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Research ArticleArticle

Characteristics of Drug Interactions with Recombinant Biogenic Amine Transporters Expressed in the Same Cell Type

Amy J. Eshleman, Marya Carmolli, Medhane Cumbay, Carey R. Martens, Kim A. Neve and Aaron Janowsky
Journal of Pharmacology and Experimental Therapeutics May 1999, 289 (2) 877-885;

Abstract

We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (K d = 1.83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k −1) was significantly lower for hSERT and the association rate (k +1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (K d = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists.

Sodium- and chloride-sensitive biogenic amine transporters are the major mechanism for terminating the neurotransmission of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) (reviewed in Amara and Kuhar, 1993). Abused drugs, such as cocaine and amphetamine, as well as therapeutic agents such as mazindol and imipramine, inhibit neurotransmitter uptake by the transporters and cause an increase in synaptic neurotransmitter concentrations. In animal models of drug abuse, the binding of cocaine and other drugs to the DAT may play a critical role in self-administration (Ritz et al., 1987). The blockade of DAT may also be involved in the reinforcing properties and abuse potential of drugs in humans (Kuhar et al., 1991; Rothman et al., 1993); indeed, the magnitude of DAT occupancy by cocaine in humans, as measured by positron emission tomography, is correlated with self-reported highs (Volkow et al., 1997). In addition, some abused substances such as amphetamine are transporter substrates and induce the release of neurotransmitter. Studies with DAT knockout mice (Giros et al., 1996) confirm that these substrates have two sites of action: release of DA from vesicular stores and via the DAT. The DAT is necessary for increased extracellular DA concentrations after amphetamine administration (Jones et al., 1998). However, other binding sites for cocaine, perhaps including the NET and SERT, play important roles in the behavioral effects of this drug because DAT knockout mice will develop cocaine-conditioned place preference, a model for assessment of drug reward (Sora et al., 1998).

An elusive goal of drug abuse pharmacotherapy has been the development of a therapeutic agent for cocaine abuse (Vocci et al., 1995). Many possible strategies are being investigated, including the development of functional antagonists such as γ-vinyl-γ-aminobutyric acid (Morgan and Dewey, 1998), the development of vaccines to cocaine (Fox, 1997), the enhancement of cocaine metabolism via administration of enzymes (Gorelick, 1997), or slow-onset, long-acting, agonist-based medications (Glowa et al., 1996). One possible method of screening for a pharmacological cocaine antagonist would be to compare the potencies of compounds for inhibition of DA uptake and cocaine analog binding under similar or identical conditions (Rothman et al., 1993; Slusher et al., 1997). A compound that has much greater potency at inhibition of binding than uptake may be a potential “DA-sparing” cocaine antagonist. To establish a screening system, we stably expressed the DAT, NET, and SERT in human embryonic kidney (HEK) 293 cells and characterized cocaine analog binding and neurotransmitter uptake. The transfected, adherent HEK 293 cells continued to take up [3H]neurotransmitter after detachment from tissue culture plates and were therefore useful for assays involving robotic pipetting and assay termination by filtration. An array of drugs, both therapeutic and those with abuse potential, was used to pharmacologically characterize the transporters as expressed in these non-neuronal cells.

Materials and Methods

[3H]DA, [3H]5-HT, [3H]NE, and [125I]RTI-55 were purchased from Du Pont-New England Nuclear (Boston, MA). Tropolone, DA, pargyline, and most other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The cloning and characterization of the hDAT cDNA used in these experiments (pcDNA1-hDAT) were described previously (Eshleman et al., 1994, 1995). The hSERT cDNA was generously supplied by Dr. Randy Blakely (Ramamoorthy et al., 1993), subcloned into pcDNA1, and transfected into HEK 293 cells through electroporation. Dr. Blakely also supplied us with HEK cells transfected with pcDNA3-hNET (HEK-hNET; Galli et al., 1995). RTI-55 and CFT were gifts from Dr. Ivy Carroll.

[125I]RTI-55 Binding.

HEK-hDAT and HEK-hSERT cells were incubated in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 5% calf bovine serum, 0.05 U penicillin/streptomycin, and puromycin (2 μg/ml). HEK-hNET cells were incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.05 U penicillin/streptomycin, and geneticin (300 μg/ml). Cells were grown until confluent on 150-mm-diameter tissue culture dishes in a humidified 10% CO2 environment at 37°C. Medium was removed from plates, cells were washed with 10 ml of PBS, lysis buffer (10 ml, 2 mM HEPES, 1 mM EDTA) was added, and plates were placed on ice for 10 min. Cells were scraped from plates and centrifuged for 20 min at 30,000g. The pellet was resuspended in 6 to 24 ml of 0.32 M sucrose with a Polytron homogenizer at setting 7 for 5 s.

Assays contained an aliquot of membrane preparation (approximately 12–30 μg protein, depending on the cell line, which resulted in binding <10% of the total radioactivity), drug, and [125I]RTI-55 (40–80 pM final concentration) in a final volume of 250 μl. Krebs-HEPES assay buffer (25 mM HEPES, 122 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1 μM pargyline, 100 μM tropolone, 2 mg glucose/ml, 0.2 mg ascorbic acid/ml, pH 7.4) was used for all assays. Specific binding was defined as the difference in binding observed in the presence and absence of 5 μM mazindol (HEK-hDAT and -NET) or 5 μM imipramine (HEK-hSERT). Membranes were preincubated with drugs at room temperature for 10 min before the addition of [125I]RTI-55, unless indicated otherwise. The reaction was incubated for 90 min at room temperature in the dark and was terminated by filtration through Wallac Filtermat A filters using a 96-well Tomtec cell harvester. Scintillation fluid (50 μl) was added to each filtered spot, and radioactivity remaining on the filter was determined using a Wallac 1205 Betaplate or 1405 microBeta scintillation counter. Competition experiments were conducted with duplicate determinations for each point. Saturation binding experiments were conducted in triplicate by diluting the specific activity of [125I]RTI-55 with unlabelled ligand ranging in concentration from 0.036 to 16 nM. The dissociation experiments were performed at 25°C after incubation with 50 pM [125I]RTI-55 for 90 min. The dissociation of the receptor-ligand complex was initiated by the addition of mazindol (5 μM), imipramine (5 μM), or RTI-55 (1 μM), as indicated, and terminated at dissociation times ranging from 1 to 120 min. TheT 1/2 and the dissociation rate constant, k −1, were determined by nonlinear regression of dissociation data. In association experiments, membranes were incubated with [125I]RTI-55 (19–400 pM) at 25°C for 1 to 90 min. For association experiments using higher concentrations of [125I]RTI-55 (1, 3, 6, and 10 nM), the specific activity of the radioligand was reduced by dilution with RTI-55. The k obsvalue at each concentration of radioligand was determined by nonlinear regression; the association rate constant,k +1, was determined from the slope of the linear regression of k obs versus the concentration of radioligand (Limbird, 1996).

[3H]Nisoxetine Binding to HEK-hNET Membranes.

Cells were prepared as described above and resuspended in 0.32 M sucrose. Assay conditions were a modification of Tejani-Butt et al. (1990) and included membranes (30–50 μg protein), [3H]nisoxetine (20 nM final concentration), drugs, and buffer (50 mM Tris, 300 mM NaCl, pH 7.4 at 4°C) in a final volume of 250 μl. Specific binding was defined as the difference in binding observed in the absence and presence of mazindol (5 μM). The assay was incubated for 2 h at 4°C and then terminated by filtration as described above. Concentrations of [3H]nisoxetine ranging from 0.1 to 10 nM were used to determine the K d value for binding to HEK-hNET cell membranes.

Concentration-Response Curves for Inhibition of Substrate Uptake by HEK-hDAT, -hSERT, and -hNET.

HEK-hDAT, -hSERT, and -hNET cells were grown on 150-mm-diameter tissue culture dishes as described. Medium was removed and plates were washed twice with Ca2+, Mg2+-free PBS. Fresh Ca2+, Mg2+-free PBS (2.5 ml) was then added to each plate and plates were placed in a 25°C water bath for 5 min. Cells were gently scraped from plates, and cell clusters were separated by trituration with a pipette for 5 to 10 aspirations and ejections.

Aliquots (50 μl) of the suspended cells were added to assay tubes containing drugs and Krebs-HEPES assay buffer in a final assay volume of 0.5 ml. Competition experiments were conducted with triplicate determinations for each point. After a 10-min preincubation in a 25°C water bath (unless indicated otherwise), [3H]neurotransmitter (20 nM final concentration; [3H]DA, [3H]5-HT, or [3H]NE; 56, 26.9, or 60 Ci/mmol, respectively) was added, and the assay was incubated for 10 min. The reaction was terminated by filtration through Wallac filtermat A filters, presoaked in 0.05% polyethylenimine, using a Tomtec cell harvester. Scintillation fluid was added to each filtered spot, and radioactivity remaining on the filters was determined as described above. Specific uptake was defined as the difference in uptake observed in the absence and presence of 5 μM mazindol (hDAT and hNET) or 5 μM imipramine (hSERT).

Data Analysis.

Prism software (GraphPad Software, San Diego, CA) was used to analyze all kinetic, saturation, and competition binding data. IC50 values were converted toK i values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Results

The association and dissociation rates of [125I]RTI-55 binding were characterized for each cell line. The T 1/2 for dissociation was 26.5 ± 5.2, 42.5 ± 6.5, and 30.1 ± 3.9 min, and the calculated k −1 values were 0.029 ± 0.004, 0.016 ± 0.003, and 0.028 ± 0.004 min−1 for HEK-hDAT, -hSERT, and -hNET, respectively (Fig. 1). In all hDAT and hSERT dissociation experiments and the great majority of hNET experiments (12 of 14 experiments), a single dissociation rate best fit the data and there was no significant improvement in fit with a two-site model. For each transporter, dissociation rates were identical regardless of whether unlabeled RTI-55 or mazindol (hDAT and hNET) or imipramine (hSERT) was used as the competing ligand. The dissociation rate for hSERT was significantly slower than that for hDAT or hNET (one-way ANOVA followed by a Tukey post-hoc analysis). The association data indicated that equilibrium was approached by 60 min with hDAT and hSERT and by 90 min with hNET at 20 and 50 pM [125I]RTI-55, the lowest concentrations tested (Fig. 2); these equilibrium times were shorter than would be predicted by the dissociation data. Association experiments were conducted with the following concentrations of [125I]RTI-55: 20, 50, 80, 220, and 400 pM for all transporters; in addition, 1, 3, 6, and 10 nM were tested for hNET. The k obs value was determined for each experiment by nonlinear regression of the data. A single association rate was the best fit to the data in most experiments (hDAT, 16 of 17 experiments; hSERT, 17 of 18 experiments; hNET, 39 of 42 experiments). The k +1 value for the three transporters, determined by linear regression of the concentration of ligand versus the k obs value (Fig. 2), was 0.160, 0.124, and 0.0029 nM−1min−1 for HEK-hDAT, -hSERT, and -hNET, respectively. Binding to HEK-hNET had a significantly slower association rate than binding to HEK-hDAT or -hSERT.K d values calculated from the kinetic data are 0.18, 0.13, and 6.8 nM for HEK-hDAT, -hSERT, and -hNET, respectively.

Figure 1
Figure 1

Dissociation of [125I]RTI-55 from HEK-hDAT, HEK-hSERT, and HEK-hNET cell membranes. Membranes were incubated with [125I]RTI-55 for 90 min before dissociation was initiated by the addition of either 5 μM mazindol or imipramine. Data shown are the mean ± S.E.M. of four experiments. Inset, linear transformation of the data.

Figure 2
Figure 2

Association of [125I]RTI-55 to membranes from HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Data are the mean ± S.E.M. of three to five experiments for each cell line at 50 PM [125I]RTI-55. Insets, determination of thek +1 for [125I]RTI-55 to each of the transporters. Association experiments were conducted at each of the following concentrations of [125I]RTI-55: 20, 50, 100, 220, and 400 pM for all transporters and at 1, 3, 6, and 10 nM for hNET. The k obs values (mean ± S.E.M.) are plotted versus the concentration of [125I]RTI-55.

Saturation equilibrium binding experiments were conducted by reducing the specific activity of [125I]RTI-55 through progressive dilution with unlabeled ligand. Figure3 shows the saturation isotherm and Scatchard plot for each of the transporters stably expressed in HEK cells: the K d andB max values are given in Table1. As the linear Scatchard plots indicate, only a single high-affinity binding site was detected with each transporter with ligand concentrations ranging from 30 pM to 17 nM.

Figure 3
Figure 3

Equilibrium saturation binding of [125I]RTI-55 to membranes from HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Data are from representative experiments. Saturation binding was conducted by diluting the specific activity of radioligand with unlabeled RTI-55 as described in Materials and Methods. Insets, Scatchard plots of the data.

View this table:
Table 1

[125I]RTI-55 equilibrium binding to HEK-hDAT, -hSERT, and -hNET cells

Time courses for [3H]neurotransmitter uptake were conducted with each cell line to determine time points within the linear portion of the uptake time curve. Both HEK-hSERT and HEK-hNET cells exhibited uptake rates that were linear for 20 to 30 min, whereas for HEK-hDAT cells, the uptake rate began to plateau after 10 min (data not shown). Thus, for all cell lines, subsequent [3H]neurotransmitter uptake assays were conducted using a 10-min incubation.

To determine whether substrates may be depleted over long preincubation times or whether short preincubation times may give an artificially reduced affinity for slowly associating drugs, the influence of preincubation time was determined using four drugs: amphetamine (a substrate), mazindol, cocaine, and desipramine (structurally dissimilar nonsubstrate inhibitors with widely differing affinity for the hDAT). Preincubation times from 1 to 30 min were tested (Table2). The only significant differences in IC50 values were between the 1- and 10-min preincubation time points for amphetamine for inhibition of [3H]DA uptake and between the 1- and 10-min preincubation time points for desipramine inhibition of [125I]RTI-55 binding to hDAT (p< .05, one-way ANOVA). A 10-min preincubation time was used in subsequent [125I]RTI-55 and [3H]nisoxetine binding assays and in [3H]neurotransmitter uptake assays for all drugs and all transporters.

View this table:
Table 2

Effect of preincubation time on IC50 values for [125I]RTI-55 binding and [3H]DA uptake

To determine the rank order of potency of an array of drugs, including substrates, abused substances, antidepressants, and other therapeutic agents, at each of the biogenic amine transporters expressed in HEK 293 cells, drugs were tested for inhibition of [125I]RTI-55 binding and [3H]neurotransmitter uptake (Table3). For hDAT, there was a good correlation between inhibitory potency at uptake and binding for all drugs; however, the correlation for nonsubstrate inhibitors was better than that for substrates (r2 = 0.633 for all drugs, r 2 = 0.921 for inhibitors,r 2 = 0.687 for substrates; Fig.4A). Substrates, including DA, 5-HT, NE, methamphetamine, d-amphetamine, and (+)-fenfluramine, were uniformly more potent at inhibition of [3H]DA uptake compared with inhibition of [125I]RTI-55 binding. For hSERT, there was an excellent correlation between inhibitory potency at uptake and binding, with high correlations for both subgroups (r 2 = 0.832 for all drugs, r 2 = 0.938 for inhibitors,r 2 = 0.936 for substrates; Fig. 4B). For hNET, the correlation between the ability of compounds to inhibit [125I]RTI-55 binding and [3H]NE uptake was less strong (r 2 = 0.444 for all drugs,r 2 = 0.839 for inhibitors,r 2 = 0.597 for substrates; Fig.5A). Due to the lower correlation in the pharmacological profile of inhibition of [125I]RTI-55 binding and [3H]NE uptake, drugs were tested for inhibition of [3H]nisoxetine binding to hNET membranes. There was excellent correlation between the potency of drugs at inhibiting [125I]RTI-55 and [3H]nisoxetine binding (r 2 = 0.874 for all drugs, Fig. 5C). However, the use of [3H]nisoxetine rather than [125I]RTI-55 gave a similar correlation between inhibition of binding and [3H]NE uptake (r 2 = 0.367 for all drugs,r 2 = 0.767 for inhibitors,r 2 = 0.667 for substrates; Fig. 5B). For both radioligands, the correlation coefficients increased when only drugs that are nonsubstrate inhibitors were included in the analysis. Several compounds had Hill slopes significantly less than unity for inhibition of [3H]DA or [3H]NE uptake (benztropine, DA, methamphetamine, 5-HT), which could indicate more than one affinity state or site for these drugs on the transporters.

View this table:
Table 3

Inhibition of [3H]neurotransmitter uptake and radioligand binding in HEK-hDAT, HEK-hSERT, and HEK-hNET cells

Figure 4
Figure 4

Correlation of inhibition of [125I]RTI-55 binding and [3H]neurotransmitter uptake. The line represents the linear regression for all drugs. A, HEK-hDAT: all drugs,p < .0001; inhibitors, p < .0001; substrates, p > .05. B, HEK-hSERT: all drugs, p < .0001; inhibitors,p < .0001; substrates, p = .002.

Figure 5
Figure 5

Correlation of inhibition of ligand binding and [3H]neurotransmitter uptake in HEK-hNET cells. The line represents the linear regression for all drugs. A, correlation of [125I]RTI-55 binding and [3H]NE uptake. All drugs, p = .001; inhibitors, p< .0001; substrates, p > .05. B, correlation of [3H]nisoxetine binding and [3H]NE uptake. All drugs, p = .008; inhibitors,p < .0001; substrates, p > .05. C, correlation of [125I]RTI-55 binding and [3H]nisoxetine binding. All drugs, p< .0001; inhibitors, p < .0001; substrates,p = .048. (+)Amph, d-amphetamine; Benz, benztropine; Bup, bupropion; Coc, cocaine; DMI, desipramine; (+)Fen, (+)-fenfluramine; GBR, GBR-12935; Meth, (+)-methamphetamine; Nix, nisoxetine; Nom, nomifensine; Nort, nortriptyline; RTI, RTI-55.

The selectivity of the transporters for the stereoisomers of two substrates was examined (Table 4). For both HEK-hDAT and -hSERT, the potency of d-amphetamine was greater than l-amphetamine for inhibition of [3H]neurotransmitter uptake (p= .03 for hDAT, p = .003 for hSERT, one-tailed t test), whereas there was no stereoselectivity for inhibition of [3H]NE uptake into HEK-hNET cells (p = .31). d-Amphetamine had higher potency than l-amphetamine for inhibition of [125I]RTI-55 binding to all three transporters (p = .04, p = .003, andp = .01 for HEK-hDAT, -hNET, and –hSERT, respectively). For the stereoisomers of fenfluramine, (+)-fenfluramine was significantly more potent than (−)-fenfluramine at inhibition of [3H]neurotransmitter uptake by all three transporters, whereas stereoselectivity of inhibition of [125I]RTI-55 binding was observed only in HEK-hSERT cells (p = .002).

View this table:
Table 4

Potency of substrate stereoisomers at HEK-hDAT, -hSERT, and hNET

Discussion

The interaction of cocaine with a unique binding site of a particular transporter may contribute to euphoria. Although many clinically useful anorectic agents or antidepressants bind to neurotransmitter transporters, they lack the psychotomimetic effects of cocaine, and some have been tested clinically as therapeutics for cocaine abuse (Gawin et al., 1989; Preston et al., 1993). Thus, an understanding of the mechanisms involved in the binding of drugs and neurotransmitters to transporters, and the sequellae of drug-transporter interaction, could indicate new directions for the development of therapeutic agents for drug abuse.

A cocaine analog, [3H]CFT, bound to two-affinity states in membranes of COS-7 cells expressing recombinant rat DAT (Boja et al., 1992a) and to a one-affinity state in membranes of COS-7 cells expressing recombinant hDAT (Eshleman et al., 1995). RTI-55 (β-CIT), a cocaine analog with high affinity for both hDAT and hSERT (Boja et al., 1992b), consistently binds to a single site in membranes from HEK 293 cells stably expressing the hDAT, hNET, or hSERT (Figs. 1-3; Table 1), in agreement with results from Qian et al. (1997) involving binding to the hSERT. RTI-55 had high affinity for hDAT (1.8 nM) and hSERT (0.98 nM), whereas the affinity for hNET (12 nM) was approximately 1 order of magnitude lower. Both dissociation and association curves (Figs. 1 and 2) were adequately described as reversible binding of RTI-55 to a homogeneous population of independent binding sites. Although the K d values determined by kinetic and saturation analyses were not identical, the rank order of affinity of RTI-55 for the transporters was maintained with both types of analyses.

The K d value obtained with HEK-hDAT cells by equilibrium binding of [125I]RTI-55 (Table 1) was identical to the lower affinity state in human striatal preparations (Little et al., 1993; Staley et al., 1994). For HEK-hSERT, the K d value was similar to the affinity of the single site in human occipital cortical membranes detected by Little et al. (1993) and midway between the affinities detected by Staley et al. (1994). Rothman et al. (1994) observed two binding sites for [125I]RTI-55 in rat caudate and a single binding site in COS cells expressing recombinant rat DAT, consistent with the present results. These differences between cell-expression systems and native tissue could be due to differences in glycosylation (Patel et al., 1993) or other posttranslational modifications such as altered steady-state phosphorylation of the transporters or other proteins that affect the affinity for the ligand.

Seven antidepressants were tested at the three transporters. Bupropion, desipramine, nisoxetine, nomifensine, and nortriptyline were most potent at inhibition of [3H]NE uptake, whereas fluoxetine and imipramine were most potent at inhibition of [3H]5-HT uptake. Except for bupropion and imipramine, this transporter selectivity is identical to that reported for inhibition of [3H]neurotransmitter uptake in rat brain membranes (Richelson and Pfenning, 1984). The transporter selectivity of antidepressants for inhibition of uptake and [125I]RTI-55 binding was similar (Table 3), except bupropion and nomifensine were more potent at inhibition of radioligand binding to HEK-hDAT than to HEK-hNET. The selectivity of antidepressants for displacement of [125I]RTI-55 binding was identical with that reported for displacement of other selective radioligands (Tatsumi et al., 1997), except for displacement by bupropion, which may indicate a ligand-specific effect.

DA and NE had higher affinity for the hNET than for the hDAT. These results are qualitatively similar to the 4- and 10-fold higher affinity of DA and NE for hNET compared with hDAT, as reported by Giros et al. (1994). Both neurotransmitters were fully efficacious at the hDAT and hNET, emphasizing the similarity of the transport mechanism by these two proteins.

Barker et al. (1994) investigated the differences between rat and human SERTs expressed in HeLa cells. The K ivalues for imipramine and desipramine in the present study were similar to values reported for hSERT (Barker et al., 1994), whereas theK i value for nortriptyline (Table 3) was similar to the value reported for rat SERT. In addition, the potency of d-amphetamine for inhibition of [3H]5-HT uptake was more similar to the value for rat SERT than for hSERT (Barker et al., 1994) but similar to that reported by Ramamoorthy et al. (1993) for hSERT.

In general, the K i values for drug inhibition of [3H]nisoxetine binding to the hNET, as well as the radioligand K dvalue, were 10-fold greater than results for rat cerebral cortical membranes (Tejani-Butt et al., 1990). Possible explanations for this consistent discrepancy may involve the difference in native versus non-neuronal systems, leading to variations in glycosylation as discussed, or in species differences in the affinity of rat and hNET.

We observed lower correlations between ligand binding and neurotransmitter uptake for hNET compared with correlations for hDAT or hSERT. The correlations between binding and uptake for hNET and hSERT were significantly different (z = 2.143, Fisher’sZ transformation; Downie and Heath, 1974), suggesting that the RTI-55 binding site on the hSERT is more closely linked to sites of drug interactions with substrate translocation. The movement of neurotransmitter across the membrane by the transporter involves an initial binding event followed by translocation of the amine across the membrane. The lower correlation between binding and uptake at hNET, when [125I]RTI-55 was used, may be related to the lower rate of association of this ligand with the hNET, suggesting that the ligand does not interact with the hNET in the same way as it does with the hDAT or hSERT. The use of one cell type stably expressing each of the transporters eliminates the variables introduced by different cell types (glycosylation differences, possible metabolism of drugs, differing populations of receptors and transport proteins).

The psychostimulants d- and l-amphetamine displayed stereoselectivity for inhibition of [3H]neurotransmitter uptake at the hDAT and hSERT but not at the hNET, which is in agreement with the findings ofGiros et al. (1994). However, stereoselectivity was observed at all transporters for inhibition of binding. For the fenfluramine isomers, stereoselectivity for inhibition of binding was observed only with hSERT, whereas stereoselectivity for inhibition of [3H]neurotransmitter uptake was observed in all three cell lines. These results suggest that stereoisomers interact differentially with binding and translocation sites in the transporter molecules.

The binding site of cocaine on the hDAT may be sufficiently different from the recognition site for DA that a drug will be able to inhibit the binding of cocaine without blocking DA uptake (Rothman et al., 1993). This hypothesis is supported by differential effects on DA uptake and cocaine ligand binding after single amino acid substitutions on the DAT (Kitayama et al., 1992), by examination of DAT/NET chimeras (Giros et al., 1994; Buck and Amara, 1995), and by some animal self-administration studies (Tella et al., 1996, but see Roberts, 1993, and Wojnicki and Glowa, 1996). One method of screening for a pharmacological cocaine antagonist would be to compare the potencies of compounds for inhibition of DA uptake and cocaine analog binding. A compound that has greater potency at inhibition of binding than uptake may be a potential “DA-sparing” cocaine antagonist. In addition, determination of the potencies of compounds at the SERT and NET would establish the selectivity of drugs.

Rothman et al. (1993) observed differences in the ratio of uptake and binding potency for one of three drugs when uptake and binding assays were conducted under identical conditions versus assays using different buffers, incubation temperatures and times, and membrane preparations. In the current report, although identical preincubation times and buffers were used, the incubation times for these inherently different experiments (equilibrium binding versus uptake during the linear portion of the time course) were different, as were the tissue preparations. A total particulate membrane preparation was used for binding due to the possible confounds of intact cells, including internalization of transporters or sequestration of drug.

Desipramine, fluoxetine, and nortriptyline were more potent at inhibition of binding than uptake at the hDAT, by factors of 6, 3, and 4, respectively (Table 3). Interestingly, Slusher et al. (1997), who tested drugs under identical conditions for [3H]DA uptake and [3H]CFT binding, also identified desipramine as having much greater potency at inhibition of binding. However, the higher potency (3 orders of magnitude) at the hNET (desipramine and nortriptyline) and hSERT (fluoxetine, Table 3) reveals the lack of selectivity for the hDAT by these antidepressants; therefore, they are unlikely candidates for therapeutic cocaine antagonists. In addition, five drugs were more potent at inhibition of binding to hSERT than at inhibition of [3H]5-HT uptake. Interestingly, all except GBR-12935 are therapeutic agents, and close analogs of GBR-12935 have potential as therapeutic agents for cocaine abuse (Glowa et al., 1996; Tella et al., 1996). No drug was more potent at inhibition of [125I]RTI-55 binding to hNET than at inhibition of [3H]NE uptake. Structural differences among the transporters may account for the varying potency ratios between the uptake and binding of a drug at the three transporters. Critical residues for the recognition of drugs may differ among the transporters, and the detection of a drug that is selective for a single transporter, with the ability to inhibit the binding of an abused substance, such as cocaine, while sparing neurotransmitter uptake may be accomplished using the assays reported here.

Acknowledgments

We thank Robert A. Johnson, Carey Teemer, and Alan Goodhue for technical assistance.

Footnotes

  • Send reprint requests to: Dr. Amy Eshleman, Research Service RD-22, Veterans Affairs Medical Center, 3710 S.W. U.S. Veterans Hospital Road, Portland, OR 97201. E-maileshleman{at}ohsu.edu

  • ↵1 This work was supported by National Institutes of Health Contracts NO1-DA-3-8303 and NO1-DA-7-8071, Department of Veterans Affairs Merit Review and Career Scientist Programs, and a Young Investigator Award from NARSAD.

  • Abbreviations:
    DA
    dopamine
    hDAT
    human dopamine transporter
    5-HT
    5-hydroxytryptamine (serotonin)
    hSERT
    human serotonin transporter
    NE
    norepinephrine
    hNET
    human norepinephrine transporter
    HEK
    human embryonic kidney 293 cells
    HEK-hDAT
    HEK-hSERT, or HEK-hNET cells, human embryonic kidney 293 cells transfected with hDAT, hSERT, or hNET cDNA, respectively
    CFT
    2β carbomethoxy-3β-(4-fluorophenyl)tropane
    GBR-12935
    1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine
    • Received September 29, 1998.
    • Accepted December 18, 1998.

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Characteristics of Drug Interactions with Recombinant Biogenic Amine Transporters Expressed in the Same Cell Type

Amy J. Eshleman, Marya Carmolli, Medhane Cumbay, Carey R. Martens, Kim A. Neve and Aaron Janowsky
Journal of Pharmacology and Experimental Therapeutics May 1, 1999, 289 (2) 877-885;
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