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Originally published In Press as doi:10.1074/jbc.M413887200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 12, 10914-10919, March 25, 2005
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Regulation of Amphetamine-stimulated Dopamine Efflux by Protein Kinase C {beta}*

L'Aurelle A. Johnson, Bipasha Guptaroy, David Lund, Susanna Shamban, and Margaret E. Gnegy{ddagger}

From the Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632

Received for publication, December 9, 2004 , and in revised form, January 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca2+-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC{delta}, had no effect. A highly specific PKC{beta} inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKC{beta}I and PKC{beta}II, but not PKC{alpha} or PKC{gamma}, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC{beta}I and PKC{beta}II but not PKC{alpha} or PKC{gamma} were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC{beta}II as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKC{alpha} or PKC{beta}I. These results suggest that classical PKC{beta}II is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The plasmalemmal dopamine transporter (DAT)1 is a pre-synaptic protein that belongs to the SLC6 family of Na+/Cl–-dependent neurotransmitter transporters. It is the primary regulator of the duration and strength of the dopamine signal in the synapse (1). DAT binds extracellular dopamine, Na+, and Cl– and transports them intracellularly, clearing dopamine from the synaptic cleft. The transporter is also the site of action of psychostimulant drugs such as amphetamine, which is a DAT substrate. Following its transport into the synaptic terminal, amphetamine stimulates a reversal of the transporter eliciting dopamine efflux. DAT is characterized as having 12 transmembrane segments and a large second extracellular loop with the N- and C-terminals located intracellularly. There are also putative phosphorylation sites for protein kinase C (PKC), protein kinase A, and Ca2+/calmodulin-stimulated protein kinase II (2). PKC has been implicated in various aspects of DAT function and regulation, such as trafficking (3), transport activity (4, 5), and direct phosphorylation (Refs. 6 and 7, and see references in Ref. 8).

Evidence strongly suggests that PKC activity is important for amphetamine-stimulated outward transport. General PKC inhibitors blocked dopamine efflux induced by amphetamine in rat striatal slices (9). Accordingly, a PKC inhibitor blocked amphetamine-mediated locomotor behavior when injected directly into rat nucleus accumbens (10). The phorbol ester PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimicked the effect of amphetamine on the reverse transport by eliciting an efflux of dopamine that was cocaine-sensitive and independent of extracellular Ca2+ (11). Finally, a deletion of the N-terminal 22 amino acids of DAT, containing the distal N-terminal serines that are strongly considered to be substrate sites of PKC (6), abrogated amphetamine-induced dopamine efflux from hDAT-HEK 293 cells (human embryonic kidney 293 cells) without altering inward transport (12). Mutation of the distal N-terminal serines of DAT to alanines similarly abolished amphetamine-stimulated dopamine efflux, whereas mutation of the N-terminal serines to aspartates restored sensitivity of the reverse transport to amphetamine.

The PKC isoform altering DAT-mediated dopamine efflux is not known, but it could be a Ca2+-sensitive isoform. Although amphetamine-induced dopamine efflux does not require extracellular Ca2+, it does require intracellular Ca2+ (13, 14). Moreover, amphetamine, acting at DAT, increases intracellular Ca2+ from thapsigargin-sensitive stores (14, 15). There are three major classes of PKC isoforms: classical, non-classical, and atypical. The classical PKC isoforms ({alpha}, {beta}1, {beta}2, {gamma}) are Ca2+- and diacylglycerol-dependent. The non-classical PKC isoforms ({delta}, {epsilon}, {eta}, {theta}) are Ca2+-independent but diacylglycerol-dependent. The atypical PKC isoforms ({zeta} and {iota}/{lambda}) are insensitive to Ca2+ and diacylglycerol. There is a limited number of inhibitors that can distinguish among the different isoforms. Gö6976 is a potent inhibitor of the classical PKC isoforms (16). LY379196, structurally similar to LY333531, is a highly selective inhibitor of the PKC{beta} isoform (17), and rottlerin is a selective inhibitor of the PKC{delta} isoform (18).

Using these selective inhibitors in combination with co-immunoprecipitation studies and the transfection of PKC isozymes into hDAT human embryonic kidney 293 (hDAT-HEK 293) cells, we investigated the role of two classical PKC isozymes in regulating outward transport of dopamine by amphetamine and their association with DAT.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Gö6976 and rottlerin were purchased from Calbiochem. d-Amphetamine, dopamine, and TPA were purchased from Sigma. LY379196 was a generous gift from Lilly. hDAT cDNA was a gift from Dr. Z. B. Pristupa, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Canada; cDNAs for PKC{alpha}, PKC{beta}I, and PKC{beta}II were generous gifts from Dr. Stephen Ferguson, John P. Robarts Research Institute, London, Ontario, Canada.

Amphetamine- or TPA-mediated Dopamine Efflux—Rat striata from female Sprague-Dawley rats (175–200 g) were sliced, weighed, and placed in chambers of a Brandel perfusion apparatus (Brandel SF-12, Gaithersburg, MD) onto Whatman GF/B filter disks. The chambers were perfused at 37 °C with oxygenated Krebs-Ringer buffer (KRB, 145 mM NaCl, 2.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, 1.0 mM MgCl2, 10 mM glucose, 0.255 mM ascorbic acid, 24.9 mM NaHCO3, 50 µM pargyline, and 1 mM tropolone) for 40 min at a rate of 100 µl/min. The slices were then perfused in the presence or absence of the drugs (130 nM Gö6976, 10 µM rottlerin, or 100 nM LY379196) for 40 min prior to the addition of a 2.5-min bolus of 3 µM amphetamine or 300 nM TPA. Eluates (500 µl, 5-min fractions) were collected into vials containing 25 µl of internal standard solution (0.05 M HClO4, 4.55 mM dihydroxybenzylamine (internal standard), 1 M metabisulfate and 0.1 M EDTA). Dopamine was measured by high performance liquid chromatography with electrochemical detection.

To measure dopamine efflux from hDAT-HEK 293 cells, cells cultured in 100-mm plates were incubated with 15 µM unlabeled dopamine for 30 min at 37 °C. Following the incubation, cells were washed two times with a Krebs-Ringer-Hepes (KRH) buffer (25 mM Hepes, pH 7.4, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 5.6 mM glucose, and 1 mM tropolone) and resuspended in 300 µl of KRH. The cells (200 µl) were placed in the Brandel superperfusion apparatus and washed for 30 min at room temperature before the addition of 3 µM amphetamine for 2.5 min. Dopamine was measured in the eluates as described above.

Synaptosome Preparation—Rat striata were homogenized in 10 volumes of homogenization buffer containing 0.32 M sucrose, 1 mM EDTA, and a mixture of protease inhibitors (Complete Mini, Roche Applied Science), pH 7.4. Homogenates were centrifuged at 1000 x g for 10 min to remove cell debris. The supernatant was saved, and the pellet was resuspended in homogenization buffer and centrifuged again. The combined supernatant fractions were centrifuged for 30 min at 15,000 x g at 4 °C. The supernatant was removed, and the pellet (P2) was resuspended in KRB for [3H]dopamine uptake assay.

[3H]Dopamine Uptake—Synaptosomes were resuspended in 1.2 ml of KRB and incubated in the presence or absence of drugs (130 nM Gö6976, 10 µM rottlerin, or 100 nM LY379196) for 40 min at 37 °C. [3H]Dopamine (330 nM, 24.4 µCi; PerkinElmer Life Sciences) was added for 3 min. [3H]Dopamine uptake was stopped with 5 ml of cold KRB; samples were filtered on GF/C filters and washed twice with 5 ml of cold KRB. Filters were dried and then counted in a Beckman LS 5801 liquid scintillation counter. Nonspecific uptake was determined using 10 µM of the DAT uptake inhibitor GBR12935. Uptake of 25 nM [3H]dopamine was measured similarly in hDAT-HEK 293 cells, which were resuspended in KRB after being triturated from the plates.

Co-immunoprecipitation—Rat striatal synaptosomes were lysed in RIPAE buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 1% SDS, and protease inhibitors) by rotating for 1 h at 4°C. Insoluble material was removed by centrifugation at 10,000 x g. Lysates (50 µg) were incubated with either anti-PKC{alpha}, anti-PKC{beta}II (both from Santa Cruz Biotechnology, Santa Cruz, CA), anti-PKC{beta}I (Sigma), anti-PKC{gamma} (BD Biosciences), or anti-DAT antibody (University of Michigan antibody core). In some experiments, anti-DAT was preincubated with 40 µg of the immunizing peptide (see "Preparation of Purified Anti-DAT") for 2 h at 4 °C. Immune complexes were bound to protein A-Sepharose (Sigma) for 30 min and pelleted at 1000 x g. After washing twice with RIPAE buffer and twice with Tris-buffered saline (Tris 10 mM, pH 7.4, and NaCl, 150 mM), proteins were eluted from protein A-Sepharose with Laemmli sample buffer and separated on 10% SDS-polyacrylamide gels.

Generation and Maintenance of Stable Cell Lines—HEK 293 were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum at 37 °C and 5% CO2. For stable transfection, the cells were plated at 80% confluence on 6-well culture plates and were transfected using Lipofectamine Plus reagent (Invitrogen) with 0.4 µg of hDAT cDNA in a pIREShyg 2 vector (Clontech) in Opti-MEM (Invitrogen) reduced serum medium according to the manufacturer's instructions. 12 h after transfection, the solution was removed, and fresh medium was added. Cells were grown for 7–8 h, and 250 µg/ml hygromycin was added to select for a stably transfected pool of cells. Stable cell lines expressing PKC isoforms (PKC{beta}I, PKC{beta}II, or PKC{alpha}) were made by transfection of 15 µg of individual cDNAs for PKC{alpha}, PKC{beta}I, or PKC{beta}II (PKC{alpha} in pEGFP-C3 vector, Clontech; PKC{beta}I in pEGFP-C1 vector, Clontech; GFP-PKC{beta}II fusion protein (19) in pBK-CMV vector, Stratagene) into hDAT-HEK cells (100-mm plates) using Lipofectamine as described above. The double transfectants were selected by growing in the presence of hygromycin and neomycin for PKC{alpha}, PKC{beta}I, and PKC{beta}II. Expression of hDAT and PKC{alpha}, PKC{beta}I, and PKC{beta}II was confirmed by Western blot analysis.

Immunoblot Analysis—Samples were resolved by SDS-PAGE on a 4–20% Tris/glycine gel and transferred to a nitrocellulose membrane. Membranes were blocked with TBST (10 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Tween 20) containing 5% casein and incubated for 1 h in TBST containing anti-rat DAT monoclonal antibody at 1:1000 dilution (mab16, generously donated by Dr. Roxanne Vaughan, University of North Dakota), anti-PKC{alpha} antibody at 1:1000, PKC{beta}I or PKC{beta}II at 1:2000 dilution in TBST. Membranes were given four 10-min washes with TBST and incubated for 1 h with anti-mouse horseradish peroxidase-tagged secondary antibody at a 1:10,000 dilution. Membranes were again washed with TBST and developed with ECL reagents (Amersham Biosciences).

Preparation of Purified Anti-DAT—A peptide corresponding to residues 42–59 of rat DAT (LTNSTLINPPQTPVEAQE) was synthesized at the Protein Structure Core, University of Michigan. Anti-peptide antibody was produced in rabbit at the Unit for Laboratory Animal Medicine, University of Michigan, following the strategy of Freed et al. (20). The peptide-specific antibody was purified using the Amino Link Plus immobilization kit from Pierce according to the manufacturer's instructions.

Biotinylation of Cell Surface Proteins—Surface expression of hDAT-HEK 293 cells and cells stably expressing hDAT + PKC{alpha}, hDAT + PKC{beta}I, and hDAT + PKC{beta}II was determined by reacting the surface proteins with sulfosuccinimidyl-2-(biotinamido)ethyl-1, 3-dithiopropionate (Pierce) using the method described by Granas et al. (21) with the following slight variations. Cells were grown to confluence on 100-mm plates, washed, removed from the plates by trituration, and resuspended in cold phosphate-buffered saline/Ca-Mg buffer, pH 7.3, before treating with sulfosuccinimidyl-2-(biotinamido)ethyl-1, 3-dithiopropionate (2.0 mg/ml) at 4 °C for 60 min in phosphate-buffered saline/Ca-Mg. The biotinylation reaction was stopped, and the samples were prepared as described by Granas et al. (21) except that only 750 µg of samples were incubated with monomeric avidin beads. The eluates were resolved by SDS-PAGE (4–20% Tris/glycine) and immunoblotted using a 1:1000 dilution of MAB369, anti-DAT (Chemicon International, Temecula, CA). Bands were visualized using goat anti-rat horseradish peroxidase-conjugated secondary antibody at 1:10,000 followed by ECL detection. Quantification of the bands was performed by the Scion Image software.

Statistics—Statistical significance was calculated using Graph Pad 3. Statistical significance among three or more groups was determined using one-way analysis of variance with post hoc Tukey-Kramer multiple comparison analysis. Two-group comparisons, such as those comparing the effect of a drug on amphetamine-stimulated dopamine efflux, were made using a two-tail Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PKC Inhibitors on Amphetamine-stimulated Dopamine Efflux—The effect of the selective PKC inhibitors Gö6976, LY379196, and rottlerin on amphetamine-stimulated dopamine efflux from rat striatal slices was investigated. The classical PKC inhibitor Gö6976 at 130 nM (IC50 of 2.3–7.9 nM) (16), reduced the amount of dopamine efflux in response to amphetamine from 0.39 ± 0.05 pmol/mg of protein in the absence of the drug to 0.13 ± 0.05 pmol/mg of protein in the presence of the drug (p < 0.05, n = 3) (Fig. 1A). Inhibition of amphetamine-stimulated dopamine efflux was also found using 13 nM Gö6976, a concentration closer to the IC50. PKC LY379196 is a highly selective inhibitor of PKC{beta} (IC50 of 30 nM) (17); we used this compound to examine the role of a single classical PKC isozyme, PKC{beta}. LY379196 demonstrates a 10-fold selectivity over other classical PKCs and 10–100-fold selectivity over non-classical PKCs. In the presence of 100 nM LY379196, amphetamine-stimulated dopamine efflux was reduced from 0.28 ± 0.08 pmol/mg of protein in the absence of drug to 0.06 ± 0.03 pmol/mg of protein (p < 0.03, n = 8) (Fig. 1B). The value for dopamine efflux in the presence of LY379196 was not different from base line (0.10 ± 0.02, n = 8). To test the activity of a novel PKC, we used rottlerin, a selective PKC{delta} inhibitor (IC50 of 2–6 µM) (18). Rottlerin, at 10 µM, did not decrease amphetamine-mediated dopamine efflux in rat striatal slices (Fig. 1C). Rottlerin exhibited a slight but non-significant stimulatory effect on efflux.



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  		FIG. 1.
The effect of isoform-specific PKC inhibitors on amphetamine-mediated dopamine efflux in rat striatal slices. Slices were incubated in the presence of 130 nM Gö6976 (selective for PKC{alpha}, PKC{beta}, PKC{gamma}) (A), 100 nM LY379196 (highly selective for PKC{beta})(B), 10 µM rottlerin (selective for PKC{delta})(C), or KRB for 40 min prior to the addition of 2.5 min of 3 µM amphetamine (AMPH). Dopamine was measured as described under "Experimental Procedures." Results are given in pmol of dopamine/mg wet weight of striatum in a 5-min fraction. Data points represent mean ± S.E. Statistical significance was determined using an unpaired two-tail Student's t test. A, fraction 11, *, p < 0.05, n = 3; B, fraction 7, *, p ≤ 0.02, n = 8; C, fraction 11, n.s., n = 3.

 
Effect of the PKC Inhibitors on [3H]Dopamine Uptake—To determine whether the PKC inhibitors prevented inward transport of substrate, [3H]dopamine uptake was assessed. Striatal synaptosomes were incubated with Gö6976, LY379196, and rottlerin at 37 °C for 40 min prior to the addition of [3H]dopamine to mimic the conditions in the efflux assay. Preincubation of the synaptosomes with 130 nM Gö6976, 100 nM LY379196, or 10 µM rottlerin had no effect on [3H]dopamine uptake as compared with vehicle controls (Fig. 2).



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  		FIG. 2.
[3H]Dopamine uptake in the presence of PKC inhibitors. Striatal synaptosomes were incubated in the presence or absence (control) of 130 nM Gö6876, 100 nM LY379196, or 10 µM rottlerin for 40 min prior to the addition of [3H]dopamine. [3H]Dopamine uptake was measured as described under "Experimental Procedures."

 
Effect of LY379196 on TPA-mediated Dopamine Efflux—We have shown previously that activation of PKC with TPA mimicked the ability of amphetamine to induce dopamine efflux from rat striatal slices (9, 11). If TPA and amphetamine are using the same signaling pathway, TPA-mediated dopamine efflux should be inhibited by the selective PKC{beta} inhibitor, LY379196, in rat striatal slices. As shown in Fig. 3, LY379196 reduced TPA-mediated dopamine efflux from 3.3 ± 0.83-fold of base-line dopamine values to 1.3 ± 0.43-fold base-line dopamine values (p < 0.05, n = 4).



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  		FIG. 3.
Effect of the selective PKC{beta} inhibitor, LY379196, on TPA-mediated dopamine efflux. Rat striatal slices were incubated with 100 nM LY379196 for 40 min prior to the addition of 300 nM TPA. For fraction 6, *p < 0.05 as determined by two-tail Student's t test.

 
PKC{beta}I and PKC{beta}II Are Associated with DAT in Rat Striatal Synaptosomes—We utilized the technique of co-immunoprecipitation to determine whether PKC{beta} was bound to DAT. DAT was immunoprecipitated using a peptide affinity-purified polyclonal antibody developed in our laboratory. The DAT immunoprecipitates were immunoblotted for PKC{alpha}, PKC{beta}I, PKC{beta}II, and PKC{gamma}. To ensure that the co-immunoprecipitation was specific for DAT, we preincubated the antibody with the DAT N-terminal antigen. The data in Fig. 4 demonstrate that both PKC{beta}I and PKC{beta}II co-immunoprecipitated with DAT in the absence but not the presence of the peptide antigen. On the contrary, neither PKC{alpha} nor PKC{gamma} co-immunoprecipitated with DAT. As shown in Fig. 4, all four forms of PKC were present in the lysate. To further test the apparent association, immunoprecipitation was conducted using antisera to the PKC isoforms, and the presence of DAT in the immunoprecipitates was tested with immunoblotting. As shown in Fig. 5, DAT co-immunoprecipitated with PKC{beta}I and PKC{beta}II but not with PKC{alpha} or PKC{gamma}. We have validated these results with two different PKC{beta}II antibodies. We also detected co-immunoprecipitation of hDAT and PKC{beta}II in hDAT-PC12 cells following immunoprecipitation with either anti-DAT or anti-PKC{beta}II (data not shown). However, we would use caution in interpreting the relative amounts of PKC{beta} isozymes that immunoprecipitated with DAT. We performed all experiments with two to three different antibodies to PKC{beta}I and PKC{beta}II, and although the results were qualitatively similar, we found that the relative amounts of the detected isozyme could vary depending on the efficacy of the antibody for immunoprecipitation or immunoblotting.



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  		FIG. 4.
Co-immunoprecipitation of DAT with PKC{beta}I and PKC{beta}II but not with PKC{alpha} or PKC{gamma}. A rat striatal synaptosomal preparation was lysed in RIPAE buffer and precleared with protein A-Sepharose as described under "Experimental Procedures." 50 µg of protein were then incubated with anti-DAT antibody preincubated with (IP+P) or without (IP) the immunogenic peptide (40 µg, corresponding to three times the concentration of antibody) as discussed under "Experimental Procedures." The immunoprecipitates were eluted into sample buffer. Eluted proteins and original lysates (Ly) were analyzed by immunoblotting (IB) for the classical PKC isozymes.

 



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  		FIG. 5.
Immunoprecipitation of striatal synaptosomes with either anti-PKC{beta}I or anti-PKC{beta}II but not anti-PKC{gamma} or anti-PKC{alpha} co-immunoprecipitates DAT. Rat striatal synaptosomes were lysed in RIPAE buffer and precleared as described under "Experimental Procedures." 50 µg of protein were incubated with either anti-PKC{alpha}, anti-PKC{beta}I, anti-PKC{beta}II immunoprecipitates or anti-PKC{gamma} antibody. The protein from the (IP) was eluted in sample buffer. Eluted proteins and original lysates (Ly) (20 µg of protein for lysate in left panel and 12.5 µg of protein in lysate in right panel) were analyzed by immunoblotting (IB) for DAT.

 
PKC{beta}II Potentiates Amphetamine-stimulated Dopamine Efflux—Because both PKC{beta}I and PKC{beta}II co-immunoprecipitated with DAT, one or both of the isozymes could be responsible for enhancing amphetamine-stimulated dopamine efflux. LY379196 does not discriminate between PKC{beta}I and PKC{beta}II. To distinguish between the two PKC{beta} isozymes we created stable hDAT-HEK 293 cell lines overexpressing PKC{alpha}, PKC{beta}I, or PKC{beta}II. As shown in Fig. 6A, hDAT-HEK 293 cells endogenously contain PKC{alpha} and PKC{beta}I but have very little PKC{beta}II. However, the stable cell lines contained high concentrations of their respective PKC isozymes. The cells were loaded with 15 µM dopamine for 30 min to measure dopamine efflux by superperfusion. The dopamine efflux in response to 3 µM amphetamine was similar in the hDAT-HEK 293 cells, the PKC{alpha}-hDAT-HEK 293 cells, and the PKC{beta}I-hDAT-HEK 293 cells (Fig. 6B). However, the dopamine efflux in response to amphetamine was much greater in the PKC{beta}II-hDAT-HEK 293 cells than in the other cell lines. The average -fold base line of amphetamine response in the PKC{beta}II-hDAT-HEK 293 cells was 2.8 ± 0.5 (n = 7), whereas that in the other lines was maximal at 1.57 ± 0.3 (for hDAT-HEK 293 cells, n = 6), 1.47 ± 0.3 (PKC{alpha}-hDAT-HEK 293 cells, n = 7), and 1.3 ± 0.2 (for PKC{beta}I-hDAT-HEK 293 cells, n = 7). Base-line values (in pmol of dopamine/5 min/mg of protein) in the untransfected hDAT-HEK 293 cells and the stable HEK 293 lines containing PKC{alpha}, PKC{beta}I, and PKC{beta}II were 9.4 ± 1.6, 12.8 ± 1.5, 9.4 ± 1.2, and 10.0 ± 1.3, respectively. The average concentration of dopamine present in the cells at the start of the perfusion was, in nmol/mg of protein: 1.6 ± 0.5 in hDAT-HEK 293 cells; 1.2 ± 0.3 in PKC{alpha}-hDAT-HEK 293 cells; 1.5 ± 0.3 in PKC{beta}I-hDAT-HEK 293 cells; and 1.9 ± 0.6 in PKC{beta}II-hDAT-HEK 293 cells (n = 6, p = 0.76, one-way analysis of variance). Similar numbers of cells were used in all assays. Therefore the increase in amphetamine-stimulated dopamine efflux in the PKC{beta}II-hDAT-HEK 293 cells was not due to a significantly greater initial concentration of dopamine in the cells. To ensure that transport of amphetamine into the different cell lines was not altered by the transfection of the PKC isoforms, we measured uptake of [3H]dopamine in the four cell lines. Uptake of 25 nM [3H]dopamine was, in pmol/3 min/mg of protein: 0.16 ± 0.02 in hDAT-HEK 293 cells; 0.22 ± 0.09 in PKC{alpha}-hDAT-HEK 293 cells; 0.19 ± 0.07 in PKC{beta}I-hDAT-HEK 293 cells; and 0.23 ± 0.1 in PKC{beta}II-hDAT-HEK 293 cells (n = 5, p = 0.7, one-way analysis of variance). To determine whether the amount of hDAT on the surface of the four cell lines was altered by transfection of the PKC isoforms, surface biotinylation of DAT was measured. As shown in Fig. 6C, the enhancement in amphetamine-stimulated dopamine efflux in PKC{beta}II-hDAT-HEK 293 cells is also not due to the presence of significantly greater levels of surface hDAT as compared with the other cell types.



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  		FIG. 6.
Effect of overexpression of PKC isoforms on amphetamine-stimulated dopamine efflux. A, PKC isoform content of untransfected (U) hDAT-HEK 293 cells and in hDAT-HEK 293 cells overexpressing (T, transfected) either PKC{alpha}, PKC{beta}I, or PKC{beta}II. B, amphetamine (AMPH, 3 µM)-stimulated dopamine efflux in hDAT-HEK cells and cells overexpressing PKC{alpha}, PKC{beta}I, and PKC{beta}II. All cells were preloaded with 15 µM dopamine for 30 min at room temperature. Cells were loaded into the superfusion apparatus, washed, and perfused with 3 µM amphetamine for 2.5 min. Results are expressed as -fold base line ± S.E. Base-line values are given under PKC{beta}II Potentiates Amphetamine-stimulated Dopamine Efflux. Analysis of variance for all groups at fraction 7, p < 0.01. In the post hoc Tukey-Kramer multiple comparisons test, results for PKC{beta} hDAT-HEK 293 cells differed from those for PKC{alpha}II hDAT-HEK 293 at p < 0.01 and for PKC{beta}I hDAT-HEK 293 and hDAT-HEK 293 cells at p < 0.05. n = 6–7. C, surface expression of hDAT in the untransfected hDAT-HEK 293 cells, PKC{alpha} hDAT-HEK 293 ({alpha}), PKC{beta}I hDAT-HEK 293 ({beta}I) and PKC{beta}II hDAT-HEK 293 ({beta}II) cells. Cells were biotinylated as described under "Experimental Procedures" and immunoblotted (IB) for hDAT. C demonstrates the biotinylated fraction (surface DAT).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We report that classical PKC isozymes regulate the mechanism of action of amphetamine in eliciting outward transport of dopamine. We made the original observations that PKC{beta} and, more specifically, PKC{beta}II, promotes amphetamine-induced outward transport through the dopamine transporter and that PKC{beta} isoforms are selectively associated with DAT. The regulation of DAT by PKC is obviously complex. We have demonstrated that brief (<3 min) incubations with TPA elicit efflux of dopamine that is blocked by cocaine but independent of extracellular Ca2+ (11). On the contrary, longer incubations with TPA (>5 min) increase inward trafficking of DAT (8). The TPA-induced internalization of DAT also involves classical PKCs; Gö6976 attenuated TPA-induced reduction of DAT-associated transport currents (4), an effect attributed to the enhanced endocytosis of DAT. Because TPA can activate many PKC isozymes, and there are four isotypes of classical PKC isozymes (PKC{alpha}, {beta}I, {beta}II, and {gamma}), different classical PKC isozymes could be mediating these two effects. Opposing regulation of cell surface transporters by different patterns of PKC activation and different PKC isozymes has been reported. Brief activation with TPA increases activity of the Na+/HCO–3 transporter in proximal convoluted tubules, whereas longer incubations decreases inward transport (22). Angiotensin II elicits biphasic effects on bicarbonate transport in the proximal tubule, and both effects appear to be mediated by PKC (22–24). Na+,K+-ATPase is another transporter that is regulated biphasically by PKC. A PKC{beta}-dependent phosphorylation of the Na+,K+-ATPase {alpha} subunit recruits the transporter to the cell surface, whereas a PKC{zeta}-dependent phosphorylation of the same subunit induces endocytosis of the transporter (25–27).

Inhibition of TPA-mediated dopamine efflux by the highly specific PKC{beta} inhibitor LY379196 strongly suggests that PKC{beta} is the specific isoform that is responsible for DAT-mediated efflux of dopamine. The effects of PKC{alpha} and PKC{gamma} on amphetamine-mediated dopamine efflux were not investigated because of the lack of selective inhibitors for these isoforms. However, the co-immunoprecipitation studies and the studies with overexpression of PKC{alpha} strongly suggest that these isozymes neither bind to DAT nor affect DAT-mediated dopamine efflux. All four classical PKC isoforms are reported to exist in the dopamine cell body areas of Sprague-Dawley rats, the strain used in these studies (28), although the predominant form was PKC{beta}I.

This study demonstrated that both the PKC{beta}I and PKC{beta}II isozymes readily co-immunoprecipitated with DAT suggesting that they associate in the terminal. We do not know whether they bind directly to DAT or whether there is an intermediary protein. The receptor for activated C kinases (RACK1) has recently been demonstrated to bind to the N terminus of DAT (29). PKC{beta}II interacts specifically with RACK1, which consequently determines the localization and functional activity of PKC{beta}II (30). The PKC{alpha}-binding protein, PICK 1 (31), has been demonstrated to form a stable complex with the PDZ domain in the C terminus of DAT (32). It is unclear at this time whether PKC{alpha} plays any role in the ability of PICK 1 to increase the number of plasma membrane DATs (32). If so, a complex of PKC{alpha}, PICK 1, and DAT may not have been retained through our immunoprecipitation protocol, or PKC{alpha} may only bind when DAT is activated. PKC{alpha} complexes with the neuronal glutamate transporter EAAC1 upon PKC activation in C6 glioma cells and rat brain synaptosomes, and this binding correlates with the PKC-dependent redistribution of EAAC1 (33). Our study suggests that PKC{beta} is associated with DAT even under "resting" conditions. Co-immunoprecipitation studies revealed complexes between protein phosphatase 2Ac and DAT as well as the norepinephrine and serotonin transporters under basal conditions (34). Therefore it appears that DAT and other transporters can exist in complexes in the membrane that include scaffolding and signal transduction molecules.

Our experiments using hDAT-HEK 293 cells transfected with PKC isozymes suggest that the PKC{beta}II isozyme is important for amphetamine-stimulated efflux of dopamine through DAT. Overexpression of hDAT-HEK 293 cells (PKC{alpha} or PKC{beta}I) did not significantly alter the amphetamine-stimulated dopamine efflux from that of untransfected HEK 293 cells. Although one could argue that overexpression of PKC{alpha} may not have a sufficient effect because the HEK 293 cells already contained some level of PKC{alpha}, that argument would not be true for PKC{beta}I. The HEK 293 cells had low levels of PKC{beta}I and almost no PKC{beta}II. Only co-transfection with PKC{beta}II significantly increased the dopamine efflux in response to amphetamine.

The mechanism by which PKC{beta} alters amphetamine-stimulated dopamine efflux is currently under investigation. PKC{beta} could be directly phosphorylating DAT and altering efflux. A PKC-dependent phosphorylation of DAT has been demonstrated at sites that appear to be the most distal N-terminal serines (6). Recently we demonstrated that phosphorylation of the N-terminal serines in DAT is required for maximal amphetamine-induced dopamine efflux (12), but [3H]dopamine uptake is not affected. An alternative explanation is that PKC{beta} could participate in the transporter vesicle recycling or maintenance of surface expression of DAT. PKC{beta} increases the surface expression of both glucose transporter 1 and rodent proximal tubule Na+,K+-ATPase (25, 26). PKC{beta}II, as opposed to PKC{beta}I, has been demonstrated to associate with the actin cytoskeleton (35) and therefore could be involved in DAT trafficking. We are presently examining this possibility.

We and others have reported that amphetamine can activate PKC in rat striatum (36–39) and that Ca2+ seems to be involved in this activation. The amphetamine-induced activation of PKC involves activation of phospholipase C and Na+/Ca2+ exchange (38). Further, amphetamine can increase intracellular Ca2+ (14, 15). However, there is no direct evidence that indicates which isoforms of PKC are activated. We demonstrated that amphetamine increases PKC activity and stimulates in vivo and in vitro phosphorylation of GAP-43 on serine-41, which is the PKC substrate site (36, 40). There is evidence that GAP-43 is phosphorylated more actively by PKC{beta}, especially PKC{beta}II, as compared with PKC{alpha} or PKC{gamma} (41), suggesting that the amphetamine-stimulated increase in GAP-43 phosphorylation could be mediated by activation of PKC{beta}II.

By elucidating which PKC isoforms are involved in regulating amphetamine-mediated dopamine efflux we can better understand the mechanism by which amphetamine acts to reverse the transport of dopamine. These studies have demonstrated that PKC{beta}II is complexed with DAT and is important in the reverse transport of dopamine through DAT.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DA11697, Pharmacological Sciences Training Grant GM07767, and Michigan Diabetes Research and Training Center Grant 5P60DK-20572 from the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, 2220E MSRB III, University of Michigan Medical School, Ann Arbor, MI 48109-0632. Tel.: 734-763-5358; Fax: 734-763-4450; E-mail: pgnegy{at}umich.edu.

1 The abbreviations used are: DAT, dopamine transporter; PKC, protein kinase C; hDAT, human DAT; hDAT-HEK-293, human DAT-transfected human embryonic kidney cells; KRB, Kreb's-Ringer buffer; KRH, Krebs-Ringer Hepes buffer; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank Stephen Fisher for valuable conversations during the preparation of this manuscript. We also thank Minjia Zhang for technical expertise and assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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