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Research ArticleNEUROPHARMACOLOGY

Dopamine D1 and D2 Receptors Influence Dopamine Transporter Synthesis and Degradation in the Rat

Heather L. Kimmel, Andrew R. Joyce, F. Ivy Carroll and Michael J. Kuhar
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 129-140;

Abstract

Neurotransmitter transporters play an important role in maintaining synaptic homeostasis and in the actions of many drugs. Utilizing a technique for measuring the kinetics (synthesis, degradation, and half-life) of the dopamine transporter (DAT) protein in the rat striatum and nucleus accumbens, we have investigated the effects of systemic administration of dopamine receptor agonists and antagonists upon DAT kinetics in these brain regions. In the striatum, the dopamine D1 receptor agonist SKF38393 and the dopamine D1 receptor antagonist SCH23390 were without effect. However, the dopamine D2 receptor agonists R-(−)-propylnorapomorphine hydrochloride (NPA) and quinpirole decreased the half-life of DAT. This effect was blocked by the dopamine D2 antagonist eticlopride, which, by itself, increased the half-life of DAT. In the nucleus accumbens, the agonist SKF38393 increased the DAT half-life, whereas the antagonist SCH23390 decreased the half-life. In contrast to the striatum, NPA and quinpirole increased the DAT half-life, which was blocked by eticlopride and by itself had no effect on DAT kinetics. Cocaine increased the half-life of DAT in both the striatum and the nucleus accumbens. The results of the present study suggest that, through dopamine receptors, dopamine indirectly influences DAT protein turnover in the striatum and in the nucleus accumbens, but in different ways.

Neurotransmitter uptake into the presynaptic terminal or glial cells is an important mechanism for terminating the action of synaptic neurotransmitters, thus maintaining neuronal homeostasis (Amara and Kuhar, 1993). Mice with a genetic deletion of the dopamine transporter (DAT) exhibit elevated levels of dopamine and decreased rates of dopamine clearance (Gainetdinov et al., 1998). These proteins responsible for uptake are sites of action for many drugs with both therapeutic and abuse potential (Amara and Pacholczyk, 1991; Giros and Caron, 1993; Kuhar, 1993; Reith et al., 1997). Changes in DAT density and function are implicated in neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and addiction (Giros and Caron, 1993; Reith et al., 1997; Miller et al., 1999). Since DAT has several important functions, it is necessary to examine the localization and regulation of these transporter proteins.

In the rat, the highest density of DAT protein is found in the striatum, where the brain dopamine levels are highest. Although DAT proteins are abundant in the plasma membrane of axons and axon terminals, they are not observed within the active zones of the synapse (Nirenberg et al., 1996; Hersch et al., 1997; Kuhar et al., 1998). This suggests that dopamine uptake occurs primarily outside of synapses and that there is a diffusion gradient of dopamine in the synapse. DAT is colocalized with dopamine D2 receptors, although the presence of one does not determine the presence of the other (Hersch et al., 1997).

The balance of protein production and degradation in the cell determines the level of DAT protein. DAT proteins are synthesized in the rough endoplasmic reticulum, then transported to Golgi bodies for post-translational modifications. From the Golgi bodies, DAT proteins are distributed to dendritic and axonal smooth endoplasmic reticula, then to the plasma membrane (Hersch et al., 1997; Nirenberg et al., 1997b). Catabolism of DAT proteins takes place by double endocytosis into endosomes and into multivesicular body vesicles (Hersch et al., 1997). Systemic administration of the DAT blocker buproprion increases DAT mRNA in the ventral tegmental area and substantia nigra of rats, suggesting that DAT mRNA expression in the brain is regulated by dopaminergic mechanisms (Petrie et al., 1998).

Changes in DAT result in alterations in dopamine neurotransmission by affecting dopamine inactivation (Reith et al., 1997). Internal cellular mechanisms have been shown to play a role in regulation of dopamine transport. For example, cAMP enhances dopamine uptake in rat hypothalamic tuberoinfundibular neurons (Kadowaki et al., 1990). Protein kinase C (PKC) activation decreases dopamine uptake by DAT in the rat (Kitayama et al., 1994) and in the human (Huff et al., 1997). Protein kinase C activation also causes a rapid sequestration of human DAT, which is then recycled rather than degraded (Huff et al., 1997;Pristupa et al., 1998; Melikian and Buckley, 1999). Conversely, protein kinase C inhibition increases DA uptake by the insertion of internalized or intracellular transporters to the plasma membrane (Pristupa et al., 1998). It has been suggested that other post-translational modifications may also contribute to the function of transporter proteins by altering the distribution of these proteins within the cell (Amara and Pacholczyk, 1991).

Previously, we developed a method for measuring the production rate, the degradation rate constant, and the half-life of the rat DAT protein using the irreversible DAT ligand, RTI-76 [3β-(3-p-chlorophenyl) tropan-2β-carboxylic acidp-isothiocyanatophenylethyl ester hydrochloride] (Kimmel et al., 2000). In that study, we determined the half-life of the DAT protein in both the rat striatum and nucleus accumbens to be about 2 days. In the present study, we sought to determine how stimulation and blockade of the D1 and D2 dopamine receptor families affect the degradation rate constant and the production rate of DAT, and thus its half-life in the rat striatum and nucleus accumbens. We administered the dopamine D1-like receptor agonist, SKF38393, the dopamine D1-like receptor antagonist, SCH23390, the dopamine D2-like receptor agonists, quinpirole, and R-(−)-propylnorapomorphine hydrochloride (NPA), or the dopamine D2-like receptor antagonist, eticlopride, systemically to rats, then determined DAT protein kinetics in the two brain regions. We also administered the psychostimulant cocaine to rats to determine the effect of an indirect dopamine agonist on the kinetics of DAT in both brain regions.

Materials and Methods

Animals.

Before surgery, male Sprague-Dawley rats (250–275 g, Charles River, Raleigh, NC) were grouped three per cage, with food and water available at all times. Animals were housed with a 12-h light/dark cycle. The animals were maintained in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH/85-23), and all experimental protocols were approved by the Institutional Animal Care and Use Committee.

Drug Treatment and Surgery.

Animals were randomly divided into seven treatment groups that were given SKF38393, SCH23390, NPA, quinpirole, eticlopride, eticlopride + quinpirole, or cocaine (n = 64 for each treatment group) (Table1). Each group of animals was treated by injections once a day for three consecutive days. On the fourth day, each group of rats (n = 64) was given a unilateral injection of saline (n = 32) or 100 nmol of RTI-76 (n = 32) in the right lateral ventricle (Fleckenstein et al., 1996). Animals were anesthetized with 400 mg/kg i.p. of chloral hydrate and placed in a stereotaxic frame. Stereotaxic coordinates used relative to bregma were: antero-posterior = −0.8, medio-lateral = −1.4, dorso-ventral = −4.0. A 25-μl Hamilton syringe was used to inject 10 μl of saline or RTI-76 solution over a 1-min period. Upon completion of the injection, the needle was kept in place for 3 min to minimize the back flow of the solution. Since RTI-76 is light- and temperature-sensitive, all manipulations of this compound were done under low-light conditions, and the containers were wrapped in foil and kept on ice.

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Table 1

Description of a drug treatment group

Following recovery from surgery, all animals received their respective saline or drug treatment every day thereafter until their sacrifice. Animals were sacrificed at 1, 2, 3, and 7 days after i.c.v. injection of RTI-76 or saline. At each day of sacrifice, 16 animals from each group were selected, so that for each of the four time points, the following treatments were used: i.c.v. saline + systemic saline (n = 4), i.c.v. saline + systemic treatment drug (n = 4), i.c.v. RTI-76 + systemic saline (n = 4), and i.c.v. RTI-76 + systemic treatment drug (n = 4). This is shown schematically in Table 1. The drug doses that were used in this study were: 3.0 mg/kg of SKF38393 (s.c.), 0.5 mg/kg of SCH23390 (i.p.), 0.1 mg/kg of NPA (i.p.), 0.3 mg/kg of quinpirole (i.p.), 0.5 mg/kg of eticlopride (i.p.), and 20 mg/kg of cocaine (i.p.). The systemic route of administration of each compound was based on those reported in the literature.

Preparation of Membranes.

Brains were removed immediately and placed in cold saline. The olfactory tubercles were removed with the aid of fine forceps, exposing the diagonal bands of Broca. A razor blade was used to cut away the frontal cortex and to dissect the nucleus accumbens from both hemispheres. The pair of nucleus accumbens was pooled for each animal, for a total weight of 10 to 20 mg. Fine forceps were used to remove the right striatum, weighing 20 to 30 mg. No differences between left and right striatum were found in this assay (data not shown). All tissues were removed rapidly and frozen on dry ice and then stored at −80°C until assayed within 4 weeks.

On the day of the assay, each tissue was weighed and then placed in the appropriate ice-cold assay buffer (see herein). The tissue was then homogenized with a Polytron (Brinkman Instruments Co., Cantiague Road, Westbury, NY) (setting 5 for 15 s). The homegenate was centrifuged for 10 min at 30,000g and the pellet suspended in buffer. The homogenate was centrifuged again and the pellet resuspended in buffer.

[3H]GBR12935 Binding.

After the final centrifugation, the tissue pellet was resuspended in buffer for a final concentration of 5.0 mg wet tissue weight/ml. Polystyrene test tubes were filled with 1.5 ml buffer (50 mM Tris-HCl, pH 7.7 at 24°C, containing 120 mM NaCl and 0.01% bovine serum albumin), 100 μl of buffer, 400 μl of radioligand (0.5, 2.0, 5.0, or 20 nM final concentration), and 40 μl of tissue for a total volume of 2.04 ml. Nonspecific binding values were generated by adding 100 μl of 100 μM mazindol (5.0 μM final) instead of buffer. Tubes were incubated at room temperature for 60 min, and the incubations were terminated by rapid filtration over Whatman GF/B filters that were pretreated with 0.05% polytheylenimine. The filters were rinsed 3 times with 4.0 ml of ice-cold buffer, and the radioactivity remaining on the filters was measured by conventional liquid spectrometry.

Drugs.

RTI-76 was obtained from the Research Triangle Institute (Research Triangle Park, NC). [3H]GBR12935 (specific activity = 53.5 Ci/mmol with label position at propylene-2,3-[3H]) was obtained from PerkinElmer Life Science Products (Boston, MA). Bovine serum albumin, chloral hydrate, eticlopride, mazindol hydrochloride, NPA, quinpirole, SCH23390, SKF38393, polyethylenimine, and potassium chloride were obtained from Sigma (St. Louis, MO). Cocaine hydrochloride was a generous gift from the National Institutes of Drug Abuse (Bethesda, MD). Sodium chloride was obtained from Fisher Scientific (Pittsburgh, PA). Tris hydrochloride and Tris base were obtained from EM Science (Cincinnati, OH).

Chloral hydrate, cocaine, eticlopride, NPA quinpirole, RTI-76, and SKF38393 were dissolved in 0.9% saline. SCH23390 was dissolved in distilled water, then diluted with 0.9% saline. Compounds used in the binding assay were dissolved in the appropriate buffer.

Binding Data Curve Fitting.

Bmax andKd values were determined in each binding assay by fitting a nonlinear least-squares analysis and by a linear Scatchard plot. Data were analyzed using GB-STAT version 6.5 (Dynamic Microsystems, Silver Spring, MD).

Calculation of DAT Kinetic Parameters.

The half-life of recovery of [3H]GBR12935 binding of DAT in the striatum and the nucleus accumbens following inactivation by RTI-76 was determined, assuming a zero-order transporter production rate and a first-order transporter degradation rate constant. The repopulation kinetics of an irreversibly inactivated transporter can be defined by the monoexponential repopulation equation (Mauger et al., 1982;Sladeczek and Bockaert, 1983):Bt=(r/k)(1−e−kt). Equation 1Here, Bt is the transporter concentration (fmol/mg of protein) at time t, ris the transporter production rate (fmol/mg of protein/h), andk is the transporter degradation rate constant (h−1). As time nears infinity, transporter recovery reaches the steady-state levels (Bss), and the terme−kt approaches 0, so eq. 1 may be restated as:Bt=Bss=r/k. Equation 2Substituting Bss for the termr/k in eq. 1,Bt=Bss(1−e−kt), then performing a logarithmic transformation results in:ln[Bss/(Bss−Bt)]=kt Equation 3where Bt <Bss.

This is an equation for a straight line (y =mx + b, where b = 0). From our experiments, where we have the Bmax in the control animals (Bss) and in the RTI-76-treated animals at each time point (Bt), we can obtain k from the slope of the plot ln [Bss/(Bss− Bt)] versus time. [3H]GBR12935 binding at DAT is used to determine Bt andBss. In some of these graphs,Bt was so close toBss that (Bss −Bt) was close to zero at the 7-day time point and could not be plotted; therefore, some graphs have three points, whereas others have four points.

The time for Bt to reach any particular percentage of control Bssvalues is dependent on k but not on r. Therefore, changes in k would change the time needed to reach a certain percentage of control, Bss. To determine the half-life of recovery, i.e., t =t1/2, letBt =Bss/2 and eq. 3 becomes ln 2 =kt1/2(t1/2 = ln 2/k = 0.693/k). Since we have the experimentally determinedBss and the calculated k, we can obtain r from restating eq. 2 as r =kBss. In these studies,Bss was taken to be the concentration of recoverable transporters (Bmaxaverage of saline controls minus residualBmax at 1 day after RTI-76 treatment).

Time Course Statistics.

The time course data were analyzed using a one-way ANOVA, followed by a Tukey's post hoc test when appropriate. Points were determined to be significantly different from saline at p < 0.05.

Results

Striatum.

The time course of recovery of [3H]GBR12935 binding to DAT in the striatum of animals that received systemic saline injections for 3 days followed by i.c.v. injection of saline or RTI-76 is shown in Fig.1A. Each drug treatment study that follows includes both of these control groups (see Table2). However, since the control groups were similar, we combined all of the data from animals that received systemic saline and i.c.v. saline into this single figure. TheBmax values of animals that received i.c.v. saline did not differ across the time points studied, so these values were pooled and are given in the last column of Table 2.

Figure 1
Figure 1

[3H]GBR12935 binding in the rat striatum following i.c.v. saline or RTI-76 during systemic treatment with saline for 3 days before i.c.v. injection and continuing until sacrifice (A). The data presented here were combined using data from each of the systemic saline control groups shown in Table 1. These data are represented in a semilogarithmic plot in (B), which has a correlation coefficient of 1.00. The slope of this line is used to determine k as described under Materials and Methods. **Different from saline at that time point,p < 0.01. ICV, intracerebroventricular.

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Table 2

Summary of DAT kinetic parameters in the striatum

Animals that received daily systemic saline injections (i.p. or s.c., determined by the route of administration of the test compound) for 3 days followed by i.c.v. administration of 100 nmol of RTI-76 exhibited an initial decrease (to 61% of control) in [3H]GBR12935 binding to striatal DAT 1 day later. Binding returned to control levels by the seventh day following RTI-76 administration. Control animals that received systemic saline for 3 days followed by i.c.v. saline did not exhibit any significant differences in [3H]GBR12935 binding between days 1, 3, 4, and 7 (Fig. 1). In the striatum, the half-life of DAT was determined to be 2.1 ± 0.05 days (mean ± S.E.M.), whereas the degradation rate constant was determined to be 0.34 ± 0.01 days−1, and the production rate was calculated to be 202 ± 6.0 fmol/mg/day (Table 2; Fig. 1B).

Neither treatment with the dopamine D1 receptor agonist SKF38393 nor with the dopamine D1 receptor antagonist SCH23390 altered DAT kinetics in the striatum (Table 2; Fig. 2). In addition, neither SKF38393 nor SCH23390 significantly altered [3H]GBR12395 binding to DAT in i.c.v. saline-treated animals on any day (Fig. 2, A to D).

Figure 2
Figure 2

[3H]GBR12935 binding in the rat striatum following i.c.v. saline or RTI-76, during systemic treatment with a dopamine D1 receptor agonist or antagonist for 3 days before i.c.v. injection and continuing until sacrifice. A, in rats treated with the dopamine D1 agonist SKF38393, RTI-76 decreased DATBmax to 77% of control on day 1. By day 7, DAT Bmax was 101% of control. A two-way ANOVA showed a significant effect of i.c.v. injection of RTI-76 [F(1,20) = 11.35, p = 0.003], but not of day [F(3,20) = 2.64, NS] nor of the interaction [F(3,20) = 1.63, NS]. C, in rats treated with the dopamine D1 receptor antagonist SCH23390, RTI-76 decreased DAT Bmax to 74% of control. By day 7, DAT Bmax was 97% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,21) = 13.68, p = 0.001], but not of day [F(3,21) = 3.03, NS] nor of the interaction [F(3,21) = 0.87, NS]. These data are represented in semilogarithmic plots in B and D. The correlation coefficients for saline and SKF38339 are 0.98 and 1.00, respectively (B). The correlation coefficients for saline and SCH23390 are 0.98 and 0.99, respectively (D). ICV, intracerebroventricular.

In contrast, treatment with the dopamine D2 receptor agonists NPA and quinpirole and with the dopamine D2 antagonist eticlopride altered DAT kinetics in the striatum (Table 2; Fig.3). Both NPA (Fig. 3B) and quinpirole (Fig. 3D) decreased the half-life of DAT in the striatum and increased the degradation and production rates (Table 2). Conversely, eticlopride (Fig. 3F) increased the half-life of DAT and decreased the degradation and production rates (Table 2). Pretreatment with eticlopride also reversed the decrease in DAT half-life observed after quinpirole (Fig. 3H; Table 2). In addition, neither NPA, quinpirole, eticlopride, nor eticlopride + quinpirole significantly altered [3H]GBR12935 binding to DAT in i.c.v. saline-treated animals on any day (Fig. 3, A, C, E, and G).

Figure 3
Figure 3

[3H]GBR12935 binding in the rat striatum following i.c.v. saline or RTI-76 during systemic treatment with a dopamine D2 receptor agonist, antagonist, or the combination for 3 days before i.c.v. injection and continuing until sacrifice. A, in rats treated with the dopamine D2 receptor agonist NPA, RTI-76 decreased DAT Bmax to 66% of control on day 1. By day 7, DAT Bmax was 103% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,21) = 27.44, p < 0.0001], of day [F(3,21) = 4.88,p = 0.01], and of the interaction [F(3,21) = 5.58), p = 0.006]. (C) In rats treated with the dopamine D2 receptor agonist quinpirole, RTI-76 decreased DAT Bmax to 54% of control on day 1. By day 7, DAT Bmax was 103% of control. A two-way ANOVA showed a significant effect of RTI-76 treatment [F(1,20) = 32.32, p< 0.0001], of day [F(3,20) = 9.97,p = 0.0003], and of the interaction [F(3,20) = 8.19, p = 0.0009]. E, in rats treated with the dopamine D2 receptor antagonist eticlopride, RTI-76 decreased DAT Bmax to 72% of control on day 1. By day 7, DAT Bmax was 94% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,18) = 47.65, p< 0.0001] and of day [F(3,18) = 3.61,p = 0.03], but not of the interaction [F(3,18) = 2.39, NS]. G, in rats treatedwith the combination of eticlopride and quinpirole, RTI-76 decreased DAT Bmax to 62% of control on day 1. By day 7, DAT Bmax was 99% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,20) = 57.99, p< 0.0001], of day [F(3,20) = 12.94,p < 0.0001], and of the interaction [F(3,20) = 6.55, p = 0.003]. These data are represented in semilogarithmic plots in B, D, F, and H. The correlation coefficients for saline and NPA are 1.00 and 1.00, respectively (B). The correlation coefficients for saline and quinpirole are 0.92 and 0.95, respectively (D). The correlation coefficients for saline and eticlopride are 0.97 and 0.97, respectively (F). The correlation coefficients for saline and eticlopride + quinpirole are 1.00 and 1.00, respectively (H). *Different from saline at that time point, p < 0.05. **Different from saline at that time point, p < 0.01. ICV, intracerebroventricular.

Administration of the nonspecific and indirect dopamine agonist cocaine increased the half-life of DAT while decreasing the DAT degradation rate constant and the production rate (Table 2; Fig.4). In addition, cocaine administration did not significantly alter [3H]GBR12935 binding to DAT in the striatum of i.c.v. saline-treated animals on any day (Fig. 4A).

Figure 4
Figure 4

[3H]GBR12935 binding in the rat striatum following i.c.v. saline or RTI-76 during systemic treatment with the nonspecific dopamine receptor agonist cocaine for 3 days before i.c.v. injection and continuing until sacrifice. A, RTI-76 decreased DAT Bmax to 58% of control on day 1. By day 7, DAT Bmax was 90% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,16) = 72.42, p < 0.0001], of day [F(3,16) = 5.87,p = 0.007], and of the interaction [F(3,16) = 6.97, p = 0.003]. These data are represented in a semilogarithmic plot in (B). The correlation coefficients for saline and cocaine are 1.00 and 0.99, respectively (B). **Different from saline at that time point,p < 0.01. ICV, intracerebroventricular.

Nucleus Accumbens.

The time course of recovery of [3H]GBR12935 binding to DAT in the nucleus accumbens of animals that received systemic saline injections for 3 days followed by i.c.v. injection of saline or RTI-76 is shown in Fig.5A. Each drug treatment study that follows includes both of these control groups (see Table3). However, since the control groups were similar, we combined all of the data from animals that received systemic saline and i.c.v. saline into this single figure. TheBmax values of animals that received i.c.v. saline did not differ across the time points studied, so these values were pooled and are given in the last column of Table 3.

Figure 5
Figure 5

[3H]GBR12935 binding in the rat nucleus accumbens following i.c.v. saline or RTI-76 during systemic treatment with saline for 3 days before i.c.v. injection and continuing until sacrifice (A). The data presented here were combined using data from each of the systemic saline control groups shown in Table 2. These data are represented in a semilogarithmic plot in (B), which has a correlation coefficient of 1.00. **Different from saline at that time point, p < 0.01. ICV, intracerebroventricular.

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Table 3

Summary of DAT kinetic parameters in the nucleus accumbens

Animals that received daily saline injections (i.p. or s.c., determined by the route of administration of the test compound) for 3 days followed by i.c.v. administration of 100 nmol of RTI-76 exhibited an initial decrease (to 55% of control) in [3H]GBR12935 binding to DAT 1 day later. Binding returned to control levels by the seventh day following RTI-76 administration. Control animals that received i.p. saline for 3 days followed by i.c.v. saline did not exhibit any significant differences in [3H]GBR12935 binding between days 1, 3, 4, and 7. For DAT in the nucleus accumbens, the half-life was calculated to be 2.1 ± 0.05 days (mean ± S.E.M.), whereas the degradation rate constant was determined to be 0.34 ± 0.01 days−1, and the production rate was calculated to be 106 ± 6.2 fmol/mg/day (Fig. 5B; Table 3).

Treatment with the dopamine D1 receptor agonist SKF38393 and with the dopamine D1 receptor antagonist SCH23390 altered DAT kinetics in the nucleus accumbens (Table 3; Fig. 6). SKF38393 (Fig. 6B) increased the half-life of DAT while decreasing the degradation rate constant and the production rate in the nucleus accumbens (Table 3). Conversely, SCH23390 (Fig. 6D) decreased the half-life of DAT while increasing the degradation rate constant and the production rate (Table 3). In addition, neither SKF38393 nor SCH23390 significantly altered [3H]GBR12935 binding to DAT in i.c.v. saline-treated animals on any day (Fig. 6, A to D).

Figure 6
Figure 6

[3H]GBR12935 binding in the rat nucleus accumbens following i.c.v. saline or RTI-76 during systemic treatment with a dopamine D1 receptor agonist or antagonist for 3 days before i.c.v. injection and continuing until sacrifice. A, in rats treated with the dopamine D1 receptor agonist SKF38393, RTI-76 decreased DATBmax to 63% of control. By day 7, DATBmax was 91% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,15) = 53.42, p < 0.001], of day [F(3,15) = 7.04,p = 0.004] and of the interaction [F(3,15) = 4.85, p = 0.015]. C, in rats treated with the dopamine D1 receptor antagonist SCH23390, RTI-76 decreased DAT Bmax to 49% of control on day 1. By day 7, DAT Bmax was 100% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,17) = 7.94, p = 0.012], but not of day [F(3,17) = 2.52, NS] or of the interaction [F(3,17) = 2.25, NS]. These data are represented in semilogarithmic plots in B and D. The correlation coefficients for saline and SKF38339 are 1.00 and 0.98, respectively (B). The correlation coefficients for saline and SCH23390 are 1.00 and 1.00, respectively (D). *Different from saline at that time point, p < 0.05. **Different from saline at that time point, p < 0.01. ICV, intracerebroventricular.

Treatment with either of the dopamine D2 receptor agonists NPA or quinpirole or with the dopamine D2 receptor antagonist eticlopride also altered DAT kinetics in the nucleus accumbens (Table 2; Fig.7). Treatment with NPA (Fig. 7B) and with quinpirole (Fig. 7D) each increased the half-life of DAT in the nucleus accumbens, while decreasing both the degradation rate constant and the production rate (Table 2). However, treatment with eticlopride (Fig. 7F), did not alter DAT kinetics in this brain region (Table 2), but pretreatment with eticlopride reversed the increase in DAT half-life observed after quinpirole (Fig. 7H; Table 2). In addition, neither NPA, quinpirole, eticlopride, nor eticlopride + quinpirole significantly altered [3H]GBR12935 binding to DAT of i.c.v. saline-treated animals on any day (Fig. 7, A, C, E, and G).

Figure 7
Figure 7

[3H]GBR12935 binding in the rat nucleus accumbens following i.c.v. saline or RTI-76 during systemic treatment with a dopamine D2 receptor agonist, antagonist, or the combination for 3 days before i.c.v. injection and continuing until sacrifice. A, in rats treated with the dopamine D2 receptor agonist NPA, RTI-76 decreased DAT Bmax to 55% of control on day 1. By day 7, DAT Bmax was 103% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,10) = 27.36, p= 0.0004], but not of day [F(3,10) = 2.67, NS] or of the interaction [F(3,10) = 2.24, NS]. C, in rats treated with the dopamine D2 receptor agonist quinpirole, RTI-76 decreased DAT Bmax to 64% of control on day 1. By day 7, DAT Bmax was 106% of control. A two-way ANOVA showed a significant effect of RTI-76 treatment [F(1,19) = 20.71, p = 0.0002], of day [F(3,19) = 5.30,p = 0.008], and of the interaction [F(3,19) = 3.54, p = 0.034]. E, in rats treated with the dopamine D2 receptor antagonist eticlopride, RTI-76 decreased DAT Bmax to 60% of control on day 1. By day 7, DAT Bmax was 95% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,15) = 27.90, p< 0.0001] and of the interaction [F(3,15) = 4.66, p = 0.017], but not of day [F(3,15) = 1.21, NS]. G, in rats treated with the combination of eticlopride and quinpirole, RTI-76 decreased DATBmax to 60% of control on day 1. By day 7, DAT Bmax was 93% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,18) = 26.27, p < 0.0001], but not of day [F(3,18) = 1.73, NS] or of the interaction [F(3,18) = 2.72, NS]. These data are represented in semilogarithmic plots in B, D, F, and H. The correlation coefficients for saline and NPA are 0.99 and 0.99, respectively (B). The correlation coefficients for saline and quinpirole are 0.95 and 0.94, respectively (D). The correlation coefficients for saline and eticlopride are 1.00 and 0.98, respectively (F). The correlation coefficients for saline and eticlopride + quinpirole are 0.98 and 1.00, respectively (H). **Different from saline at that time point, p < 0.01. ICV, intracerebroventricular.

Administration of the nonspecific and indirect dopamine agonist cocaine increased the half-life of DAT in the nucleus accumbens while decreasing the DAT degradation rate constant and the production rate (Table 2; Fig. 8). In addition, cocaine did not significantly alter [3H]GBR12935 binding to DAT in i.c.v. saline-treated animals on any day (Fig. 8A).

Figure 8
Figure 8

[3H]GBR12935 binding in the rat nucleus accumbens following i.c.v. saline or RTI-76 during systemic treatment with the nonspecific dopamine receptor agonist cocaine for 3 days before i.c.v. injection and continuing until sacrifice. A, RTI-76 decreased DAT Bmax to 58% of control on day 1. By day 7, DAT Bmax was 87% of control. A two-way ANOVA showed a significant effect of i.c.v. injection [F(1,13) = 78.86, p < 0.0001], of day [F(3,13) = 5.47,p = 0.012], but not of the interaction [F(3,13) = 2.27, NS]. These data are represented in a semilogarithmic plot in B. The correlation coefficients for saline and cocaine are 0.98 and 0.96, respectively (B). *Different from saline at that time point, p < 0.05. **Different from saline at that time point, p < 0.01. ICV, intracerebroventricular.

Discussion

In this study, we confirmed previous results in which we determined that the half-life of DAT in the rat striatum and nucleus accumbens was approximately 2 days (Kimmel et al., 2000). The degradation rate constant was similar for the two regions, but the synthesis rate was higher in the striatum, reflecting a higher density of DAT in this brain region (see Tables 1 and 2). The synthesis rate and the degradation rate constant measured here reflect the summations of complex processes. The calculated overall synthesis rate includes DAT protein synthesis, axonal flow, and insertion at the nerve terminal, whereas the experimentally determined degradation rate constant reflects retrieval from the cell membrane, degradation, and retrograde transport. Although we show dopamine receptors clearly influence DAT kinetics, the precise mechanism by which synthesis and degradation are affected is not yet clear.

Acute and repeated stimulation of dopamine receptors can influence dopamine uptake by DAT in the striatum and the nucleus accumbens. In vitro rotating disk electrode voltametry studies using rat striatal synaptosomal suspensions showed that acute administration of the dopamine D2 receptor agonist, quinpirole, increased dopamine uptake (Meier-gerd et al., 1993). Repeated systemic administration of the dopamine D2 receptor antagonist, pimozide, blocked the up-regulation of dopamine uptake and release induced by repeated cocaine administration in the rat (Parsons et al., 1993). The absence of dopamine receptors can also influence DAT function, as shown by dopamine D2 receptor-deficient mice, which exhibit decreased striatal DAT uptake (Dickinson et al., 1999). Although these studies suggest that dopamine receptor stimulation can influence DAT function, the present study is the first to show that DA receptors can influence the synthesis rate and degradation rate constant of DAT proteins. In these experiments, dopamine receptor agonists and/or antagonists were administered systemically for 3 days before the i.c.v. injection of RTI-76 and continued until sacrifice. Thus, the receptor stimulation or inhibition was repeated, but only once daily, during the study. Although RTI-76 binds to DAT (Carroll et al., 1992) and presumably increases dopamine levels, recent studies in our laboratory show that the dose of RTI-76 used in this study (100 nmol) does not produce locomotor activity, although doses that are 25-fold higher did (Kimmel et al., 2001). Thus, the dose of RTI-76 used in this study is not a behaviorally active dose, and although we did not measure dopamine levels, it is unlikely that they were increased to a functionally significant degree.

The effects of the dopamine agonists and antagonists within each region were very consistent, but differed between the two regions. In the striatum, DAT half-life was decreased by both dopamine D2 receptor agonists, and increased by the dopamine D2 receptor antagonist. Furthermore, the agonist-induced change in DAT kinetics was inhibited by the coadministration of an antagonist. Dopamine D1 receptor agonists and antagonists had no effect on DAT kinetics in the striatum. In contrast, in the nucleus accumbens, the dopamine D1 receptor agonist increased the half-life of DAT, whereas the dopamine D1 receptor antagonist decreased it. Two dopamine D2 receptor agonists increased DAT half-life, an effect that was inhibited by coadministration with a dopamine D2 receptor antagonist. Alone, the dopamine D2 receptor antagonist had no effect on DAT half-life. Despite the different regional effects on DAT induced by the dopamine receptor-related compounds, the DAT inhibitor, cocaine, which is a nonspecific and indirect dopamine receptor agonist, increased DAT half-life in both brain regions. The finding that some antagonists alone can alter DAT half-life suggests that endogenous dopamine tone affects DAT kinetics.

The strikingly different effects of dopamine D1 and D2 receptor-selective compounds on DAT kinetics between the two brain regions are presumably due to differences in dopamine receptor localization and/or function in the nigrostriatal and mesolimbic pathways. The dopamine D1 receptor is the most widespread of all the dopamine receptors and is generally expressed at a higher level in the brain than the other dopamine receptors. The mRNA for this receptor is found in many brain regions, including the striatum and nucleus accumbens (Vallone et al., 2000). In particular, the striatum of the rat, monkey, and human is characterized by dopamine D1 receptor-rich patches that are also rich in dopamine and tyrosine hydroxylase (Graybiel, 1984, 1990). In the striatum of the rat, DAT is localized in the plasma membrane of axons and terminals, along with D2 dopamine receptors, but not with D1 dopamine receptors (Hersch et al., 1997). However, the nucleus accumbens shell contains patches of dopamine D1 receptors that do not contain high levels of dopamine D2 receptors or DAT. Areas containing high levels of dopamine D2 receptors and DAT surround these patches. In the core, however, dopamine D1 receptor-rich compartments are also rich in dopamine D2 receptors, but low in dopamine and tyrosine hydroxylase (Jansson et al., 1999). Within these brain regions, the majority of D1 and D2 receptors are found postsynaptically, although D2 receptors are also found presynaptically in dopamine neurons, where they function as autoreceptors (Levey et al., 1993; Sesack et al., 1994; Yung et al., 1995). The influence of D2 receptor ligands on DAT turnover may be partially explained by the colocalization of D2 receptors with DAT on the presynaptic membrane, as described previously.

Moreover, the signal transduction pathways are different for dopamine D1 versus dopamine D2 receptors; dopamine D1 receptor stimulation activates intracellular adenylate cyclase whereas dopamine D2 receptor stimulation inhibits adenylate cyclase (Sibley et al., 1993; Jaber et al., 1996). In mammalian species, the DAT protein has several sites for phosphorylation by cAMP-dependent protein kinase A and PKC (Shimada et al., 1991; Giros and Caron, 1993). Activation of PKC inhibits DAT activity in mammalian cells by phosphorylation of DAT (Copeland et al., 1996; Huff et al., 1997; Vaughan et al., 1997; Zhang et al., 1997), which then causes a rapid internalization of the protein from the cell membrane (Pristupa et al., 1998; Daniels and Amara, 1999; Melikian and Buckley, 1999). In contrast, the inhibition of protein kinase A or PKC produces an increase in dopamine uptake by the insertion of internalized or intracellular transporters to the plasma membrane (Pristupa et al., 1998). Thus, stimulation or inhibition of second messenger systems can influence DAT protein localization and, perhaps, levels. Further experiments are necessary to determine by which pathways dopamine D1 and D2 receptor stimulation influences DAT turnover in the brain regions studied.

Whereas the difference in second messenger coupling might explain the different effects of the dopamine D1- and D2-receptor selective drugs on DAT kinetics within one region, the mechanism underlying the regional differences are not clear. However, if the signal transduction pathways are dissimilar in these brain regions, this difference, coupled with the differences in the cellular location of dopamine D2 receptors in these two regions, might begin to explain how DAT proteins could be differentially regulated in these two brain regions. In slices of rat striatum, dopamine D2 receptors mediate a selective inhibition of dopamine D1 receptor-stimulated cAMP accumulation, but in the nucleus accumbens, dopamine D2 receptors are not coupled to dopamine D1 receptor-linked adenylate cyclase (Kelly and Nahorski, 1987).

In addition to the disparity in the localization of the dopamine receptors in the striatum and nucleus accumbens, DAT density is significantly lower in the nerve terminals of the nucleus accumbens than in those of the striatum (Cass et al., 1992; Nirenberg et al., 1996, 1997a). As a result, dopamine is cleared from the synapse more quickly in the striatum than in the nucleus accumbens, presumably due to the increased density of DAT (Cass and Gerhardt, 1995). However, it is unclear how this discrepancy in DAT density would account for the different effects of dopaminergic compounds on DAT kinetics. One issue to consider is that the dopamine D1- and D2-receptor ligands were administered systemically to the animals, which could recruit many differing influences on the two different DA containing pathways. Thus, trying to determine the mechanisms underlying these receptor-mediated differences can be difficult. Future experiments using more localized administration of the drugs into brain or using an appropriate cell line may be useful.

Although the results clearly show that receptors can influence the synthesis rate, degradation rate constant, and half-life of DAT, the physiological significance of the receptor-mediated influence is not clear, since significant changes in the density of DAT were not observed following treatment with any of the dopaminergic ligands. Despite the lack of a clear physiological role of this regulation, the present results suggest an important relationship between dopamine receptor activation and the kinetics of the DAT protein.

Acknowledgments

We acknowledge the expert technical assistance of April B. Chang and Steven C. Payne.

Footnotes

  • This research was supported by the National Institutes of Health Grants RR00165, DA00418, DA10732, and DA005935.

  • Abbreviations:
    DAT
    dopamine transporter
    DA
    dopamine
    RTI-76
    3β-(3-p-chlorophenyl) tropan-2β-carboxylic acid p-isothiocyanato-phenylethyl ester hydrochloride
    NPA
    R-(−)-propylnorapomorphine hydrochloride
    i.c.v.
    intracerebroventricular
    ANOVA
    analysis of variance
    PKC
    protein kinase C
    • Received November 16, 2000.
    • Accepted April 4, 2001.

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Research ArticleNEUROPHARMACOLOGY

Dopamine D1 and D2 Receptors Influence Dopamine Transporter Synthesis and Degradation in the Rat

Heather L. Kimmel, Andrew R. Joyce, F. Ivy Carroll and Michael J. Kuhar
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 129-140;
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