomes, the concentration and temporal characteristics of the non-selective TK inhibitor genistein on [3H]DA uptake were first investigated. Genistein (10–50 μmol/L) decreased specific uptake of [3H]DA into synaptosomes in a concentration- and time-dependent manner (Figs 1a and b). Pre-incubation with either 25 or 50 μmol/L genistein for 12 min at 37°C significantly reduced [3H]DA uptake by 35% or 52%, respectively (Fig. 1a). Pre-incubation of the synaptosomes with 50 μmol/L genistein for 5–30 min rapidly and significantly reduced [3H]DA uptake (Fig. 1b). Within 5 min of exposure, 50 μmol/L genistein significantly reduced [3H]DA uptake by 20%, reaching a maximal inhibition of 53% by 12 min. At 20 min, 50 μmol/L genistein still significantly reduced [3H]DA uptake by 26%. However, after a 30-min pre-incubation with 50 μmol/L genistein, [3H]DA uptake no longer differed significantly from control.

image

Figure 1.  Pre-treatment with the tyrosine kinase inhibitors genistein and tyrphostin 23 reduced specific uptake of [3H]DA into rat dorsal striatal synaptosomes in a concentration- and time-dependent manner that was non-additive. In each set of experiments, following the specified pre-treatment and washing by centrifugation, specific uptake of 0.5 nmol/L [3H]DA was assayed for 3 min at 37°C. Non-specific uptake was defined in the presence of 1 mmol/L cocaine. The horizontal dashed line delineates the level of control. (a) Log-dose effect curves for synaptosomes (n = 3–11) pre-incubated with genistein (10, 25, 50 μmol/L) for 12 min or tyrphostin 23 (25, 50, 100, 200 μmol/L) for 15 min at 37°C. (b) Time course of genistein- and tyrphostin 23-mediated down-regulation of [3H]DA uptake. Synaptosomes (n = 4–9) were pre-incubated with 50 μmol/L genistein or 200 μmol/L tyrphostin 23 for various times at 37°C. Separate synaptosomal preparations were used for each incubation time point. (c) Non-additivity of genistein and tyrphostin 23. Synaptosomes (n = 3) were pre-treated with 50 μmol/L genistein and/or 200 μmol/L tyrphostin 23 either alone, or in combination, for 15 min at 37°C. *p < 0.05 compared with control, post hoc Holm-Sidak test.

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In addition to its inhibition of TKs (Akiyama et al. 1987), genistein can affect many intracellular activities, including apoptosis, estrogen signaling and the formation and maintenance of actin-based protrusions such as growth cones (Goldberg and Wu 1995; Kuiper et al. 1998; Linford et al. 2001). To rule out the involvement of non-TK actions of genistein, the effects of the structural analogs daidzein and genistin on [3H]DA uptake were studied. Like genistein, daidzein also activates estrogen receptors, but it does not affect TKs (Kuiper et al. 1998; Linford et al. 2001). At a concentration (100 μmol/L) previously used to examine genistein’s potential effects on estrogen receptors (Law et al. 2000; Linford et al. 2001), daidzein did not significantly reduce specific [3H]DA uptake (94 ± 9% of control; n = 3). Genistin is an inactive analog of genistein at TKs. Genistin (50 μmol/L) also did not significantly affect specific [3H]DA uptake (102 ± 9% of control; n = 3).

To compare geinstein’s effects on specific [3H]DA uptake into synaptosomes to those of a selective TK inhibitor, the effects of pre-incubation with tyrphostin 23 on specific [3H]DA uptake were examined. Tyrphostin 23 (25–200 μmol/L) also significantly decreased [3H]DA uptake into STR synaptosomes in a concentration- and time-dependent manner (Figs 1a and b). Pre-incubation for 15 min at 37°C with 100 or 200 μmol/L tyrphostin 23 significantly reduced [3H]DA uptake by 25% or 40%, respectively (Fig. 1a). Pre-incubation of the synaptosomes with 200 μmol/L tyrphostin 23 for 5–30 min rapidly and significantly reduced [3H]DA uptake (Fig. 1b). Within 5 min of exposure, 200 μmol/L tyrphostin 23 reduced [3H]DA uptake by 48%, reaching a maximal inhibition of 59% by 15 min. In contrast with genistein, the level of DAT inhibition remained stable following a 30-min exposure to 200 μmol/L tyrphostin 23. As a further test of genistein’s mechanism of action, synaptosomes were pre-incubated with both 50 μmol/L genistein and 200 μmol/L tyrphostin 23. If genistein affected [3H]DA uptake via non-TK pathways, then pre-incubation of the synaptosomes with genistein and tyrphostin 23 should reduce specific [3H]DA uptake to a greater extent than with either drug alone. In these experiments, pre-treatment of STR synaptosomes with 50 μmol/L genistein or 200 μmol/L tyrphostin 23 significantly reduced [3H]DA uptake by 40% or 53%, respectively (Fig. 1c). [3H]DA uptake into synaptosomes pre-exposed to both TK inhibitors was significantly reduced by 57%, but this reduction was not different from that induced by either inhibitor alone. Together, our results strongly argue that genistein’s effect on [3H]DA uptake was through selective inhibition of TKs.

Next, kinetic analyses were performed to determine if TK inhibitor-induced decreases in [3H]DA uptake were due to a reduction in Vmax or Km. Pre-treatment with either 50 μmol/L genistein (12 min; Fig. 2a and Table 1) or 200 μmol/L tyrphostin 23 (15 min; Table 1) significantly reduced Vmax by 29% or 70%, respectively, versus control. Neither drug significantly altered Km.

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Figure 2.  Pre-treatment of dorsal striatal synaptosomes with tyrosine kinase inhibitors decreased the maximal velocity of specific [3H]DA uptake and dopamine transporter (DAT) cell surface expression. (a) Synaptosomes (n = 3) were pre-treated with 50 μmol/L genistein and uptake was measured as in Fig. 1, with the exception that a range of unlabeled dopamine concentrations (5–500 nmol/L) and 0.5 nmol/L [3H]DA were used. See Table 1 for Km and Vmax values, determined by non-linear regression analysis. (b and c) Synaptosomes (n = 3–5) were pre-treated with 50 μmol/L genistein or 200 μmol/L tyrphostin 23 for 15 min at 37°C; then samples were split and used for DAT biotinylation (see Materials and methods) and [3H]DA uptake (see Fig. 1) assays. DAT cell surface expression was calculated as a ratio of biotinylated/total DAT for each sample. (b) Representative blot of the total (T) and biotinylated (B) DAT signals across control- and TK inhibitor-treated conditions. (c) TK inhibitor-induced reductions in DAT cell surface expression. The horizontal dashed line delineates the level of control. Aliquots from each condition were assayed for specific [3H]DA uptake to confirm a significant drug-induced reduction in [3H]DA uptake: genistein, 72 ± 5% of control; tyrphostin 23, 66 ± 10% of control. *p < 0.05 compared with control, post hoc Holm-Sidak test.

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Table 1.   Effect of an acute pre-incubation with tyrosine kinase inhibitors on kinetic parameters of specific [3H]DA uptake into dorsal striatal synaptosomes
ConditionKm (nmol/L)Vmax (pmol/min/mg protein)
  1. *p < 0.05, compared with control. n = 3–4. See Materials and methods for details.

Control63 ± 1185 ± 3
50 μmol/L Genistein52 ± 460 ± 8*
Control100 ± 1057 ± 14
200 μmol/L Tyrphostin 2382 ± 1418 ± 7*

To insure that the TK inhibitors were not affecting synaptosomal membrane potential or ion gradients in general, uptake of 10 nmol/L [3H]alanine was measured following pre-incubation of STR synaptosomes with either 50 μmol/L genistein or 200 μmol/L tyrphostin 23 under the same conditions used to study [3H]DA uptake. Neither drug significantly altered [3H]alanine uptake, compared with control (genistein, 84 ± 6% of control and tyrphostin 23, 97 ± 6% of control; n = 3).

Regulation of dopamine transporter cell surface expression by tyrosine kinase inhibitors

To examine if the reduction in Vmax induced by TK inhibitors was because of loss of DAT from the synaptosomal plasma membrane, we used a surface biotinylation assay. In these experiments, total (biotinylated and non-biotinylated) and biotinylated DATs were measured in synaptosomes pre-treated with either 50 μmol/L genistein or 200 μmol/L tyrphostin 23 (Figs 2b and c). In addition, an aliquot of the same synaptosomal preparation was used to determine the drug-induced decrease in [3H]DA uptake. Genistein and tyrphostin 23 significantly reduced specific [3H]DA uptake by 28% and 34%, respectively, compared with control (Fig. 2 legend). Coincident with the reduction in [3H]DA uptake, genistein and tyrphostin 23 significantly decreased DAT cell surface expression by 23% and 27%, respectively (Fig. 2c). Examination of the percent of biotinylated/total DAT under control conditions revealed that at least 28 ± 5% of DAT was located at the cell surface (data not shown). The western blots were re-probed for the intracellular protein PP2A. PP2A was detected only in the synaptosomal samples containing non-biotinylated proteins (data not shown), confirming that intracellular proteins were not biotinylated.

Regulation of phospho p44/p42 MAPK levels by tyrosine kinase inhibitors

Constitutive and stimulated TK activity can result in the activation of multiple downstream effectors, including the p44/p42 MAPK (ERK 1/2) isoforms of the MAPK signaling cascade. Thus, we next examined if the MAPK pathway is involved in the reduction of [3H]DA uptake by TK inhibitors. Similar to a previous report (Moron et al. 2003), we observed phospho p44/p42 MAPK in vehicle-treated STR synaptosomes (Fig. 3a). A 15-min pre-exposure of synaptosomes to either 50 μmol/L genistein or 200 μmol/L tyrphostin 23 significantly decreased the levels of phospho MAPK, compared with control. Genistein and tyrphostin 23 reduced phospho levels of the p44 isoform by 45% and 68%, respectively. Additionally, genistein and tyrphostin 23 reduced phospho levels of the p42 isoform by 45% and 63%, respectively (Figs 3a, b). In contrast, neither TK inhibitor significantly altered levels of total MAPK. These decreases in phospho MAPK levels paralleled significant decreases in specific [3H]DA uptake in samples of the same synaptosomal tissue. Pre-treatment of STR synaptosomes with 50 μmol/L genistein or 200 μmol/L tyrphostin 23 reduced [3H]DA uptake by 28% and 55%, respectively, compared with control (Fig. 3 legend).

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Figure 3.  Pre-treatment of dorsal striatal synaptosomes with either genistein or tyrphostin 23 decreased phospho p44/p42 MAPK levels. Synaptosomes (n = 3–7, except p44 isoform pre-treated with genistein, n = 2, mean ± SD shown) were pre-treated with 50 μmol/L genistein or 200 μmol/L tyrphostin 23 for 15 min at 37°C. Synaptosomes were then either centrifuged and resuspended in lysis buffer for western blot analysis or used to measure specific [3H]DA uptake, as described in Fig. 1. Blots were probed for both total and phospho p44/p42 mitogen-activated protein kinase (MAPK), and phospho MAPK levels were normalized to total MAPK levels. (a) Representative blot of phospho p44/p42 MAPK under control- and drug-treated conditions. (b) Summary of the drug-induced changes in phospho MAPK levels. The horizontal dashed line delineates the level of control. Significant reductions in specific [3H]DA uptake after pre-treatment of: genistein, 72 ± 3% of control; tyrphostin 23, 45 ± 3% of control. *p < 0.05 compared with control, post hoc Holm-Sidak test.

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Tyrosine kinase agonists and [3H]DA uptake into dorsal straital synaptosomes

Since inhibition of TKs leads to decreased [3H]DA uptake into STR synaptosomes, we investigated whether TK activation by BDNF increased uptake of [3H]DA. Initial experiments pre-treating synaptosomes with 10–200 ng/mL BDNF resulted in robust increases in specific uptake of [3H]DA (178 ± 17% of control; n = 3) that was TK-, MEK 1/2-, and PI3K-dependent (data not shown). However, as we further repeated these initial experiments, we observed that BDNF’s effects were inconsistent, ranging from no change in uptake to increases as large as 100% above control levels. BDNF up-regulation of [3H]DA uptake could be opposed by BDNF-stimulated release of DA (Blochl and Sirrenberg 1996), which could rapidly down-regulate [3H]DA uptake (Chi and Reith 2003). If DA release were antagonizing BDNF’s facilitatory effects, we reasoned that depletion of vesicular DA might unmask the growth factor’s effects. Pre-treatment of rats with 5 mg/kg reserpine reduces STR DA levels by ∼95% 15–17 h post-injection (Corrodi and Fuxe 1967). However, this reserpine pre-treatment 24 h prior to synaptosomal preparation and 10 ng/mL BDNF pre-treatment failed to resolve the inconsistencies in BDNF’s effects on [3H]DA uptake into STR synaptosomes (104 ± 10% of control, n = 2; mean ± SD).

Initial characterization of primary mesencephalic-striatal co-cultured neurons

In order to better control the DAT milieu, our remaining studies were carried out in primary mesencephalic and striatal neurons from E15 rat embryos co-cultured together for 7–9 DIV. BDNF has been reported to affect DA release in mesencephalic neurons (Blochl and Sirrenberg 1996). In the present study, variability in BDNF regulation of [3H]DA uptake into primary mesencephalic neurons was still observed. However, it was not as great as that observed in synaptosomes. In particular, we observed consistent BDNF-induced increases in specific [3H]DA uptake when the neuronal co-cultures were serum-deprived for 2 h (see below).

To verify that DA neurons (identified by TH immunocytochemistry) in the primary mesencephalic-striatal neuronal co-cultures expressed TrkB receptors, double fluorescent immunocytochemistry for TH (Figs 4a and d) and TrkB (Figs 4b and e) was performed on neurons at 7-9 DIV. We found that while TrkB + neurons far outnumbered TH + neurons, every TH + neuron was also immunopositive for TrkB (Figs 4c and f).

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Figure 4.  Tyrosine hydroxylase (TH)-positive embryonic rat mesencephalic neurons expressed TrkB receptors. Fluorescent immunocytochemistry was used to detect TH and TrkB receptors in dissociated mesencephalic-striatal neurons co-cultured for 7–9 DIV and incubated with mouse anti-TH and rabbit anti-TrkB primary antibodies, followed by visualization of TH with CY3-labeled anti-mouse secondary antibodies (a, d) and TrkB with FITC-labeled anti-rabbit secondary antibodies (b, e). See Materials and methods for details. Merging of the TH and TrkB immunoreactivities are displayed in c and f. Yellow neurons indicate co-expression of TH and TrkB receptors. Images were collected at either 10X (a–c) or 63X (d–f) magnification. Scale bars: 100 μm (a–c), 10 μm (d–f).

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To test DAT function and regulation in serum-containing co-cultures of mesencephalic-striatal neurons, specific [3H]DA uptake was measured following drug pre-treatments similar to those used to assay DAT function in STR synaptosomes. First, however, phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC was tested. Pre-incubation for 30 min at 37°C with 100 nmol/L or 1 μmol/L PMA significantly reduced [3H]DA specific uptake into mesencephalic neurons by 24% or 32%, respectively (Fig. 5a). Besides PKC, TKs also regulated [3H]DA uptake into the mesencephalic neurons. Specific uptake of [3H]DA was reduced by TK inhibitors in a concentration-dependent manner (Fig. 5b). Specifically, a 30-min pre-treatment with genistein (25 or 50 μmol/L) significantly reduced [3H]DA uptake by 74% or 71%, respectively. Likewise, tyrphostin 23 (10–50 μmol/L) pre-treatment for 30 min also significantly reduced [3H]DA uptake by 36–67%. As a positive control, we used western blot analysis to examine the biochemical effect of the TK inhibitors on phosphoproteins in the neuronal cultures. Three different tyrosine-phosphorylated proteins (∼55, 120, 155 kDa) were detected in the cultures, with both TK inhibitors decreasing the 155 kDa tyrosine-phosphorylated protein (data not shown).

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Figure 5.  Protein kinase C activation, as well as selective tyrosine kinase (TK), mitogen-activated protein kinase kinase (MEK) 1/2, and phosphatidylinositol 3-kinase (PI3K) inhibition, decreased specific [3H]DA uptake into dissociated mesencephalic neurons. In each set of experiments, following pre-incubation with drug(s) for 30 min at 37°C in serum-containing media, specific uptake of 50 or 100 nmol/L [3H]DA was assayed for 10 min at 37°C. Non-specific uptake was defined in the presence of 1 mmol/L cocaine. (a) Mesencephalic-striatal neuronal co-cultures (5–7 DIV) were pre-incubated with phorbol 12-myristate 13-acetate (PMA). (b) Log concentration-effect for pre-incubation of neurons (7–9 DIV) with genistein or tyrphostin 23. (c) Neurons (7–9 DIV) were pre-incubated with the MEK 1/2 inhibitor U0126 or PI3K inhibitor LY294002. (d) To demonstrate that the MEK 1/2 and PI3K inhibitors decreased constitutive kinase activity, neurons (6 DIV) were pre-incubated with 10 μmol/L U0126 or LY294002 for 30 min at 37°C in serum-containing media and prepared for western blot analysis of phospho and total MAPK or Akt, respectively (see Materials and methods). The horizontal dashed line delineates the level of control. For uptake experiments, n = 3–7 (PMA and all inhibitors, except 500 nmol/L of each TK inhibitor and 1-10 μmol/L genistein, n = 2, mean ± SD shown) separate experiments, each done in duplicate wells. For western blot analysis, n = 1. *p < 0.05 compared with respective control, post hoc Holm-Sidak test. #p < 0.05 compared with 10 μmol/L U0126, post hoc Holm-Sidak test.

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The MEK 1/2 inhibitor U0126 and PI3K inhibitor LY294002 also decreased specific uptake of [3H]DA into the mesencephalic neurons in a concentration-dependent manner (Fig. 5c). A 30-min pre-treatment with U0126 (0.5–10 μmol/L) significantly reduced uptake by 9–37%, whereas a similar pre-treatment with LY294002 (0.5–10 μmol/L) significantly reduced uptake by 17–57%. Furthermore, combined pre-treatment with 10 μmol/L of each inhibitor significantly decreased [3H]DA uptake by 60%, which was statistically greater than the effect produced by 10 μmol/L U0126 alone, but not by 10 μmol/L LY294002 (Fig. 5c). To confirm biochemically the pharmacological action of the MEK 1/2 and PI3K inhibitors, western blot analyses of mesencephalic neurons were performed to measure phospho p44/p42 MAPK and Akt, respectively. A 30-min pre-treatment with 10 μmol/L U0126 or LY294002 markedly reduced phospho p44/p42 MAPK or Akt, respectively (Fig. 5d).

Tyrosine kinase agonists and [3H]DA uptake into primary mesencephalic-striatal co-cultured neurons

To begin to examine the role of TK activation in the regulation of [3H]DA uptake into rat primary co-cultures of mesencephalic-striatal neurons, the concentration and temporal characteristics of BDNF’s effects were investigated at 7–9 DIV in serum-deprived neurons. Following a 2-h serum deprivation, a 30-min pre-incubation with 100 ng/mL BDNF significantly increased specific uptake of [3H]DA into mesencephalic neurons by 38% (Fig. 6a). At concentrations less than 100 ng/mL, BDNF exerted variable effects on [3H]DA uptake, whereas at 200 ng/mL, BDNF did not further increase uptake (data not shown). In contrast with the 30-min pre-treatment, pre-incubating the neurons with 100 ng/mL BDNF for 10 or 20 min produced smaller non-significant increases in [3H]DA uptake (Fig. 6a). The BDNF-mediated up-regulation of [3H]DA uptake was blocked by the TrkB receptor antagonist K252a (100 nmol/L), which had no effect on its own (Fig. 6b). Pre-incubation with 100 ng/mL BDNF and 500 nmol/L of either U0126 or LY294002 also completely blocked BDNF’s up-regulation of [3H]DA uptake (Fig. 6b), suggesting that the MAPK and PI3K cascades are involved in the BDNF-induced up-regulation observed after a 2-h serum deprivation. Under this serum deprivation (2 h) condition, none of the inhibitors by themselves altered [3H]DA uptake, versus control (Fig. 6b). To examine if the BDNF-induced increase in [3H]DA uptake was because of a change in DAT velocity or affinity, kinetic analyses were performed (Fig. 6c; Table 2). At 100 ng/mL, BDNF pre-treatment for 30 min significantly increased not only Vmax by 27%, but also Km by 142% (decreased affinity), versus control.

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Figure 6.  Time course, signaling cascades, and kinetic analysis for brain-derived neurotrophic factor (BDNF)-mediated up-regulation of specific [3H]DA uptake into dissociated mesencephalic neurons that were serum-deprived for 2 h. Specific uptake of 50 or 100 nmol/L [3H]DA was measured, as described in Fig. 5. The horizontal dashed line delineates the level of control. (a) Following a 2-h serum deprivation, mesencephalic-striatal neuronal co-cultures (7–9 DIV) were pre-incubated with 100 ng/mL BDNF for various times at 37°C. (b) After serum deprivation, neuronal co-cultures (7–9 DIV) were pre-incubated with 100 ng/mL BDNF, 100 nmol/L K252a (TrkB antagonist), 500 nmol/L U0126 (MEK1/2 inhibitor), or 500 nmol/L LY294002 (PI3K inhibitor) alone or in combination for 30 min at 37°C. (c) A representative kinetic analysis curve determined with serum-deprived neuronal co-cultures (7–9 DIV) after pre-treatment with 100 ng/mL BDNF for 30 min at 37°C and uptake measured as in Fig. 5, with the exception that a range of unlabeled dopamine concentrations (200–4000 nmol/L) and 50–100 nmol/L [3H]DA were used. See Table 2 for Km and Vmax values, determined by non-linear regression analysis. n = 3–20 separate experiments, each done in duplicate wells. *p < 0.05 compared with control, post hoc Holm-Sidak test.

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Table 2.   Effect of a 30-min pre-incubation with brain-derived neurotrophic factor on kinetic parameters of specific [3H]DA uptake into serum-deprived (2 h) mesencephalic-striatal neuronal co-cultures
ConditionKm (nmol/L)Vmax (pmol/min/mg protein)
  1. *p < 0.05, compared with control. n = 5. See Materials and methods for details.

Control395 ± 471.8 ± 0.2
100 ng/mL BDNF941 ± 187*2.2 ± 0.2*

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we found that the TK inhibitors genistein and tyrphostin 23 rapidly reduced [3H]DA uptake into both rat STR synaptosomes and dissociated mesencephalic neurons in a concentration-dependent manner. Similar pre-treatment with these inhibitors also diminished the amount of phospho p42/p44 MAPK detected in STR synaptosomes. Genistein and tyrphostin 23 selectively reduced maximal DAT velocity, which is consistent with our observation of fewer DATs on the plasma membrane following pre-treatment with the TK inhibitors. Conversely, in serum-deprived mesencephalic neurons, BDNF activation of TrkB receptors rapidly up-regulated [3H]DA uptake via an increase in Vmax. Surprisingly, however, BDNF also reduced DAT affinity, suggesting more complex effects. The BDNF-mediated up-regulation of [3H]DA uptake required both MEK 1/2 and PI3K pathways. Together, our results support a role for TKs and rapid effects of BDNF in DAT trafficking in mesostriatal DA neurons.

Tyrosine kinase regulation of dopamine transporter in dorsal striatal synaptosomes

Recent studies have focused attention on the rapid regulation of DAT function and corresponding changes in transporter cell surface expression (Melikian 2004; Zahniser and Sorkin 2004). Here, we found that the TK inhibitors genistein and tyrphostin 23 decreased DAT uptake velocity within 5 min, reaching maximal inhibition by ∼15 min. Kinetic analyses and biotinylation experiments revealed that the TK inhibitors’ reduction of the DAT Vmax was due to reduced DAT cell surface expression. Yet to be determined is whether this change in surface expression is the result of an increased rate of DAT endocytosis and/or decreased DAT exocytosis. Biotinylation of the DAT also revealed that 28% of the transporter is localized to the STR synaptosomal cell surface, suggesting a large pool of intracellular transporters exists under basal conditions. However, this value is likely an under-representation since biotinylation of cell surface proteins does not occur at 100% efficiency. Indeed, Law et al. (2000) found that 60% of GAT1, another SLC6 transporter, is on the cell surface of dissociated hippocampal neurons under basal conditions.

The intracellular signaling pathways involved in positively regulating basal [3H]DA uptake are the p44/p42, but not p38, MAPK isoforms (Lin et al. 2003; Moron et al. 2003). Consistent with these findings, we observed a high level of phospho p44/p42 MAPK in vehicle-treated STR synaptosomes; this was decreased by genistein and tyrphostin 23, concomitant with decreases in [3H]DA uptake. Taken together, our results demonstrate that DAT activity, and thereby cell surface expression, in rat STR synaptosomes is under the constitutive control of TKs, suggesting that TKs are basally activated, possibly by endogenous growth factors. Furthermore, the TK inhibitors may exert this control over DAT activity through a reduction in p44/p42 MAPK activity. These results not only confirm, but also extend the studies of DAT regulation by TK inhibitors in mouse STR homogenates (Simon et al. 1997) and DAT-expressing oocytes (Doolen and Zahniser 2001). Furthermore, these data are in agreement with reports of TK inhibitors reducing the cell surface expression and activity of GAT1 and NET, transporters closely related to DAT (Law et al. 2000; Apparsundaram et al. 2001). Future studies will examine the role of the PI3K/Akt pathway in the regulation of synaptosomal DAT activity by TK inhibitors.

We did not observe a consistent effect of TK activation by BDNF on [3H]DA uptake into STR synaptosomes. Although BDNF’s regulation of [3H]DA uptake was not affected by depletion of vesicular DA (see Results), other factors might have contributed to the observed variability. For instance, the p75 neurotrophin receptor, which BDNF also activates, can mediate opposing cellular functions (Huang and Reichardt 2003). Alternatively, variable basal TK activation and/or protein tyrosine phosphatase activity could alter BDNF/TrkB signaling. Thus, the variable response of STR synaptosomes to BDNF was likely due to multiple interactions between BDNF-mediated signaling pathways and the biochemical milieu.

Tyrosine kinase regulation of dopamine transporter in primary mesencephalic-striatal co-cultured neurons

Inhibition or activation of TKs and the subsequent effects on [3H]DA uptake were further examined in primary mesencephalic neurons co-cultured with striatal neurons for 7–9 DIV. We found that [3H]DA uptake was decreased in a concentration-dependent manner by a PKC activator or inhibitors of the TK, MEK 1/2, and PI3K pathways. These findings indicated that the DAT was not only functionally active, but also regulated in the primary mesencephalic neurons in a manner similar to previous reports using STR synaptosomes and DAT-expressing heterologous cells (Zahniser and Doolen 2001; Carvelli et al. 2002; Lin et al. 2003; Moron et al. 2003; Garcia et al. 2005). In addition, we utilized mesencephalic neurons and their well-defined biochemical environment to provide information about potential DAT regulation following TK activation with the growth factor BDNF.

Brain-derived neurotrophic factor has well-known long-term effects on DA neurons, promoting their survival, differentiation, and activity (Hyman et al. 1994; Shen et al. 1994). Genetic ablation of one of the BDNF alleles in mice has further emphasized the importance of BDNF for the normal development and function of DA neurons and DA-dependent behaviors (Horger et al. 1999; Dluzen et al. 2001; Hall et al. 2003). Undeniably, DA neurons are well-positioned to generate and respond to BDNF neurotrophic signaling. Both BDNF and TrkB mRNAs and proteins are expressed in DA neurons (Seroogy et al. 1994; Numan and Seroogy 1999). Indeed, our immunocytochemical experiments confirmed that every TH-expressing neuron in the mesencephalic cultures also expressed TrkB receptors. Nearly all of the studies examining BDNF-mediated regulation of DA neuronal health and function have utilized chronic BDNF treatments. Under these treatment regimens, DAT activity is also increased and is correlated with an increased number of DA neurons (Knusel et al. 1991; Hyman et al. 1994).

In contrast to most studies, our work examined DAT regulation following a brief exposure to BDNF and supports the finding of Blochl and Sirrenberg (1996) that 100 ng/mL BDNF increases [3H]DA uptake in a TrkB receptor-dependent manner. Here, we also found that a 30-min pre-treatment of the mesencephalic neurons with BDNF increased both Vmax and Km. Mathematically, an increase in Km could result in a larger Vmax; however, this explanation seems unlikely because BDNF also increased uptake of 50 nmol/L [3H]DA, a concentration below its Km. BDNF’s dual effects of increasing transporter velocity while reducing its affinity for DA raise the possibility that TK inhibitors are not simply blocking TrkB receptor signaling. Future studies will examine this question in more detail, using insulin regulation of the DAT and other SLC6 transporters (see below) as a guide for how BDNF may regulate the DAT. The BDNF-mediated increase in [3H]DA uptake was dependent on both MEK 1/2 and PI3K pathways. Likewise, Goggi et al. (2003) found that both MEK 1/2 and PI3K pathways were essential for BDNF-mediated increases in K+-stimulated DA release in STR slices and synaptosomes. Furthermore, Feng et al. (1999) found that a 30-min pre-treatment with 50 ng/mL BDNF (a concentration similar to that used in the present study) increased phospho p44/p42 MAPK levels in mesencephalic neurons. While the involvement of p38 MAPK in basal and BDNF-mediated DAT regulation was not examined here, others have reported that this MAPK isoform either has no effect (Lin et al. 2003; Moron et al. 2003) or reduces constitutive DAT activity (Zhu et al. 2005a). In sum, our results demonstrate that the MEK 1/2 and PI3K pathways are essential for TK-mediated up-regulation of DAT activity in mesencephalic neurons. The involvement of both kinases may result from their participation in distinct components of DAT regulation, such as phosphorylation versus endocytosis.

Similar to DAT, NET and GAT1 activity are increased following activation of TKs by insulin or BDNF, respectively (Law et al. 2000; Apparsundaram et al. 2001). Furthermore, TK activation also up-regulates SERT activity and cell surface expression (Prasad et al. 1997; Gil et al. 2003), although Mossner et al. (2000) found that BDNF decreases transporter activity. Insulin’s effect on NET activity is dependent on PI3K and p38 MAPK activity and independent of MEK 1/2 activation (Apparsundaram et al. 2001). Moreover, basal SERT (but not NET) activity is regulated by p38 MAPK, but not MEK 1/2 activity (Apparsundaram et al. 2001; Samuvel et al. 2005; Zhu et al. 2005a). Despite these reports of TK activators increasing DAT, NET, GAT1, and SERT activity, the underlying mechanism(s) responsible for the regulation appears to differ from one transporter to another. For instance, insulin’s up-regulation of NET activity is due to catalytic activation, rather than transporter trafficking (Apparsundaram et al. 2001). Conversely, insulin-induced up-regulation of DAT activity is due to increased transporter cell surface expression (Carvelli et al. 2002; Garcia et al. 2005). Thus, while it is clear that TKs regulate the activity of the SLC6 neurotransmitter transporters, there are obvious distinctions in the underlying mechanism and intracellular signaling pathways involved.

The mechanism(s) by which TKs and their potential downstream effectors MAPK and/or PI3K directly or indirectly regulate DAT function and trafficking is unknown. Examination of consensus tyrosine phosphorylation sites on the SLC6 transporters has yielded few positive results. For example, Simon et al. (1997) detected no phosphotyrosine residues where mouse STR DAT should migrate (60–80 kDa) during SDS-PAGE. Furthermore, NET’s tyrosine residues are not phosphorylated by insulin (Apparsundaram et al. 2001). The exception is GAT1; BDNF does increase tyrosine phosphorylation of GAT1, whereas TK inhibition induces the opposite effect, resulting in GAT1 internalization (Law et al. 2000). Interestingly, TKs may act in balanced opposition to PKC to fine-tune GAT1’s phosphorylation state, and thereby its function (Quick et al. 2004). Alternatively, TKs could phosphorylate a DAT accessory protein, as is the case for GAT1 with PKC and Munc-18 (Beckman et al. 1998).

Understanding TK regulation of DA uptake will increase not only understanding of how normal DA neurotransmission is controlled, but also may provide insights into treatments for CNS disorders where DA signaling is affected, such as drug addiction. Amphetamine and cocaine produce neural and behavioral adaptations that are intimately linked to the elevated extracellular DA levels these drugs induce. Interestingly, amphetamine’s and cocaine’s DA-mediated behavioral effects depend on insulin and BDNF, respectively (Marshall 1978; Horger et al. 1999; Hall et al. 2003; Owens et al. 2005). Beyond enhancing cocaine-induced locomotion, BDNF also potentiates the associative learning between cocaine and cocaine-associated cues that leads to drug-seeking behavior, and ultimately relapse (Horger et al. 1999; Grimm et al. 2003; Hall et al. 2003; Pu et al. 2006). Recent reports that insulin reverses amphetamine’s effects on DAT activity and trafficking (Carvelli et al. 2002; Garcia et al. 2005) and our finding that BDNF up-regulated DAT activity suggest that these TK-activating growth factors may exert a homeostatic influence to counter abnormal DA neurotransmission induced by psychostimulants by transiently reducing extracellular DA and thereby synaptic activity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. Susan Jones and Ms. Cynthia Hutt (University of Colorado at Denver and Health Sciences Center) for training in the preparation of the primary neuronal cultures; Drs. Jun Zhu and Linda Dwoskin (University of Kentucky) for suggestions with the DAT biotinylation assays; and Dr. Roxanne Vaughan (University of North Dakota) for the generous gift of the DAT monoclonal antibody. This work was supported by NIH DA 14204, DA 04216, NS 07083, DA 16860, and DA 15050.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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