Expression and regulation of the human dopamine transporter in a neuronal cell line

Molecular Brain Research 59 Ž1998. 66–73

Research report

Expression and regulation of the human dopamine transporter in a neuronal cell line Lian Zhang a , Lawrence W. Elmer a

c

b,c

, Karley Y. Little

a,b,)

Department of Psychiatry, UniÕersity of Michigan, Ann Arbor, MI, USA b Ann Arbor VAMC, Ann Arbor, MI 48105, USA Department of Neurology, UniÕersity of Michigan, Ann Arbor, MI, USA Accepted 2 June 1998

Abstract Human cocaine users exhibit increased striatal w3 HxWIN35428 binding to the dopamine transporter ŽDAT.. However, the nature of the changes induced in the DAT are complex and may not result from a simple increase in number of DAT molecules. To better understand the regulation of DAT inhibitor binding sites and their relationship to the overall process of dopamine uptake, a neuronal model system expressing the human DAT has been developed. Initial experiments were attempted with native dopaminergic neurons so as to allow examination of DAT interactions with vesicular release and storage mechanisms. Dissociated fetal rat mesencephalic neurons, of various ages and mixtures with target cells, were grown to confluence. However, w3 HxWIN35428 binding was of low affinity at all levels of maturity. Following this, a simpler model was assessed, using DAT cDNA transfected into neuroblastoma-derived Neuro2A cells. Initially, no specific and little non-specific w3 HxWIN35428 or w3 Hxparoxetine binding was found in non-transfected cells. After transfection with the human DAT inserted in the pcDNA vector, both DAT binding and dopamine uptake were significantly and stably present. Treatment with Žy.cocaine, 10y6 M for 24 h, increased DAT binding and uptake, which did not occur in parallel COS-7 experiments. Other experiments with Neuro2A cells also found that dopamine uptake was down-regulated by treatment with a PKC activator. These results suggest that the transfected Neuro2A neurons should be useful for ongoing experiments examining the regulation of the DAT by assorted treatments. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dopamine transporter regulation uptake w3 HxWIN35428

1. Introduction Human cocaine users demonstrate marked increase in striatal dopamine transporter ŽDAT. binding sites in experiments performed in brain tissue collected at autopsy w23,36x. Other experiments utilizing different paradigms have also found that the DAT function is upregulated by chronic cocaine exposure w13,19,41x. However, results have differed based on the radioligand utilized w13,15,41x, among several possible confounding factors, suggesting involvement of a complex mechanism involving conformational

Abbreviations: DAT, dopamine transporter; MAT, monoamine transporter; PKC, protein kinase C; N2A, Neuro2A neuron; PMA, phorbol 12-myristate b-acetate; ED, embryonic day ) Corresponding author. Psychobiology Laboratoryr116-A, Ann Arbor V.A.M.C., 2215 Fuller Rd., Ann Arbor, MI 48105, USA. Fax: q1-313769-7410; E-mail: [email protected] 0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 3 8 - 7

andror stoichiometric changes in the DAT protein. Although human tissue can be used to characterize binding changes in the DAT in situ, DAT-expressing cell models make it possible to evaluate the dynamics of plasticity changes, as well as parallel changes in dopamine uptake, which are difficult in frozen human tissue because of variable decay and logistical problems. In addition to questions about the nature of regulatory changes, a number of important issues remain unresolved concerning the number of binding sites on the DAT w22,23,25,33,36x, and the relationships between dopamine, DAT inhibitors, sodium, chlorine, and potassium binding sites on the DAT w2,33,40x, which may be best dissected using cell systems. Initially, we focused our efforts on fetal rat mesencephalic cultures which might allow examination of the interactions between the DAT and vesicular storage and release of dopamine. Others have described atypical DAT binding properties in these types of cell cultures which we

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

attempted to overcome. Despite our efforts to broaden this model’s applicability, binding affinity was problematic, with w3 HxWIN35428 K d’s consistently less than 1 mM Ždata not shown.. PC 12 neurons, which are derived from a rat pheochromcytoma line were also considered as a potential model system, but these cells avidly transport dopamine through the norepinephrine transporter w14x. Because of these problems, a simpler model system was developed involving transfection with DAT cDNA. Previous investigators have characterized hDAT function after expression in several types of non-neuronal cells w7,10,30,35x. However, our initial experiments using the hDAT-transfected COS-7 found no effects of cocaine treatment on DAT binding and uptake ŽFig. 4.. Because of potential differences in regulation and function between neuronal and non-neuronal cells, we further evaluated a neuronally-derived model system. Our evaluative criteria included vigorous and selective dopamine uptake, high affinity DAT inhibitor binding, and low non-specific binding, with quantitative characteristics closely resembling that found in human brain. In the experiments described below, we discovered that hDAT-transfected N2A neurons displayed DAT binding and uptake features that were similar to that reported in human striatum, as well as regulation after exposure to cocaine or a PKC activator. Although the rat fetal dopaminergic model was problematic, data describing its characteristics are reported because of its interesting features, which might warrant further investigation.

2. Materials and methods 2.1. Dopamine cell cultures Rat fetal mesencephalic neurons were obtained by sacrificing timed pregnant Sprague–Dawley rats by cervical dislocation using a protocol approved by the University of Michigan Animal Use Committee. Embryos at days E18 and E19 were then removed under sterile conditions. Dissection of the embryonic brain was performed and separate removal of the ventral mesencephalic region as well as the striatal region was performed under the dissecting microscope. Co-cultures of ventral mesencephalic and striatal neurons were performed following modification of a protocol developed for spinal cord and dorsal root ganglion neurons. The tissue fragments were incubated for 1 h in the presence of Dispase Ž100 units mly1 . followed by trituration of the tissue fragments with a sterile 10 ml pipette. One milliliter of 1000 units mly1 of DNAse was added and the cells were incubated for an additional 30 min at 378. Tissue fragments and cells were pelleted at 1000 = g for 5 min. The pellets were re-suspended in HBSS Žwithout calcium or magnesium. and triturated 10 times through a Pasteur pipette. The tissue fragments were

67

allowed to settle, and the supernatant was collected in a separate tube. This procedure was repeated 2 to 3 times using progressively smaller flame-polished Pasteur pipettes, until no tissue fragments remained. Cell counts were then performed of trypan blue excluding cells. Equal portions of the striatal and ventral mesencephalic neurons were combined and re-pelleted at 1000 = g for 5 min. The combined cells were re-suspended at concentrations of either 300,000 per ml, 500,000 per ml or 700,000 per ml in the plating media in an initial study, which suggested that the highest concentration was best to optimize characterization of DAT binding. Plating media consisted of Neurobasal media and the B27 supplement ŽGibco-BRL. supplemented with 15 mM glutamate Žmanufacturer’s recommendations.. One ml of this cell suspension was pipetted into each of the 24 wells of precoated Costar plates. ŽCostar plates were precoated with 100 ml of 10 mg mly1 of poly-D-lysine and a 1 to 20 dilution of Matrigel, and allowed to dry overnight.. Cells were maintained in a 5% CO 2 atmosphere at 378 for the times indicated. Cells were given partial changes of NeurobasalrB27 without added glutamate at approximately one week intervals. 2.2. hDAT-transfected cell cultures Neuro2A cells were obtained from ATCC ŽBethesda, MD.. They were grown in Opti-MEMI with 10% fetal bovine serum ŽFBS. in 75-cm2 flask. After reaching confluence Žafter about 3 days of growth., the cells were collected by trypsinization and transfected with cDNAhDAT Žsee Section 2.2.1.. Transfected neurons were selected for long term culture by treatment with geneticin through four generations over 28 days. After selection, cells were grown to confluence and then distributed in 24 well plates and allowed to re-attach for 24 h before assay. COS-7 cells were obtained from ATCC ŽBethesda, MD.. They were grown in Dulbecco’s modification of Eagle’s medium ŽDMEM. with 10% fetal bovine serum ŽFBS. in 75-cm2 flask. After reaching confluence Žafter about 3 days of growth., the cells were collected by trypsinization and transfected with cDNA-hDAT Žsee Section 2.2.1.. Cells were then distributed in 24 well plates and allowed to grow for 2–3 days before assay. 2.2.1. cDNA transfection The human DAT cDNA was provided by Znedek Pristupa ŽUniversity of Toronto., cloned into the pcDNA3 plasmid. This vector replicates in Escherichia coli and includes an AMP resistance gene and SV40 origin of replication. Transfection of Neuro2A cells and COS-7 with cDNA-hDAT by electroporation was performed using a BTX Electroporation system 600 ŽBiotechnologies and Experimental Research, San Diego, CA.. Initially, cells were trypsinized and collected by spinning at 1000 = g for 10 min at room temperature. The supernatant was carefully

68

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

poured out of the tube, and the undisturbed cell pellet was subsequently re-suspended in phosphate-buffered saline ŽPBS. at a density of ; 8 = 10 6 cells mly1. Forty micrograms of cDNA-hDAT was added to 0.36 ml Neuro2A cells in PBS and transferred to a 2-mm gap cuvette, which was then incubated on ice for 10 min before and after electroporation. A single 8 ms pulse at a field strength of 1.0 kV cmy1 was used for the transfection. Neuro2A cells then were re-suspended in Opti-MEM Ir10% FBSr1% Penicillin–Streptomycin. The transfection of COS-7 cells were carried out similarly to the transfection of Neuro2A cell described above with the following changes. For COS-7 cells, 20 mg of hDAT-cDNA Ž1 mg mly1 . was mixed with 0.7 ml COS-7 cells in PBS and delivered to a 4-mm gap cuvette, which was incubated on ice for 5 min. Recipient cells were subjected to a single 10 ms pulse at a field strength of 0.75 kV cmy1 and incubated on ice for another 5 min. Cells were then re-suspended in DMEMr10% FBS and seeded into 24-well culture plates Ž1.0 ml welly1 ., which contained about 200,000 cells welly1 3 days after transfection. 2.3. Uptake and radioligand binding assays 2.3.1. [3H]dopamine uptake assays For dopamine uptake studies, the culture medium was removed from 24-well plates. Cells in each well were washed with 1 ml Krebs Phosphate ŽKP. buffer at room temperature. w3 Hxdopamine was added to wells containing KP buffer to reach a final concentration of 10 nM and a total volume of 0.5 ml, followed by incubation for 5 min Župtake linear. at 378C on a plate shaker. Uptake was terminated by removing assay medium immediately after incubation with a Brandel 48 well cell harvester, followed

by two washes with 1 ml ice-cold KP buffer. Cells were dissolved in 0.8 ml of 1% SDS. The entire liquid content of each well was transferred to a scintillation vial and assayed for radioactivity with 5 ml of cytoscint cocktail ŽICN, EcoLite. by liquid scintillation counting in Beckman model LS 6500 IC spectrometer. Non-specific uptake was defined with 30 mM cocaine. The cells in one or two wells of each plate were dissolved with 1 ml 1 N NaOH, and the concentration of protein determined ŽBio-Rad, St. Louis, MO.. Approximately 0.2 mg of protein was obtained from one well. Computer analysis of binding and uptake data were performed using Prism Software ŽGraphPAD Software, San Diego, CA.. 2.3.2. [3H]WIN35428 and [3H]paroxetine binding assays w3 HxWIN35428 and w3 Hx paroxetine binding assays were performed using a 0.32 M sucrose and 10 mM Na 2 HPO4 buffer. Intact cells were incubated with w3 HxWIN35428 Ž2 nM final concentration. or w3 Hxparoxetine Ž1 nM final concentration. for 1 h at 28C on a shaker, followed by mechanical scraping and rapid filtration over Whatman GFrB glass fiber filters presoaked in 0.01% polyethylenimine using a Brandel 48 well cell harvester. After several washes with sucrose–phosphate buffer, the filter was put in a scintillation vial, which was assayed with 4 ml of cytoscint cocktail. Non-specific binding was defined with 30 mM cocaine. 2.3.3. (y)Cocaine treatment Both N2A cells and COS-7 cells were grown to confluence, then treated with fresh media containing either Žy. cocaine, 10y6 M, or fresh media only. Twenty-four hours later, cells were washed and treated as described above for

Fig. 1. w3 HxWIN35428 binding to dispersed rat fetal mesencephalic cells. ŽA. Cells were dissected and plated as described in the Methods section, then allowed to mature for varying times. Specific binding was relatively low, but increased slightly up to 8 days. There was substantially more binding Žat a radioligand concentration 3 nM. using a phosphate–sucrose buffer compared to a TrisrNaCl buffer. There were tree replicate flasks for each condition. ŽB. Saturation experiments by homologous displacement Ž n s 3. showed that binding was of low affinity, ) 1 mM, at each time interval. A representative experiment is shown.

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

69

Fig. 2. w3 HxWIN35428 binding to Neuro2A ŽN2A. neurons. ŽA. Saturation experiments performed with homologous displacement found that binding was saturable and of high affinity in hDAT-transfected, but not non-transfected neurons. The curve shown is representative of five separate experiments, which together yielded a K d of 18.6 q 0.55 nM. ŽB. Competition for N2A binding displayed the appropriate rank order for the dopamine transporter. The results shown are representative of 3–5 independent experiments. All were best fit with one-site models, except for the cocaine displacement curve, which variably displayed a small low affinity component. Data from an experiment demonstrating this phenomenon is shown.

uptake and binding assays. Cell densities per well were measured and found to be equivalent in cocaine-treated and control cells. 2.3.4. PMA treatment N2A cells were pre-incubated for 15 min at room temperature with varying concentrations Ž0.1 to 100 mM. of phorbol 12-myristate b-acetate ŽPMA. after a prelimi-

nary time study indicated that effects were maximal at this time. Following this, w3 Hxdopamine uptake was performed as described above. 3. Results Fig. 1 displays the results obtained in fetal mesencephalic neurons culture, which were allowed to mature

Fig. 3. w3 HxDopamine uptake in hDAT-transfected N2A and COS-7 cells. ŽA. Uptake in N2A cells was of high affinity. Because the K m was lower than previously reported in cell systems, dopamine uptake was also examined in COS-7 cells, which displayed a higher K m . Results are calculated from 3–5 experiments. ŽB. Inhibition of uptake by various monoamine uptake inhibitors displayed a dopaminergic-selective profile. Three or four independent experiments were performed.

70

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

for up to 14 days. At each time point assessed, specific binding was relatively low Žbetween 1.5 and 2 = nonspecific, see Fig. 1A.. This figure also displays results obtained using a Tris Ž50 mM.rNaCl Ž120 mM. buffer which displayed even less specific binding. Fig. 1B displays the results of a saturation experiment performed after 8 days of incubation, which was typical for each of the incubation time points. w3 HxWIN35428 binding consistently displayed low affinity Ž K d’s greater than 1 mm.. In contrast, w3 HxWIN35428 binding in hDAT-transected N2A neurons displayed a strong signal vs. non-specific binding, which was of high affinity. Further control experiments examining w3 HxWIN35428 and w3 Hxparoxetine binding to non-transfected neurons found virtually no specific binding. Fig. 2A shows the binding found in transfected N2A neurons. Fig. 2B also shows the results of competition experiments performed with cocaine and other monoamine transporter-selective ligands. Of note, there was a second low affinity component Ž K d approximately 10–20 mm. variably present in cocaine competition experiments, one of which is displayed in the graph. Binding to this component varied between 0 and 30% of total binding. A two site model was preferred in two of six cocaine experiments. Binding of the remaining selective competitors was monophasic, and of the expected rank order for the DAT. Paralleling the binding experiments, dopamine uptake experiments found high affinity dopamine uptake Ž K m s 433 nM, Fig. 3A. which was inhibited with high affinity by dopaminergic-selective inhibitors, but not serotonergic or noradrenergic inhibitors ŽFig. 3B.. Fig. 3A also includes for comparison the results found with transiently transfected COS-7 cells. In these cells, the K m for dopamine uptake was somewhat higher: 1.6 = 10y6 mM.

Fig. 5. Effect of protein kinase activator ŽPMA. on w3 Hxdopamine uptake. As shown above, doses of PMA of 1 mM or higher significantly depressed w3 Hxdopamine uptake Ž10 nM concentration.. ) p- 0.01, )) p - 0.001, significantly different from controls, one-way ANOVA.

Cocaine treatment of N2A neurons elicited a marked upregulation of DAT binding and uptake, which was not present in COS-7 cells Žsee Fig. 4.. w3 HxWIN35428 binding in six independent experiments was 35.1 " 8.1% higher than in controls. w3 Hxdopamine uptake similarly was 46.7 = 15.1% higher than controls in four independent experiments Ž p - 0.05, t-test, two-tailed.. Experiments performed in hDAT-transfected N2A neurons also found a dose-related effect of PMA treatment on dopamine uptake in the range of 0.1 to 100 mm Žsee Fig. 5.. As shown in Table 1, binding to the DAT in N2A cells, as assessed with w3 HxWIN35428 was similar to parameters previously determined in human striatum w22–24x. Cocaine inhibition of w3 HxWIN35428 binding was similar: 231 " 22

Table 1 Pharmacological features of hDAT-expressing Neuro2A neurons Neuro2A neurons

Fig. 4. Comparison of effect of cocaine treatment on COS-7 and Neuro2A cells. Both treatment groups were exposed to cocaine, 1 mM for 24 h, while controls received only buffer. Specific binding was assessed with w3 HxWIN35428, 3 nM concentration. Binding was significantly elevated in cocaine treated Neuro2A cells Ž) p- 0.01, t-test, two-tailed..

Binding w3 HxWIN35428 K d w3 HxWIN35428 Bma x Cocaine IC 50 Dopamine IC 50 GBR12935 IC 50 Nisoxetine IC 50 Citalopram IC 50

18.6"0.55 nM 2.51"0.26 pmol mgy1 protein 104"30 nM 1230"170 nM) 14.3"1.5 nM 219"21 nM 2.5"1.1 mM

Uptake w3 Hxdopamine uptake, K m w3 Hxdopamine uptake, Vma x Cocaine IC 50 WIN35428 IC 50 GBR12935 IC 50 Nisoxetine IC 50 Citalopram IC 50

433"8.8 nM 5.53"2.1 pmol miny1 mgy1 protein 95"4.7 nM 20.7"2.0 nM 7.92"0.65 nM 197"51 nM 210"72 mM

Results represent an average from 4–7 independent N2A experiments. )Actual IC 50 may be slightly lower due to intracellular sequestration of dopamine.

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

nM in human brain, 104 " 30 nM in N2A cells. IC 50 for dopamine was lower in the human striatum, 653 q 43 nM vs. 1230 " 170 nM in N2A. Dopamine uptake parameters were not available in human striatum, but in N2A cells dopamine uptake was saturable and of high affinity. Dopamine selective ligands inhibited both w3 HxWIN35428 binding and dopamine uptake in the expected rank order: GBR12935 IC 50 for binding was 14.3 " 1.5 nM and 7.92 " 0.65 nM for uptake, nisoxetine Žnorepinephrine selective. IC 50 for binding 219 q 2l nM, IC 50 for uptake 197 " 51 nM. Citalopram Žserotonin selective. was not potent: IC 50 for binding was 2.5 " 1.1 mM, and IC 50 for uptake ) 100 mM. WIN35428 was potent at blocking uptake with an IC 50 of 20.7 " 2.0 nM.

4. Discussion Based on the present data, it appears that the Neuro2A cells are a good model system, in contrast to the rat fetal mesencephalic culture. hDAT-expressing N2A neurons were found to demonstrate appropriate binding and uptake characteristics, which were similar to hDAT characteristics in human striatum. Our results in N2A cells are comparable to the results previously reported in other types of hDAT-expressing cell cultures, except that K m for dopamine uptake appeared nearer to that found in fresh rat striatum w1,3x. To determine if this was related to uptake methods idiosyncratic to this laboratory, we compared N2A results to those found in hDAT expressing COS-7 cells. In COS-7 cells, we detected a K m for dopamine uptake of 1.6 mM Žsee Fig. 3A, which was similar to that reported by Giros et al. w10x Ž1.2 mM, in Ltk-fibroblasts., Pristupa et al. w30x Ž1.8 mM, COS-7., and Eshleman et al. w7x Ž1.2 mM, COS-7.. Thus it appears that the affinity for dopamine uptake is somewhat higher in the N2A system. Most significantly, we found that hDAT binding and uptake could be regulated by cocaine exposure in the N2A neurons, in contrast to COS-7 cells. In addition to regulation by cocaine treatment, the hDAT-transfected N2A cells also demonstrated regulation by a PKC activator, which has been previously shown to occur in striatal preparations w5,39x and other transfected cell systems w16,42x. Somewhat surprisingly, the regulatory processes that we observed in the N2A cell were independent of the presence of dopamine. Whether these examples of DAT regulation in N2A neurons occur through the same mechanisms that occur in intact striatum is uncertain, but the N2A system clearly appears to better model DAT regulation than COS-7 cells. It has been shown that glycosylation of the DAT differs between different brain regions w21,28x which may be related to regional differences in function w9x. Glycosylation patterns and other post-translational phenomena also likely vary between different types of cell cultures. Post-

71

translational events can markedly influence binding and functional characteristics of receptors w18,27x and recent experiments have demonstrated that protein kinase C ŽPKC.-mediated regulation of uptake by a related monoamine transporter—the serotonin transporter, involves changes in membranal insertion w32x. Specialized compartmental trafficking may depend on post translational modifications which could be critical in understanding DAT plasticity. Although it is not clear if post translational mechanisms are involved in regulating expression and function of the DAT in N2A neurons, studies with glycosidases comparing hDAT-expressing N2A neurons and COS-7 cells might prove interesting. Previous investigators have found that Neuro2A cells display many normal neuronal characteristics. They robustly differentiate into mature neurons, even more readily than human neuroblastoma w34,37x. Both neuronal sprouting and neurite development occur in the Neuro2A cell line and can be manipulated by expression of mutant gangliosides which are readily introduced by transfection w11,20x. Minana et al. w26x have also shown that PKC is highly active in these neurons, playing an important role in regulating differentiation, and that this PKC activity can be regulated by PMA and H7. Further, it has also been shown that Neuro2A cells demonstrate cAMP-inducible protein expression w17x, guanyl cyclase activity coupled to nitric oxide syntheses w4x, and typical endogenous muscarinic receptors which are functionally coupled to adenyl cyclase w6x. Although rat fetal mesencephalic cultures were problematic, our results confirm and extend previous investigators findings that rat fetal mesencephalic cells display a dissociation in maturation between dopamine uptake and cocaine and cocaine analog binding w12,38x. In both of the earlier studies, dopamine uptake was present in neurons harvested from more immature embryos, while only low affinity cocaine or cocaine-analog binding was detectable. Two factors have appeared critical in explaining low affinity cocaine binding: fetal age w8,31x, and the presence of appropriate striatal target cells, which have appeared to promote maturation w29x. In the present experiments, we used mature ED19 fetuses, mixed the midbrain cells with striatal cells, and allowed the cultures up to 2 weeks to mature. We varied several parameters including length of culture after dissociation and plating, fractional component of mesencephalic and striatal cell, as well as buffer Ždata not all shown.. Despite these manipulations, we continued to find an absence of adult-like binding of the cocaine-analog w3 HxWIN35428. It thus appears that high affinity binding does not appear before birth. No previous binding experiments have examined embryos in the later range that we did, or included striatal target cells. Although we did not explore w3 HxWIN35428 binding after birth, others have shown that high affinity cocaine binding sites are present at 5 days postnatal w12x. In contrast, dopamine uptake is active by the ED15 stage w31x.

72

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73

This dissociation may be of interest to those examining the effect of maternal cocaine use on fetuses in utero, suggesting that immature brains might be somewhat immune to the effects of administered cocaine. The presence of normal dopamine uptake, but not high affinity cocaine binding also appears relevant to those involved in a search for pharmacological agents that block cocaine binding to the DAT but do not disturb normal dopamine uptake. It appears that dissociation of uptake and cocaine binding naturally occurs during a limited stage in development. Further work exploring this phenomenon might elucidate the mechanisms involved and the structural implications for designing therapeutic agents. Also, interestingly, others have noted that the low affinity fetal cocaine binding appears insensitive to sodium, which is necessary for cocaine or cocaine analog binding in the adult DAT w12x. This low affinity binding fraction may not be properly inserted in the plasma membrane. Based on the results described above, it appears that hDAT-expressing N2A neurons are a promising system for evaluating the regulation of dopamine uptake, as well as other interesting functional and structural questions that remain about the dopamine transporter. However, other types of neuronal cells might be equally or even more appropriate than the N2A line. At this point, however, further experiments examining the regulation of DAT function by cocaine treatment appear warranted.

Acknowledgements This work was supported by an Ann Arbor Veteran’s Affairs Medical Center VERAM Grant and NIH award DA09491.

w7x

w8x

w9x

w10x

w11x

w12x

w13x

w14x

w15x w16x

w17x

w18x

w19x

References w1x B.A. Bennett, C.H. Wichems, C.K. Hollingsworth, H.M.L. Davies, C. Thornley, T. Sexton, S.R. Childers, Novel 2-substituted cocaine analogs: uptake and ligand binding studies at dopamine, serotonin, and norepinephrine transport sites in the rat brain, J. Pharmacol. Exp. Ther. 272 Ž1995. 1176–1186. w2x J.J. Bonnett, S. Benmansour, J.M. Vaugeois, J. Costentin, Ionic requirements for the specific binding of w3 HxGBR12873 to a site associated with the dopamine uptake carrier, J. Neurochem. 50 Ž1988. 759–765. w3x F.I. Carroll, P. Kotian, A. Dehghani, J.L. Gray, M.A. Kuzemko, K.A. Parham, P. Abraham, A.H. Lewin, J.W. Boja, M.J. Kuhar, X Cocaine and 3b-Ž4 -substituted phenyl. tropane-2b-carboxylic acid ester and amide analogues. New high-affinity and selective compounds for the dopamine transporter, J. Med. Chem. 38 Ž1995. 379–388. w4x S. Chaki, T. Inagami, New signaling mechanism of angiotensin II in neuroblastoma Neuro2A cells: activation of soluble guanylyl cyclase via nitric oxide synthesis, Mol. Pharmacol. 43 Ž1993. 603–608. w5x B.J. Copeland, V. Voelsberg, N.H. Neff, M. Hadjiconstantinou, Protein kinase C activators decrease dopamine uptake into striatal synaptosomes, J. Pharmacol. Exp. Ther. 277 Ž1996. 1527–1532. w6x A. Edwards, M. Gillard, E. Merler, Characterization of muscarinic

w20x

w21x

w22x

w23x

w24x

w25x

receptors: type M2 Žsubtype B. on Neuro2A neuroblastoma cells, J. Recept. Res. 9 Ž1989. 259–270. A.J. Eshleman, R.L. Neve, A. Janowsky, K.A. Neve, Characterization of a recombinant human dopamine transporter in multiple cell lines, J. Pharmacol. Exp. Ther. 274 Ž1995. 276–283. M.L. Fiszman, A. Zuddas, M.I. Masana, J.L. Barker, U. Porzio, Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurons, J. Neurochem. 56 Ž1991. 392–399. P.A. Garris, R.M. Wightman, Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study, J. Neurosci. 14 Ž1994. 442–450. B. Giros, S. El Mestikawy, N. Godinot, K. Zheng, H. Han, T. Yang-Feng, M.G. Caron, Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter, Mol. Pharmacol. 42 Ž1992. 383–390. Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca2qrcalmodulin-dependent protein kinase II in Neuro2A and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 Ž1993. 559–567. M. Grilli, A.G. Wright, I. Hanbauer, Characterization of w3 Hxdopamine uptake sites and w3 Hxcocaine recognition sites in primary cultures of mesencephalic neurons during in vitro development, J. Neurochem. 56 Ž1991. 2108–2115. A. Hitri, K.Y. Little, E.H. Ellinwood, Effect of cocaine on dopamine transporter receptors depends on routes of chronic cocaine administration, Neuropsychopharmacology 14 Ž1996. 205–210. K. Houben, K. Dardashti, B.D. Howard, PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters, Neurochem. Res. 19 Ž1994. 743–751. Y. Hurd, M. Herkenham, Molecular alterations in the neostriatum of human cocaine addicts, Synapse 13 Ž1993. 357–369. S. Kitayama, T. Dohi, G.R. Uhl, Phorbol esters alter functions of the expressed dopamine transporter, Eur. J. Pharmacol. 268 Ž1994. 115–119. E. Klann, K.R. Shelton, A lead-associated nuclear protein which increases in maturing brain and in differentiating neuroblastoma 2A cells exposed to cyclic AMP-elevating agents, Exp. Brain Res. 57 Ž1990. 71–75. B.K. Kobilka, The role of cytosolic and membrane factors in processing of the human b-2 adrenergic receptor following translocation and glycosylation in a cell-free system, J. Biol. Chem. 265 Ž1990. 7610–7618. J.M. Koff, L. Shuster, L.G. Miller, Chronic cocaine administration is associated with behavioral sensitization and time-dependent changes in striatal dopamine transporter binding, J. Pharmacol. Exp. Ther. 268 Ž1994. 277–282. N. Kojima, N. Kurosawa, T. Nishi, N. Hanai, S. Tsuji, Induction of cholinergic differentiation with neurite sprouting by de novo biosynthesis and expression of GD3 and b-series gangliosides in Neuro2A cells, J. Biol. Chem. 269 Ž1994. 30451–30456. R. Lew, R. Vaughan, R. Simantov, A. Wilson, M.J. Kuhar, Dopamine transporters in the nucleus accumbens and the striatum have different apparent molecular weights, Synapse 8 Ž1991. 152–153. K.Y. Little, J.A. Kirkman, F.I. Carroll, G.R. Breese, G.E. Duncan, w125 IxRTI-55 binding to cocaine-specific dopaminergic and serotonergic uptake sites in the human brain, J. Neurochem. 61 Ž1993. 1996–2006. K.Y. Little, J.A. Kirkman, F.I. Carroll, T.B. Clark, G.E. Duncan, Cocaine use increases w3 H xWIN35428 binding sites in human striatum, Brain Res. 628 Ž1993. 17–25. K.Y. Little, F.I. Carroll, B.J. Cassin, Characterization and localization of w125 IxRTI-121 binding sites in human striatum and medial temporal lobe, J. Pharmacol. Exp. Ther. 274 Ž1995. 1473–1483. B.K. Madras, R.D. Spealman, M.A. Fahey, J.L. Neumeyer, J.K. Saha, R.A. Milius, Cocaine receptors labeled by w3 Hx2bcarbomethoxy-3b-Ž4-fluorophenyl. tropane, Mol. Pharmacol. 36 Ž1989. 518–524.

L. Zhang et al.r Molecular Brain Research 59 (1998) 66–73 w26x A.D. Minana, V. Felipo, S. Grisolia, Inhibition of protein kinase C induces differentiation in Neuro2A cells, Proc. Natl. Acad. Sci. USA 87 Ž1990. 4335–4339. w27x B.F. O’Dowd, M. Hnatowich, M.G. Caron, R.J. Lefkowitz, M. Bouvier, Palmitolytion of the human b2-adrenergic receptor, J. Biol. Chem. 264 Ž1989. 7564–7569. w28x A. Patel, G. Uhl, M.J. Kuhar, Species differences in dopamine transporters: postmortem changes and glycosylation differences, J. Neurochem. 61 Ž1993. 496–500. w29x C. Perrone-Capamo, A. Tino, U. Porzio, Target cells modulate dopamine transporter gene expression during brain development, Dev. Neurosci. 5 Ž1994. 1145–1148. w30x Z. Pristupa, J. Wilson, B. Hoffman, S. Kish, H. Niznik, Pharmacological heterogeneity of the cloned and native human dopamine transporter: disassociation of w3 H xWIN35428 and w3 H xGBR12935 binding, Mol. Pharmacol. 45 Ž1994. 125–135. w31x A. Prochiantz, U. Porzio, A. Kato, B. Berger, J. Glowinski, In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells, Proc. Natl. Acad. Sci. USA 76 Ž1979. 5387–5391. w32x Y. Quin, A. Galli, S. Ramamoorthy, S. Risso, L.J. Defelice, R.D. Blakely, Protein kinease c activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression, J. Neurosci. 17 Ž1997. 45–57. w33x M.E.A. Reith, L.L. Coffey, w3 HxWIN 35,428 binding to the dopamine uptake carrier: II. Effect of membrane fractionation procedure and freezing, J. Neurosci. Meth. 51 Ž1994. 31–38. w34x W. Shi, M.N. Gould, Induction of differentiation in Neuro2A cells by the monoterpene perillyl alcohol, Cancer Letters 95 Ž1995. 1–6. w35x S. Shimada, S. Kitayama, C. Lin, A. Patel, E. Nanthakumar, P. Gregor, M. Kuhar, G. Uhl, Cloning and expression of a cocaine-sen-

w36x

w37x

w38x

w39x

w40x

w41x

w42x

73

sitive dopamine transporter complementary DNA, Science 254 Ž1991. 576–578. J.K. Staley, L. Hearn, J. Ruttenber, C.V. Wetli, D.C. Mash, High affinity cocaine recognition sites on the dopamine transporter are elevated in fatal cocaine overdose victims, J. Pharmacol. Exp. Ther. 271 Ž1994. 1678–1685. S. Tsuji, T. Yamashita, M. Tanaka, Y. Nagai, Synthetic sialyl compounds as well as natural gangliosides induce neuritogenesis in a mouse neuroblastoma cell line ŽNeuro2A., J. Neurochem. 50 Ž1988. 414–423. M. Valchar, I. Hanbauer, Comparison of w3 HxWIN35428 binding, a marker for dopamine transporter, in embryonic mesencephalic neuronal cultures with striatal membranes of adult rats, J. Neurochem. 60 Ž1993. 469–476. R.A. Vaughan, R.A. Huff, G.R. Uhl, M.J. Kuhar, Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes, J. Biol. Chem. 272 Ž1997. 15541–15546. S.C. Wall, R.B. Innis, G. Rudnick, Binding of the cocaine analog 2b-carboxymethoxy-3b-Ž4-w125 Ixiodphenyl.tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2b-carboxymethoxy-3b-Ž4-w125 Ixiodphenyl.tropane binding, Mol. Pharmacol. 43 Ž1993. 264–270. J.M. Wilson, J.N. Nobrega, M.E. Carroll, H.B. Niznik, K. Shannak, S.T. Lac, Z.B. Pristupa, L.M. Dixon, S.J. Kish, Heterogeneous subregional binding patterns of w3 H xWIN35428 and 3Hw3 HxGBR12935 are differentially regulated by chronic cocaine selfadministration, J. Neurosci. 14 Ž1994. 1121–1130. L. Zhang, L.L. Coffey, M.E.A. Reith, Regulation of the functional activity of the human dopamine transporter by protein kinase C, Biochem. Pharmacol. 53 Ž1997. 677–688.