[17] Inhibition of [3H]dopamine translocation and [3H]cocaine analog binding: A potential screening device for cocaine antagonists

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[ 17]

[ 17]

Inhibition of [3H]Dopamine Translocation and [3H]Cocaine Analog Binding: A Potential Screening Device for Cocaine Antagonists

By MAARTEN

E . A. REITH, f E N XU, F. IVY CARROLL, a n d N1AN-HANG CHEN

Introduction There is considerable interest in developing a cocaine antagonist for use as an adjunct in the treatment of cocaine addiction or as a counteractive medication to combat acute central overstimulation. Because the dopamine transporter in the plasma membrane is one of the targets considered to be crucial for its centrally stimulatory activity, 1 it would be logical to conceive of a compound that interferes with the action of cocaine at the transporter level. Antagonism of cocaine action could be accommodated in a model depicting separate sites of action for uptake blockers and substrate recognition. In the case of the serotonin transporter, early work with radiolabeled imipramine led to proposals involving separate sites 2'3 perhaps with an endogenous ligand 4 acting only on the blocker site to explain how upregulated imipramine binding sites could cause reduced neuronal uptake of serotoninJ The possibility of an endogenous ligand has been also entertained for the dopamine transporter, 6'7 but such compounds have remained elusive. 8-1° In a model assuming separate blocker and substrate recognition 1M. W. Fischman and C.-E. Johanson, in "Pharmacological Aspects of Drug Dependence: Towards an Integrated Neurobehavioral Approach" (C. R. Schuster and M. J. Kuhar, Eds.), p. 159 (1996). M. S. Briley, S. Z. Langer, R. Raisman, D. Sechter, and E. Zarifian, Science 209, 303 (1980). 3 N. Brunello, D. M. Chuang, and E. Costa, Science 215, 1112 (1982). 4 E. Costa, M. L. Barbaccia, O. Gandolfi, and D. M. Chuang, in "New Vistas in Depression" (S. Z. Langer, R. Takahashi, T. Segawa, and M. Briley, Eds.), p. 31. Pergamon Press, Oxford, 1985. 5 M. L. Barbaccia, O. Gandolfi, D. M. Chuang, and E. Costa, Proc. Natl. Acad. Sci. USA 80, 5134 (1983). 6 I. Hanbauer, L. T. Kennedy, M. C. Missale, and E. C. Bruckwick, in "New Vistas in Depression" (S. Z. Langer, R. Takahashi, T. Segawa, and M. Briley, Eds.), p. 41. Pergamon Press, Oxford, 1985. 7 M. E. A. Reith, N I D A Res. Monogr. 88, 23 (1988). s M. E. A. Reith, H. Sershen, and A. Lajtha, Neurochem. Res. 5, 1291 (1980). 9 C. R. Lee, A. M. Galzin, M. A. Taranger, and S. Z. Langer, Biochem. PharrnacoL 36, 945 (1987).

METHODS IN ENZYMOLOGY, VOL. 296

Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 0076-6879/98 $25.00

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sites, it is possible to envision an antagonist that binds to the blocker binding site but does not induce the required conformational change to prevent substrate binding and translocation; this antagonist then could interfere with the binding of a blocker such as cocaine.7 Although equilibrium binding experiments with radioligands for the dopamine transporter and uptake inhibition studies have generally supported the involvement of common binding domains for cocaine and dopamine, 11-14 there is also evidence for the additional involvement of separate domains from studies on kinetic modeling of uptake data collected by rotating disk voltammetry, 15 thermodynamic analysis of radioligand binding] 6 protection against action of sulfhydryl reagents at blocker binding sites, 17 19 and construction of transporter chimeras 2°'2~ or mutants with site-directed alterations. 22 All data taken together support the existence of a common binding domain in the recognition of cocaine and dopamine, as well as separate domains. It is possible that a cocaine antagonist could be developed that only interferes with a cocaine binding domain that is not involved in the recognition of dopamine, thereby preventing the action of cocaine without disturbing dopamine uptake itself. ~'22'23 Although the conformational model as advanced by Saadouni et al. ~4could also accommodate the existence of a cocaine antagonist at the dopamine transporter level, no direct evidence for this model beyond thermodynamic analysis is available. The group of Rothman has

m F. Artigas, E. Martinez, and A. Adell, Eur. Y. Pharmacol. 181, 9 (1990). 11 E. Richelson and M. Pfenning, Eur. J. Pharmacol. 104, 277 (1984). 12 B. K. Krueger, J. Neurochem. 55, 260 (1990). 13 M. E. A. Reith, B. De Costa, K. C. Rice, and A. E. Jacobson, Eur. J. Pharmacol. 227, 417 (1992). 14 S. Saadouni, F. Refahi-Lyamani, J. Costentin, and J. J. Bonnet, Eur. J. Pharmacol. 268, 187 (1994). ~5j. S. McElvain and J. O. Schenk, Biochem. Pharmacol. 43, 2189 (1992). 16 j. j. Bonnet, S. Benmansour, J. Costentin, E. M. Parker, and L. X. Cubeddu, J. Pharmacol. Exp. Ther. 253, 1206 (1990). 17 K. M. Johnson, J. S. Bergmann, and A. P. Kozikowski, Eur. J. Pharmacol. 227, 411 (1992). 18 M. E. A. Reith, C. Xu, and L. L. Coffey, Biochem. Pharmacol. 52, 1435 (1996). 19 C. Xu, L. L. Coffey, and M. E. A. Reith, Naunyn-Schmiedeberg's Arch. Pharmacol. 355, 64 (1997). 20 K. J. Buck and S. G. Amara, Proc. Natl. Acad. Sci. USA 91, 12584 (1994). 21 S. Povlock and S. G. Amara, in "Neurotransmitter Transporters: Structure, Function, and Regulation" (M. E. A. Reith, Ed.), p. 1. Humana Press, Totowa, New Jersey, 1996. 22 S. Kitayama, S. Shimada, H. Xu, L. Markham, D. M. Donovan, and G. R. Uhl, Proc. Natl. Acad. Sci. USA 89, 7782 (1992). 23 F. I. Carroll, A. H. Lewin, and M. J. Kuhar, in "Neurotransmitter Transporters: Structure, Function, and Regulation" (M. E. A. Reith, Ed.), p. 263. Humana Press, Totowa, New Jersey, 1996.

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reported the possibility of "partial agonism" in the context of effects on extracellular dopamine by G B R 12909 in awake animals24; in vivo interrelating systems may be required for this intriguing phenomenon as in vitro approaches have not been able to show partial agonismY There is an in vitro observation demonstrating a moderate antagonistic effect of (7amethoxy)cocaine against cocaine-induced dopamine uptake blockade. 26

Impact of Assay Conditions The general approach in screening for a potential cocaine antagonist consists of assessing the inhibitory effect of a test compound on the translocation of dopamine by the dopamine transporter, usually in rat striatal synaptosomal preparations, as well as its inhibitory effect on the binding of a radiolabeled cocaine analog. The aim is to find a compound that potently inhibits binding without affecting dopamine uptake. Routinely, such assays are performed under conditions optimized for the intended measure, uptake, or binding. However, it is known that experimental conditions can greatly affect the observed characteristics of uptake or binding phenomena as well as the potency of inhibitors. For instance, dopamine uptake rates depend on the assay conditionsS '2s and inhibitors will be more or less active as a function of ionic content of the incubation mixture. 29-31 Binding of cocaine analogs has been shown to vary depending on the conditions used to generate the brain preparation and on the assay buffer, 32,33and conditions also affect observed potencies of inhibitors. 31'34"35 One important condition, not usually taken into account, is the pH of the assay buffer. An example of this, observed in our laboratory, is the impact of pH on the dependency of the shape of the Na+-dependency curve of the binding of 3H-labeled 2fl-carbomethoxy-3fl-(4-fluorophenyl)tropane (WIN 24 M. H. Baumann, G. U. Char, B. R. De Costa, K. C. Rice, and R. B. Rothman, J. Pharmacol. Exp. Ther. 271, 1216 (1994). 25 A. N. Gifford, J. S. Bergmann, and K. M. Johnson, Drug Alcohol Depend. 32, 65 (1993). 26 D. Simoni, J. Stoelwinder, A. P. Kozikowski, K. M. Johnson, J. S. Bergmann, and R. G. Ball, J. Med. Chem. 36, 3975 (1993). 27 R. P. Shank, C. R. Schneider, and J. J. Tighe, J. Neurochem. 49, 381 (1987). 28 I. Zimanyi, A. Lajtha, and M. E. A. Reith, Naunyn Schmiedebergs Arch. Pharmacol. 340, 626 (1989). 29 S. C. Wall, R. B. Innis, and G. Rudnick, Mol. PharmacoL 43, 264 (1993). 3o N. Amejdki-Chab, J. Costentin, and J. J. Bonnet, J. Neurochem. 58, 793 (1992). 31 R. B. Rothman, K. M. Becketts, L. R. Radesca, B. R. De Costa, K. C. Rice, F. I. Carroll, and C. M. Dersch, Life Sci. 53, PL267 (1993). 32 M. E. A. Reith and L. L. Coffey, J. Neurochem. 61, 167 (1993). 33 L. L. Coffey and M. E. A. Reith, J. Neurosci. Methods 51, 23 (1994). 34 D. O. Calligaro and M. E. Eldefrawi, Membr. Biochem. 7, 87 (1987). 38 C. Xu and M. E. A. Reith, J. Pharmacol. Exp. Ther. 282, 920 (1997).

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35,428), a phenyltropane analog of cocaine, to rat striatal membrane (Fig. 1). At pH 7.4 in the presence of 0.32 M sucrose, used in many dopamine transporter studies to enhance binding, a peak of [3H]WlN35,428 binding was observed at [Na ÷] - 3 0 - 5 0 mM along with a reduction in binding at higher [Na +] (Fig. 1A). When sucrose was not present, binding was much lower with a plateau at 30 mM continuing up to 200 mM Na +. The stimulatory effect of sucrose was more pronounced at low rather than high [Na +]

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FJG. 1. Na + dependency (A and C) and saturation analysis (B and D) of [3H]WIN 35,428 binding at p H 7.4 and 7.9 in the presence (©) and absence (B) of sucrose. (A, C) [3H]WIN 35,428 binding to the rat striatal membranes was measured with varying concentrations of Na + from 0 to 200 mM. Binding is expressed as % of the binding at 50 m M Na" without sucrose measured with the same membrane preparation on the same day. Values are mean _+ SEM of three independent experiments carried out in triplicate. The average binding at 50 m M Na + without sucrose was 880 fmol/mg of protein. (B, D) The radioligand was present at 3.5 nM at pH 7.4 and 1.5 nM at pH 7.9, and increasing concentrations of unlabeled WIN 35,428 were added up to 500 nM; nonspecific binding was defined with 30/~M cocaine. The straight line represents the best fit chosen by the L I G A N D program. Both panels show a typical experiment that was assayed in triplicate. The experiment was carried out three times with independent striatal membrane preparations (for averages see text). P < 0.05 compared with absence of sucrose at same [Na +] (Student's t-test).

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resulting in a changed shape of the Na + curve. A similar effect was also observed at pH 7.9 (Fig. 1C), although the binding peak appeared at a lower [Na +] - 1 0 - 3 0 mM both with and without sucrose. In addition, a reduction in binding at higher [Na +] was evident both in the absence and, more strongly, in the presence of sucrose. For pH 7.4, the effect of 0.32 M sucrose was tested on the binding constants at 18 mM Na +, on the ascending portion of the Na ÷ curve (Fig. 1B). The presence of sucrose in the assay buffer reduced the Ko value from 18 -+ 2 to 11 ___1 nM (P < 0.05, Student's t-test) (average __+SEM for 3 independent experiments) without affecting the Brnax value (9 -+ 1.0 compared with 8 _+0.3 pmol/mg of protein) (Fig. 1B). The application of the one-site binding model to [3H]WIN 35,428 binding data under the present conditions is discussed in detail in our previous studies. 21'36 At pH 7.9 and 30 mM Na + (peak binding in the Na + curve), again the presence of 0.32 M sucrose reduced the Kd from 12 __+ 1 to 6 ___ 1 nM without affecting the Bmax (6 --- 0.9 compared with 7 _ 0.7 pmol/mg of protein) (P < 0.05) (Fig. 1D). Thus, both pH and sucrose are important factors determining the shape of the Na + curve. At a relatively higher pH (7.9) the binding peak tends to occur at 10-30 mM Na ÷ and inhibition by higher [Na +] is observed with or without sucrose, whereas at a relatively lower pH (7.4) the binding peak is shifted rightwardly to - 3 0 - 5 0 mM Na + and no inhibition by higher [Na +] is seen in the absence of sucrose. This puts a different light on our previously proposed distinction between cocaine-/ methyl phenidate-like ligands and GBR-/mazindol-like ligands, inhibited and not inhibited, respectively, by high [Na+]. 32 In most studies on [3H]cocaine and [3H]WIN 35,428 binding reviewed by us previously32either the pH was ->7.7 or sucrose was present, favoring the occurrence of high [Na ÷] inhibition, whereas in most studies on [3H]GBR 12935/12783 and [3H]mazindol binding the pH was -<7.6 and sucrose was absent, favoring the occurrence of a Na + plateau. In searching for a cocaine antagonist, it is important to reduce the chance of finding false positives, inhibiting binding more than uptake, and minimize the risk of missing potential lead compounds, inhibiting uptake more than binding. Given the impact of conditions described above, it is therefore important to carry out the two assays under comparable experimental conditions, as discussed later.

Kinetic Considerations in Comparing Uptake and Binding There are special kinetic features that need to be considered in comparing uptake and binding assays. For example, uptake needs to be studied 36 C. Xu, L. L. Coffey, and M. E. A. Reith, Biochem. Pharmacol. 49, 339 (1995).

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in the initial velocity phase (i.e., duing a short time period) whereas binding is ideally assessed on equilibration (i.e., after a relatively longer time period). Because some inhibitors need time to equilibrate, a short uptake assay can therefore yield a higher ICs0 for uptake inhibition than the ICs0 for binding inhibition observed in a longer binding assay, erroneously leading to the conclusion that the compound is a "partial agonist." The method we have adopted for rat striatal synaptosomes circumvents these problems, making use of a rapidly equilibrating ligand for translocation. [3H]dopamine itself, and a rapidly equilibrating radioligand for binding, [3H]WIN 35,428. In the uptake assay, the inhibitor is added at time 0, [3H]dopamine is added at exactly 7 min, and termination occurs at exactly 8 min; in the binding assay, inhibitor and [3H]WIN 35,428 are present from time 0, and termination occurs at 8 min. Consideration of the rate constants involved of radioligands and inhibitor demonstrates that both assays "catch" the inhibitor at the same time point of its approach toward equilibrium (7-8 min) so that uptake and binding potencies are comparable in that respect even if the inhibitor has not fully equilibrated (for full details, see our previous paper36). Furthermore, both [3H]dopamine and [3H]WIN 35,428 can be considered to be at equilibrium with their binding sites on the transporter under the allotted times in the assays. 36 An important study in this context is that of the group of Rothman et aL 31 pioneering the approach of carrying out uptake and binding assays under identical conditions. In their study, uptake of [3H]dopamine was compared with binding of 125I-labeled 2B-carbomethoxy-3/3-(4-iodophenyl)tropane (RTI-55), another phenyltropane analog of cocaine. [125I]RTI-55 equilibrates appreciably slower than [3H]WIN 35,428, and the timing approach is therefore necessarily different. Rothman et aL 31 preincubated the inhibitor for 1 hr, and then conducted either the uptake or binding assay for an extra 20 min. Although in this design [125I]RTI-55 will not equilibrate in the time allowed, the inhibitory potencies of test compounds will be comparable in the uptake and binding assays as long as the concentration of [125I]RTI-55 is smaller than its K~ so that the inhibitor equilibrium is not shifted appreciably. Results Obtained u n d e r Identical Conditions for Uptake and Binding Uptake ([3H]dopamine) and binding ([3H]WIN 35,428) inhibitory potencies were obtained pairwise with rat striatal synaptosomal preparations on the same experimental day under the conditions described above for cocaine, WIN 35,428, benztropine, nomifensine, mazindol, BTCP, and GBR 12909 (Fig. 2). The data for GBR 12909 were subject to appreciable variation between days, with uptake and binding ICs0 values changing in tandem, presumably because of variable adsorption onto the walls of tubes of the

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TRANSPORT ASSAYS AND KINETIC ANALYSES

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Fro. 2. IC50values of compounds in inhibiting [3H]WlN 35,428 binding and [3H]dopamine uptake under identical conditions, Each point represents a paired observation of both the binding and uptake IC50obtained with the same membrane preparation and the same stocks of drugs. The data were obtained with 8 min for [3H]WIN 35,428 binding and 1 min for [3H]dopamine uptake. The solid straight line represents a theoretical line describing a perfect one-to-one relationship between IC50values for binding and uptake. The dashed line represents the results of least squares linear regression analysis of the data (r = 0.97, n = 10, P < 0.001). (Reprinted in adapted form by permission of the publisher from "Translocation of dopamine and binding of WIN 35,428 [2/3-carbomethoxy-3/3-(4-fluorophenyl)tropane] measured under identical conditions in rat striatal synaptosomal preparations: inhibition by various blockers," M. E. A. Reith, L. L. Coffey, and C. Xu, Biochemical Pharmacology 49, No. 3, pp. 339-350. Copyright 1995 by Elsevier Science Inc.) i, Cocaine; 2, WIN 35,428;3, benztropine; 4, nomifensine; 5, mazindol; 6, BTCP; and 7, GBR 12909.

stock solutions (for discussion see Ref. 36). T h e correlation line c o m p o s e d of all points (Fig. 2, b r o k e n line, r = 0.97, n = 10, P < 0.001) was displaced to the left c o m p a r e d with the theoretical line depicting a perfect one-toone ratio (solid straight line). Binding over u p t a k e IC50 ratios were on the average 2.3, indicating a m o r e p o t e n t u p t a k e inhibitory activity. Several reasons for this are discussed in o u r previous report, 36 including the possibility of a spare r e c e p t o r reserve speculated to derive f r o m transporter dimers that carry two binding sites for W I N 35,428, of which only one needs to be occupied for preventing u p t a k e by that dimer. T h e same experimental a p p r o a c h was taken to reassess the potencies of three p h e n y l t r o p a n e analogs of cocaine that had b e e n d e m o n s t r a t e d previously to be stronger in inhibiting [ 3 H ] W I N 35,428 binding than [3H]dop a m i n e u p t a k e u n d e r assay conditions optimized for the respective m e a s u r e (Table I). W h e n assayed u n d e r identical b i n d i n g / u p t a k e conditions, the c o m p o u n d s did not differ f r o m W I N 35,428 itself in their binding over u p t a k e IC50 ratio, again being approximately 1.4-2.

[ 17]

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TABLE I INHIBITION OF [ 3 H ] W I N 35,428 BINDING AND [3H]DoPAMINE UPTAKE UNDER NONIDENTICAL AND IDENTICAL CONDITIONS

Identical conditions Nonidentical conditions, binding over uptake Compound

IC5o (ratio) °

W I N 35,428 RTI-32 RTI-114 RTI-121

1.0 0.24 0.23 0.15

± 0.2 ± 0.04 _+ 0.02 ± 0.02

Binding

Uptake Binding over

IC5oh'' (nM)

IC5ob'~

Hill

(nM)

Hill

u p t a k e IC5o

(ratio) a

21.1 + 3.2 5.8 _ 0.2 3.7 2.8 ± 0.2

1.1 _+ 0.1 1.1 ± 0.1 1.2 1.2 ± 0.1

14.2 ± 1.2 2.9 ± 0.3 2.6 2.2 ± 1.0

1.0 ± 0.1 1.0 _+ 0.1 1.0 1.1 -+ 0.0

1.5 _+ 0.1 2.0 ± 0.2 1.4 1.7 -+ 0.8

" Computed from mean -2_ S E M of binding and uptake data of Carroll and colleagues. 39 b Average for 2 independent experiments each carried out in triplicate ( ± S E M = range × 0.5 because n = 2), except for RTI-114, which was examined in one experiment. ' There are significant different differences (two-way A N O V A ) between drugs [factor A , F(3,13) = 38.47, P < 0.0005] and assays [factor B, binding/uptake, F(1,13) = 6.41, P < 0.05], but there was no interaction [F(3,13) = 1.54, P = 0.30]. d Average of ratios obtained in 2 independent experiments ( ± S E M = r a n g e x 0.5 b e c a u s e n = 2);

each ratio was calculated per experiment with the parallel binding and uptake assays; RTI-ll4 was examined in one experiment.

Experimental Protocols General Considerations

Ideally, the binding and uptake assays are performed in parallel on the same P2 preparation with the same stocks of test compounds. This ensures a valid comparison between binding and uptake results. It does require the participation of at least two or three people (see proceeding text) in the experiment, because the striatal suspensions, once prepared, need to be processed for binding and uptake in a timely fashion. When uptake experiments are carried out by single filtration, incubations are staggered in time; we routinely start each incubation 1 min apart, and 60 assays will therefore result in the striatal suspension sitting on ice between 5 and 10 min for the first tube, and 65-70 min for the last tube. Over that time frame, uptake activity only decreases marginally, for which one can control by repeating total and nonspecific conditions in the second half of the set of incubations. This approach will require two people handling the uptake assays (one for starting preincubation/incubation and one for stopping and filtering), or if a harvester is used, one individual. An additional person needs to participate to run the binding assays which are routinely harvested in most labs.

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Protocols

1. Set up tubes for binding and uptake assays with every condition in triplicate. The following description is for binding assays carried out in a total volume of 0.2 ml in 1-ml ministrip tubes (Skatron, Sterling, VA), for harvesting with a Skatron miniharvester, and uptake assays in a total volume of 0.4 ml in borsosilicate culture tubes (12 × 75 ram), for workup by the single filtration assay. Adjust details for other assay designs. For instance, one might want to assay for binding in 96-well plates, and terminate with a harvester that has the 96-well format. Or one might terminate the uptake assays with a harvester with pins in a format of 24 or 48 positions as the standard racks that hold the 12 × 75-mm tubes. We have measured [3H]dopamine uptake (in parallel with binding), in sets of 24 assay mixtures by harvesting in the Brandel (Gaithersburg, MD) cell harvester with Whatman (Clifton, NJ) GF/C filters. In that case, incubations with drug and [3H]dopamine were started with less than 5 sec between samples. Immediately after addition of ice-cold stop solution on the same time schedule, all 24 samples were filtered collectively. This procedure results in a variable waiting time (0-2 min) between adding a stop solution and filtering the mixture, but in control experiments (data not shown) it was found that there was no loss of accumulated [3H]dopamine taken up during waiting times up to 6 min, the longest interval tested. With GF/C filters the filtration by the Brandel harvester was more rapid than with GF/F filters, and the uptake results were the same. The final concentrations of all components, in both binding and uptake assays, are 73 mM NaC1, 3 mM KC1, 0.7 mM MgSO4, 6 mM glucose, 0.6 mM CaC12, 0.006 mM nialamide, 0.08 M sucrose, 18 mM Na +, and 10 mM phosphate from a mixture of primary and secondary phosphate buffer (giving a pH of 7.4 at room temperature), approximately 0.25 mg (for binding) or 0.12 mg (for uptake) of P2 protein per ml assay medium, and test drug (a total of 8 concentrations evenly spaced around the IC50). The radioactive [3H]WlN 35,428 stock for the binding assays can be prepared at this point. The stock should be prepared such that addition of 20-/xl aliquots to the binding assays results in a final concentration of 4 nM radioactive [3H]WlN 35,428 (approximately 80 Ci/mmol, DuPont---New England Nuclear, Boston, MA). For binding (uptake) measurements, the tubes are filled with 20 (0)/xl [3H]WIN35,428 stock, 120 (240)/xl buffer stock, and 10 (20)/xl test drug and kept on ice. 2. Dissect striatal tissue from rats (choose a strain, age, and gender and use same in followup experiments). Homogenize in 15 volumes of ice-cold 0.32 M sucrose in a glass homogenizer with a motor-driven Teflon pestle (7 strokes up and down at 800 rpm). Rinse the homogenizer and pestle with 30 volumes of 0.32 M sucrose. Combine this fluid and the homogenate,

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and centrifugate at 1000 g for 10 min at 0-4 °. Centrifuge the supernatant fraction subsequently at 17,000 g for 20 rain at 0-4 °. 3. Prepare the dopamine radioisotope stocks while the striatal preparation is taken through the final centrifuge spin. The stock should be prepared such that addition of 40-/xl aliquots to the uptake assays results in 4 nM [3H]dopamine (from supplier, specific activity 30-35 Ci/mmol) along with 46 nM unlabeled dopamine. Isotope solutions that come from the supplier with a gas other than air in the vial are accessed through the cap with a Hamilton syringe in a manner that does not introduce air in the container. Keep the prepared stocks on ice, and shield the dopamine-containing stock from light by wrapping the tube with aluminum foil. 4. Take the tube(s) with the striatal preparation(s) from the centrifuge, and homogenize the resulting pellet (P2) in approximately 0.04 ml of 0.32 M sucrose per mg of initial tissue weight with the glass-Teflon homogenizer (not with a Polytron or similarly forceful device); dilute part of this homogenate 2-fold for the uptake experiments. At this stage of the experiment, initiation of the actual binding and uptake assays is approximately 10 min away. Binding assays are handled in multiples of 12 (harvester), and uptake tubes individually. Transfer the first set of assay tubes (#1-12 for binding and #1 for uptake) to a waterbath at 25° so that tubes are allowed to become equilibrated at that temperature; set the shaker at a moderate setting (for example, 50 rpm). The goal is to warm up each tube for approximately 10 min prior to adding brain membranes (see proceeding text; the smallersized binding tubes may require less time to warm up). Transfer next sets of binding tubes to the waterbath at l-rain intervals allowing completion of subsequent steps (see proceeding text); transfer uptake tubes sequentially at 1-min intervals. For the binding assays, after prewarming in the bath, add 50 /xl P2 suspension followed by gentle vortexing. Because a multiple of 12 tubes need to be harvested at the end of the incubation, make additions sequentially as fast as possible, which can be done within half a minute for all additions to all tubes; the use of a multichannel pipette will enhance the process. Incubate the assay mixture at 25° for 8 rain. Terminate the reaction by the addition of an excess of ice-cold wash buffer (73 mM NaC1, 3 mM KC1, 0.7 mM MgSO4, 6 mM glucose, 0.6 mM CaC12, 30 mM Na ÷, and 17 mM phosphate from a mixture of primary and secondary phosphate buffer, giving a pH of 7.4 at room temperature) and filtration over Skatron receptor binding filter mats (glass fiber filter, 1-/xm retention, No. 11734, equivalent to Whatman GF/B) with the Skatron miniharvester. Because this binding assay is of the equilibrium type, a difference of 0.5 rain in total incubation time between the first and last tube of each set of 12 has little or no impact on the final results. For the uptake assays, approximately 10 min after prewarming in the

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bath, add 100/xl P2 suspension followed by gentle vortexing. Incubate the assay mixture at 25 ° for 7 rain and add, exactly at 7 rain, 40/zl radioligand stock. At exactly 8 min, terminate the reaction by the addition of an excess of ice-cold wash buffer (see previous text) and filtration over Whatman GF/F glass fiber filters with a single-manifold Millipore filtration apparatus. All filters are pretreated with 0.05% (w/v) poly(L-lysine) (molecular weight 15,000-30,000) (other groups have successfully used polyethyleneimine instead of polylysine for these radioligands). After the first filtration, filters are washed three times with 1 (binding) or 4 (uptake) ml of ice-cold wash buffer, and asssayed for radioactivity by liquid scintillation counting. Filters are cut individually and counted in separate scintillation vials. These counting procedures can be simplified if a scintillation counter is available that accepts cassettes containing filter mats with assayed material in designated spots. Nonspecific binding or uptake is defined with 100/xM cocaine. Nonspecific binding or uptake is usually no more than 4% of the total value determined in the absence of inhibitor. 5. Data analysis can be done in many ways with a number of software packages available. We routinely compute ICs0 values and pseudo-Hill numbers with the equation of the ALLFIT program of De Lean et aL 37 entered into the Microsoft ORIGIN curve-fitting and plotting software. We usually run this nonlinear regression program with total and nonspecific binding (uptake) centered as constants; if there are enough data points of low levels of test compound showing little or no inhibition, total binding values are better estimated by letting that parameter float. When WIN 35,428 or dopamine is the inhibiting compound in the binding or uptake assays, respectively, the data can be analyzed with the nonlinear computer fitting program LIGAND. 3s In this case, we routinely use data files in which nonspecific uptake (binding) (N1) has not been subtracted; the results obtained by entering N1 as a constant in the fitting procedures are usually close to estimates obtained by having N1 float, and generally we choose the former approach, relying on a pharmacological definition of nonspecific binding for the target site.

37 A. DeLean, P. J. Munson, and D. Rodbard, Am. J. Physiol. 235, E97 (1978). 38 p. j. Munson and D. Rodbard, A n a l Biochem. 107~ 220 (1980). 39 F. I. Carroll, P. Kotian, A. Dehghani, J. L. Gray, M. A. Kuzemko, K. A. Parham, P. Abraham, A. H. Lewin, J. W. Boja, and M. J. Kuhar, J. Med. Chem. 38, 379 (1995).

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Acknowledgment We would like to thank the National Institute on Drug Abuse (DA 08379 to M.E.A.R.) and National Natural Sciences Foundation of China (39570812 to N.-H.C.) for support, and Lori L. Coffey for dedicated participation in developing the described protocols in our laboratory.

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and

Drug Vesicle

Binding

Kinetics

in Membrane

Preparation

By HEINZ

BONISCH

Introduction Molecular cloning has shown that neurotransmitter transporters of the plasma m e m b r a n e f o r m at least two families. 1-3 One family consists of the sodium- and chloride-dependent transporters, and m e m b e r s of this family include the transporters for the m o n o a m i n e s dopamine, norepinephrine (NE), and serotonin and those for a series of amino acids such as y-aminobutyric acid ( G A B A ) or glycine. M e m b e r s of the second family are the sodium- and potassium-dependent excitatory amino acid transporters such as glutamate or aspartate. Transport of neurotransmitters is driven by the t r a n s m e m b r a n e Na ÷ gradient, established by the Na+,K+-ATPase, and the process is analogous to other secondary active transport systems such as the intestinal Na+/glucose transporter. However, unlike Na+/glucose transport, additional ions are required for transport of most of the neurotransmitters, such as extracellular C F and intracellular K +. Even before the cloning of neurotransmitter transporters, studies on plasma m e m b r a n e vesicles had contributed significantly to our present knowledge on energetic and pharmacological properties of m a n y neurotransmitter transport systems. For example, plasma m e m b r a n e vesicles have been used to study transport and/or binding characteristics of the transporters for G A B A , 4's

1 S. G. Amara and M. J. Kuhar, Annu. Rev. Neurosci. 16, 73 (1993). 2 B. Borowsky and B. J. Hoffman, Int. Rev. Neurobiol. 38, 139 (1995). 3 M. E. A. Reith, "Neurotransmitter Transporters." Humana Press, Totowa, New Jersey, 1997. 4 g. I. Kanner, Biochemistry 17, 1207 (1978). 5 R. Roskoski, J. Neurochem. 36, 544 (1981).

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