Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1749-1753, April 1987 Biochemistry Uptake of and related by cultured chromaffin cells: Characterization of cocaine-sensitive and -insensitive plasma membrane transport sites DIPAK K. BANERJEE*tt, RUDOLF A. LUTZ§¶, MARK A. LEVINE*, DAVID RODBARD§, AND HARVEY B. POLLARD* *Laboratory of Cell Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, §Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Diseases, National Institutes of Health, Bethesda, MD 20892; and TDepartment of Biochemistry and Nutrition, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936-5067 Communicated by Bernhard Witkop, September 23, 1986

ABSTRACT Norepinephrine and its closely related ana- ited by reserpine. Both the ATPase(s) (14, 15) and the logues, and epinephrine, are transported into chro- transport site(s) (16, 17) have been purified and functionally maffin cells in culture by two distinct types of sites on the reconstituted. At a more chemical level, the specificity ofthe plasma membrane: one is sensitive to cocaine while the other is uptake system for different analogues has also not. The cocaine-sensitive site has a high affinity for catechol- been extensively studied by us as well as by others (18-21). amines and depends on sodium in the medium. The apparent In the present paper we have used our detailed knowledge Km for norepinephrine uptake by the cocaine-sensitive site is 5.8 of the chromaffin granule transport system to direct our IM when determined in the presence of 118 mM NaCl, investigation into the biochemistry and pharmacology of the obtained using nonlinear least-square curve fitting. Detailed catecholamine transport system in the chromaffin cell plasma kinetic analysis has also shown cocaine to be a competitive membrane. Utilizing the particular sensitivity of the plasma inhibitor of norepinephrine uptake with an apparent Ki of ca. membrane transport system to cocaine and related drugs, we 1 ,.M. This site is blocked by a series oftricyclic have found evidence for multiple catecholamine transport drugs with relative potencies characteristic of norepinephrine sites at the cell surface, only one of which proved to be transport sites in neurons. In contrast, the cocaine-insensitive cocaine-sensitive. We have also further delineated the sub- site(s) have a low affinity for norepinephrine (apparent strate specificities of the plasma membrane and chromaffin Km, =88 ,uM) and are also able to transport catecholamine granule membrane transport systems." analogues such as dimethylepinephrine and isoproterenol, which have bulky groups attached to the amine moiety. MATERIALS AND METHODS Transport of norepinephrine at both sites is blocked by low temperature, by mitochondrial uncouplers, and by other Cell Preparation and Culture. Chromaffin cells were pre- metabolic inhibitors. Both of these transport sites in the pared and cultured from bovine adrenal glands as described chromaffin cell plasma membrane, therefore, appear to be (21). Over a 48-hr incubation period nonchromaffin cells (22) different from the well-characterized catecholamine transport and some chromaffin cells attached to flask surfaces, leaving sites in the chromaffin granule membrane on the basis of most chromaffin cells clustered together and in suspension substrate specificity and their sensitivity to inhibitors. (23). After 48 hr, cells were harvested by centrifugation over a density step gradient (1.03 g/ml) composed of isotonic Chromaffin cells and catecholaminergic nerve endings take metrizamide diluted 1:7 in standard release medium (SRM) or up catecholamines such as norepinephrine by a specific 10% (vol/vol) Ficoll-Paque in SRM, and then resuspended in sodium-dependent transport process localized in the plasma 10 ml of SRM before use in the transport assay. (SRM, 118 membrane (1-5). This capacity to take up catecholamines has mM NaCl/1.2 mM MgSO4/6.7 mM KCl/10 mM glucose/2.2 been extensively used to load chromaffin cells with radiola- mM CaCl2/25 mM Hepes*NaOH, pH 7.4.) beled catecholamines to simplify measurement of secretion Assay ofCatecholamine Transport. Chromaffin cell suspen- (6). Nonetheless, the uptake process in chromaffin cells has sions were maintained at 37°C for 15 min. Transport was received surprisingly little attention, particularly in view of initiated by mixing 50-,ul aliquots of cells with 350 ,ul of its functional importance (1, 2). In more heterogeneous reaction mixture containing (R)-[3H]norepinephrine (5 uCi/ nervous tissue, this transport system serves to terminate ml; 1 Ci = 37 GBq) or other labeled species and various neurotransmission (3-5) and is the likely site of action of a amounts of unlabeled compounds dissolved in SRM or wide variety ofpsychoactive drugs, including cocaine and the modifications of SRM, as indicated. Experiments were per- tricyclic antidepressents (7, 8). Homogenous preparations of formed according to a simple sedimentation procedure de- chromaffin cells can, therefore, provide an ideal model vised by us for transport analysis (24). Results are reported system for studying the details of these important functions. as mean of quadruplicate measurements ± SEM. In a typical In contrast to the paucity of studies on catecholamine experiment, 10,000 cpm accumulated in the cells in 10 min; transport systems in the chromaffin cell plasma membrane, cells in the blank, in which the entire reaction mixture was the catecholamine transport system in the chromaffin granule maintained on ice, accumulated 300-400 cpm. In the absence membrane, located within the chromaffin cell, has been well of cells, the blank was ca. 30-40 cpm, essentially the studied. The granule membrane has an ATP-dnven proton pump that energizes uptake of norepinephnne and related VPresent address: Medizinisch-Chemisches Zentrallaboratorium, catecholamines (9-13) through a transport site that is inhib- Kantonsspital, Winterthur, Switzerland CH-8401. *To whom requests for reprints should be addressed at the Univer- sity of Puerto Rico. The publication costs of this article were defrayed in part by page charge Part of this work was presented in abstract form at the Federation payment. This article must therefore be hereby marked "advertisement" ofAmerican Societies for Experimental Biology, June 3-7, 1984, St. in accordance with 18 U.S.C. §1734 solely to indicate this fact. Louis, MO.

Downloaded by guest on September 29, 2021 1749 1750 Biochemistry: Banedjee et al. Proc. Natl. Acad Sci. USA 84 (1987)

0.30 the uptake curves were linear for up to 10 min, and data from 10-min incubations were used as initial velocities in the remainder of the study. To further substantiate this choice, 0.25 F we compared uptake data for 2 min and 10 min for calculation t01 FIG. 1. Time course of (R)- ._ [3H]norepinephrine uptake. ofan apparent Km. There were no differences; the Km at 2 min E 0.20 _ , The time courses of uptake at was 14.4 ± 2.3 ,uM, while the Km at 10 min was 13.7 ± 4 AuM. from Fig. 1 was that while sub- E different (R)-norepinephrine An additional observation A concentration were studied. stantial uptake could be observed at (R)-norepinephrine 0. 0.15F The external (R)-norepineph- concentrations as low as 1 ,.M, progressive increases in the Q rne concentrations were as fol- observed between 10 and 20 -WD velocity of uptake could still be 0. lows: curve a, 1 jiM; curve b, 2 ,M (R)-norepinephrine. This broad substrate concentration 0.1 F curve c, 5 AM; curve d, 8 CL AuM; dependence led us to investigate effects of specific inhibitors 0. AiM; curve e, 10 AiM; curve f, transport on uptake of(R)-[3H]norepineph- .a 4 20 The curves were satis- ofnorepinephrine 0 AiM. rine by these cells. z 0.05 F factorily linear by regression -_ analysis for the first 10 min (r > As shown in Fig. 2A, cocaine inhibited (R)-norepinephrine 0.98). The linear portions are uptake profoundly at low norepinephrine concentrations, but 0 shown in solid lines while the less so at higher concentrations. A Lineweaver-Burk plot of 0 10 20 30 nonlinear portions are shown these data (Fig. 2B) showed that the curve could be resolved Time in Min. in dashed lines. into a cocaine-sensitive component, with an apparent Km of 5.8 AM and a cocaine-insensitive component with an appar- background. The purity of the transported norepinephrine in ent Km of 88 AM. The cocaine-insensitive component was the cell was analyzed by HPLC as described (25). This further analyzed at norepinephrine concentrations of 0-200 procedure showed that 100%o of the cell-associated radioac- AM (Fig. 2C), and an apparent Km of 103.5 ± 26.8 /hM was tivity was norepinephrine only. Representative samples of found. This was consistent with the previous determination supernatants were also collected to measure epinephrine and at lower substrate concentrations (Fig. 2B). norepinephrine in the medium by the method of Anton and Action of Metabolic Inhibitors, Ion-Depletion, and Specific Sayre (26). Protein concentration was determined by Drugs and Substrates on Cocaine-Sensitive Transport. Uptake Bradford assay, as described for application to chromaffin of norepinephrine by the cocaine-sensitive site in chromaffin tissue using bovine serum albumin as the standard (27). cells was found to be blocked by low temperature, by Statistical Analysis and Mathematical Modeling. Data were metabolic inhibitors such as trichlorotrifluorobenzene, a analyzed using programs ALLFIT (28) and ENZYME (29)**, potent mitochondrial uncoupler, sodium azide, and ouabain employing iterative reweighting (30) and standard statistical (Table 1). Similar observations were made on the low-affinity methods (31). Program ENZYME (29) was used to discrim- (cocaine-insensitive) uptake site as well (data not shown). alternative models of inhibi- However, only the cocaine-sensitive, high-affinity site was inate between competitive and inhibited by partial depletion of sodium from the medium tion (32). (Table 1). Lowering the potassium concentration of the medium, thereby hyperpolarizing the cell, also had an inhib- RESULTS itory effect, but equivalent reductions in Mg2+ or Ca2+ were Uptake of (R)-[3H]Norepinephrine by Cells. A time course without significant effect. of (R)-[3H]norepinephrine uptake into chromaffin cells was drugs, known to be inhibitors of measured over a 30-min period in concentrations of (R)- high-affinity uptake of norepinephrine in nerve terminals norepinephrine ranging from 1 to 20 As shown in Fig. 1, (33), were also found to inhibit only cocaine-sensitive (R)- AiM. norepinephrine uptake. As shown in Table 2, desipramine was more potent than , which was in turn more **Programs ALLFIT and ENZYME are available from the authors in a form suitable for the DEC-10, HP-85, and IBM-PC, as well as potent than , which was in turn more potent than compatible microcomputers. chlorimipramine or . Conversely, zimilidine, a

0.30 0.40

0.24 0.32 . IC.-2 0.18 I 2 0.24 *E0.c 0 E- *~ E 0.12 D 0.16

c 0.06 0.08

o o 0 5 10 15 20 0 50 100 150 200

[NorepinephrineJ, jM 1/ [Norepinephrinel, MM1 [Norepinephrinel, jM

FIG. 2. Kinetics of (R)- Hjnorepinephrine uptake as a function of external norepinephrine concentration in the presence and absence of 50 /AM cocaine. (A) Substrate-velocity plots of norepinephrine uptake. *, Total uptake; A, uptake in presence of 50 1AM cocaine; o, cocaine-sensitive uptake. (B) Lineweaver-Burk plot ofdata inA. (C) Substrate-velocity and Lineweaver-Burk (Inset) analysis ofnorepinephrine uptake through the cocaine-insensitive site. In A, B, and C, predicted curves are superimposed on the experimental data using parameters from program ENZYME (29). Downloaded by guest on September 29, 2021 Biochemistry: Banedee et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1751 Table 1. Cocaine-sensitive uptake of (R)-norepinephrine in the Table 3. Inhibition of cocaine-sensitive (R)-norepinephrine presence of various inhibitors and ion-depleted media transport by catecholamines and related compounds % of cocaine-sensitive Additive IC50, UM Culture conditions (R)-norepinephrine uptake Dopamine 3.3 370C 100.0 ± 7.7 (R)-Epinephrine 11.5 OOC O* (R,S)- 3.8 TTFB (5 ,IM) 18.8 ± 0.9 (R,S)-Isoproterenol 40.0 Sodium azide (10 ,uM) 44.6 ± 2.1 (R,S)-Dimethylepinephrine 97.5 Ouabain (10 juM) 63.0 ± 1.6 6-Hydroxydopamine 450.0 Na+-depleted (14.75 mM)t 32.9 ± 0.6 (l)-Dopa 50.0 K+-depleted (0.59 mM)t 51.5 ± 0.9 (I)-Tyrosine Mg2+-depleted (0.15 mM)t 96.7 ± 2.3 The (R)-norepinephrine concentration was 1.0 ,uM, and cocaine Ca2l-depleted (0.275 mM)t 85.3 ± 5.8 blocked 90o of the observed uptake. IC50 values were calculated The concentration of (R)-norepinephrine was 1.0 jLM, and the from logit-log plots. observed cocaine-sensitive uptake was 84% of the total. The time of accumulation was 10 min. The experiments were done in quadru- hypothesis, the effects ofdifferent concentrations of cocaine plicate, and the data are presented as mean ± SEM. TTFB, on transport of two well-defined concentrations of (R)- tri-chlorotrifluorobenzene. norepinephrine were examined. As shown in Fig. 3A, in- *Transport was defined as the difference between uptake at 370C and inhibited that at 0C. The uptake at 0C was <10% of the total. creasing concentrations of cocaine progressively tSodium chloride in the assay medium was replaced iso-osmotically uptake of (R)-norepinephrine. It appears that cocaine be- with sucrose or reduced as done for other ions. The final concen- haves as a competitive inhibitor, with Ki of 1.2 ILM (Fig. 3B). tration of the specified ion is in parentheses. The Lineweaver-Burk analysis, Dixon plot, and nonlinear least-squares analysis (program ENZYME) all indicated that the data for cocaine (Ki, 1.2 ,uM) were more consistent with blocker of serotonin transport in nerve endings, was much a competitive than a noncompetitive or uncompetitive model. less potent, as was reserpine, the specific inhibitor of the Thus, we concluded that the high-affinity site, defined by chromaffin granule catecholamine transporter. cocaine sensitivity, had affinity for both norepinephrine and Inhibition of the cocaine-sensitive norepinephrine trans- cocaine and was distinct from the low-affinity uptake site. porter was also observed by a variety of catecholamines and Transport of Other Catecholamnes into Chromaffin Cells. related substances listed in Table 3. Particularly potent The existence of at least two classes of uptake sites for inhibition was observed for dopamine and octopamine, an norepinephrine in the chromaffin cell led us to examine the analogue of norepinephrine having one ring hydroxyl group substrate specificity ofboth classes of sites using a variety of in the para position. Epinephrine was somewhat less potent, catecholamines and their analogues. As shown in Table 3, the whereas the neurotoxin 6-hydroxydopamine was a poor cocaine-sensitive transport of (R)-norepinephrine was inhib- inhibitor. ited by a series of related compounds with varying efficien- cies as defined by IC50. However, dopamine and epinephrine Kinetic Analysis ofCocaine Inhibition of(R)-Norepinephrine were selected for closer study because of their physiological Transport. In the case of nervous tissue, it is presumed that importance, and isoproterenol and dimethylepinephrine were cocaine acts directly on the high-affinity norepinephrine selected because of previous extensive studies on their transport site to block norepinephrine uptake. If this were transport through the chromaffm granule membrane (19). also true in chromaffin cells, then the mechanism of cocaine Studies on transport of labeled dopamine and epinephrine inhibition ought to be classically competitive. To test this were performed following the procedure exactly as described for norepinephrine uptake. We found that both cocaine- Table 2. Inhibition of cocaine-sensitive, high-affinity sensitive and cocaine-insensitive uptake sites existed forboth (R)-norepinephrine uptake by tricyclic amines. As shown in Table 4, the cocaine-sensitive transport and other drugs system for dopamine and epinephrine had similar Km values, IC5o, nM in the 2-3 tuM range, comparable to that for norepinephrine. Drug Logit-log plot ALLFIT program However, the transport ofdopamine and epinephrine through the cocaine-insensitive transport sites could not be statisti- Tricyclic antidepressants cally distinguished from a diffusion-limited process when Desipramine* 130 130 ± 26 analyzed by the program ENZYME (Table 4). A further Imipramine 520 460 ± 80 Doxepin 800 810 ± 180 difference between the high- and low-affinity sites, shared by Chlorimipramine 930 1050 ± 180 all three substrates, was the fact that only the activities ofthe Amitriptyline 980 1000 ± 170 high-affinity, cocaine-sensitive sites were dependent on so- Zimilidine 4000t 4700 ± 1800 dium. Thus, these data were consistent with the possibility Norzimilidine 700t 560 ± 310 that dopamine, norepinephrine, and epinephrine were trans- Reserpine 2150 1660 ± 360* ported by the same, or at least closely related sets of, high- and low-affinity sites on the chromaffin cell plasma mem- The IC5o concentrations were estimated from logit-log plots over concentrations spanning the dose that gave 50% inhibition and by use brane. of program ALLFIT. Standard errors are estimated within the We then turned our attention to the transport properties of experiments. dimethylepinephrine and isoproterenol. In the chromaffin *The slopes of the logit-log plots for all tricyclic antidepressants granule transport system, isoproterenol is a competitive were indistinguishable (1.44 ± 0.23). inhibitor of norepinephrine transport (19) and a substrate for tThe slopes of the logit-log plots for zimilidine and norzimilidine transport in its own right (19, 34). In contrast, dimethylepi- were 0.52 _ 0.10, appreciably flatter from those of the tricyclic antidepressants (1.44 + 0.23). nephrine, a permanently charged quaternary catecholamine, tReserpine behaved as a classical competitive inhibitor as defined by is a competitive inhibitor of norepinephrine transport but is the kinetics described in this paper, in spite ofits poor potency. This itself not transported (19). In the case of transport across the value was obtained using program ENZYME (29). chromaffin cell plasma membrane, however, both isoproter- Downloaded by guest on September 29, 2021 1752 Biochemistry: Banerjee et al. Proc. Natl. Acad. Sci. USA 84 (1987)

50 I-

FIG. 3. Dixon analysis of inhibition of , E (R)-[3H]norepinephrine uptake by cocaine in chromaffin cells. (A) Substrate-velocity plots of norepinephrine uptake at the follow- ing cocaine concentrations: curve a, none; curve b, 1 jLM; curve c, 5 ttM; curve d, 10 tLM; curve e, 20 ,uM. (B) Dixon plot of the data from A. Predicted curves are superim- posed on the experimental data points using parameters from program ENZYME. NE, [Norepinephrinel, IM [Cocaine], pM norepinephrine.

enol and dimethylepinephrine were taken up by a cocaine- transport site. Indeed, (R,S)-[3H]isoproterenol was found to insensitive low-affinity uptake site (Table 4). be taken up by a saturable mechanism (Km, 14.9 AM; Vmax, The mechanism of inhibition of norepinephrine transport 6.2 pmol/mg of protein per min), but, as expected, was by dimethylepinephrine was analyzed by Dixon analysis insensitive to cocaine (data not shown). Therefore, it appears using the program ENZYME, the same approach as used for that the bulkiness of the group around the nitrogen in both other substrates. The data indicated that the inhibition of dimethylepinephrine and isoproterenol may be sufficient to norepinephrine uptake by dimethylepinephrine occurred via exclude the molecules as substrates for the high-affinity, a "pure classic noncompetitive" model (3) with an apparent cocaine-sensitive transport site in chromaffin cells. Ki of91 ,M. This result was consistent with the concept that dimethylepinephrine did not interact directly with the high- DISCUSSION affinity cocaine-sensitive transport site. To test this conclu- sion further, the uptake of radiolabeled (R,S)-[3H]dimethyl- The major conclusion of this study is that the catecholamine epinephrine into chromaffin cells was studied. As shown in transport mechanism in the plasma membrane of chromaffin Fig. 4A, dimethylepinephrine was taken up into cells by a cells is qualitatively and quantitatively different from the saturable but low-affinity mechanism with a Km of 154 AM catecholamine transport mechanism in the chromaffin gran- (Fig. 4B). Cocaine (50 AM) was without effect on uptake (for ule membrane. While there is only one carrier-mediated example, see open circle in Fig. 4A). Thus, the inhibition data transport mechanism in the chromaffin granule, the plasma and the transport data were consistent, with regard to the site membrane contains at least two. The high-affinity class of of dimethylepinephrine action being separate from the co- sites is sensitive to cocaine and to a variety of tricyclic caine-sensitive norepinephrine transport site. antidepressant drugs. The order of potency of these drugs- Dimethylepinephrine has both a bulky group of three desipramine > imipramine > doxepin > chlorimipramine, carbons around the nitrogen moiety, as well as a permanent amitriptyline-is identical to their order of potency for positive charge due to the quaternary nitrogen. Either of inhibition of norepinephrine uptake at noradrenergic nerve these properties could be responsible for the lack of inter- endings (34). In contrast, these drugs have only low potency action ofdimethylepinephrine with the high-affinity transport on chromaffin granule transport, an effect being observed site. We, therefore, examined the transport properties of only at concentrations of >30 ILM (4). Consistent with this isoproterenol. Isoproterenol is a secondary amine, as is pharmacologic difference is the fact that reserpine, a specific epinephrine, but as dimethylepinephrine, it also has three inhibitor of the chromaffin granule transporter with a Ki of carbons in proximity to the nitrogen. Thus, if the charge on 1-10 nM, was also found to be a competitive inhibitor of the the nitrogen were the only critical factor, one might then cocaine-sensitive plasma membrane transport site, but with expect isoproterenol to behave as a "classical competitive" a calculated K, of 1.7 tLM. This value is similar to that referred inhibitor of catecholamine uptake. However, isoproterenol to by Iversen for type 1 uptake in rat heart (4). also appeared to be a pure classic noncompetitive inhibitor of In the chromaffin granule system dimethylepinephrine is a the high-affinity, cocaine-sensitive norepinephrine uptake competitive inhibitor of norepinephrine transport into the site with a Ki of56 AM. It thus seemed that isoproterenol also granule, but itself is not a substrate for transport. Isoproter- did not interact directly with the high-affinity norepinephrine enol, however, is both a competitive inhibitor and a substrate Table 4. Summary of kinetic constants and pharmacologic data for the uptake of catecholamine analogues Total uptake Cocaine-sensitive uptake Cocaine-insensitive uptake Substrate Km VM. Km Vanr Km V=, (R)-Norepinephrine 13.7 ± 3.4 51.4 ± 9.4 5.8 ± 1.0 23.8 ± 2.7 87.6 ± 15.9 50.7 ± 13.5 Dopamine 16.3 ± 6.8 192.3 ± 52.1 2.4 ± 1.2 33.2 ± 6.7 Indeterminate* (R)-Epinephrine 72.5 ± 13.5 111.6 ± 16.6 2.2 ± 1.5 50.9 ± 9.1 Indeterminate* (R,S)-Dimethylepinephrine 154.0 ± 57.2 40.4 ± 10.1 None Same as total uptake (R,S)-Isoproterenol 14.9 ± 6.8 6.2 ± 1.8 None Same as total uptake Km is in ,uM, and Vmax is in pmol/min per mg of protein. Values are expressed as the mean ± SEM of four determinations. *Cocaine-insensitive component was too close to the diffusion-limited process to make quantitative determination of these values using the program ENZYME. Downloaded by guest on September 29, 2021 Biochemistry: Banedee et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1753

50 E E 40 _ C e

Y c, 30F .90X 3.a FIG. 4. Kinetics of (R,S)-[3H]dimethyl- 20F epinephrine uptake as a function of external _ (R,S)-dimethylepinephrine. (A) Substrate- 0 velocity plot of (R,S)-dimethylepinephrine E 10 1 0 uptake. (B) Lineweaver-Burk plot of data in .E Km = 154MM A. *, Uptake in absence of cocaine; o, Vmax = 40.4 pmol/Min/mg Protein uptake in presence of 50 juM cocaine. The 0 I I 'lines are constructed with the computer 0 0.025 0.05 0.075 0.1 predicted values following the program EN- ZYME, and then the experimental data are [Dimethylepinephrine], aM /lDimethylepinephrine], pM1 plotted.

(19). Thus, the positive charge and not the size of the bulky 15. Scherman, D., Jamdon, P. & Henry, J. P. (1983) Proc. Natl. group around the amine moiety seemed to be responsible for Acad. Sci. USA 80, 584-588. the inability of dimethylepinephrine to be transported across 16. Gabizon, R., Yetinsen, R. T. & Schuldiner, S. (1982) J. Biol. the granule membrane. Conversely, in case of the cocaine- Chem. 257, 15145-15152. sensitive on the 17. Knoth, J., Handloser, K. & Njus, D. (1980) Biochemistry 19, transporter plasma membrane, bulky sub- 2938-2942. stitution around the nitrogen atom seems to abolish the 18. Knoth, J., Isaacs, J. M. & Njus, D. (1981) J. Biol. Chem. 256, interaction with the transport site altogether. This strongly 6541-6543. suggests that the topography of the cocaine-sensitive cate- 19. Ramu, A., Levine, M. A. & Pollard, H. B. (1983) Proc. Natl. cholamines binding site on the plasma membrane is qualita- Acad. Sci. USA 80, 2107-2111. tively different from that of the analogous site on the granule 20. Scherman, D. & Henry, J. P. (1981) Eur. J. Biochem. 116, membrane transporter. 535-539. 21. Pollard, H. B., Pazoles, C. J., Creutz, C. E., Scott, J. H., 1. Koenigsberg, R. L. & Trifaro, J. M. (1980) Neuroscience 5, Zinder, 0. & Hotchkiss, A. (1984) J. Biol. Chem. 259, 1547-1556. 1114-1121. 22. Banedjee, D. K., Ornberg, R. L., Youdim, M. B. H., Held- 2. Role, L. W. & Perlman, R. L. (1983) Neuroscience 10, man, E. & Pollard, H. B. (1985) Proc. Natl. Acad. Sci. USA 987-996. 82, 4702-4706. 3. Iversen, L. L. (1967) The Uptake and Storage of 23. Waymire, J. C., Waymire, K. G., Boehme, R., Noritake, D. & Noradrenaline in Sympathetic Nerves (Cambridge Univ. Wardell, J. (1977) in Structure and Function of Monoamine Press, Cambridge, UK). Enzymes, eds. Usdin, E., Weiner, N. & Youdim, M. B. H. 4. Iversen, L. L. (1976) in Handbook of Psychopharmacology, (Dekker, New York). eds. Iversen, L. L., Iversen, S. D. & Snyder, S. H. (Plenum, 24. Levine, M. A. & Pollard, H. B. (1983) FEBS Lett. 158, New York), Vol. 3, pp. 340-381. 134-138. 5. Paton, D. M. (1976) The Mechanisms of Neuronal and 25. Levine, M. (1986) J. Biol. Chem. 261, 7347-7356. Extraneuronal Transport of Catecholamines (Raven, New 26. Anton, A. H. & Sayre, D. F. (1962) J. Pharmacol. Exp. Ther. York). 138, 360-375. 6. Role, L. W. & Perlman, R. L. (1983) Neuroscience 10, 27. Pollard, H. B., Menard, R., Brandt, H. A., Pazoles, C. J., 979-985. Creutz, C. E. & Ramu, A. (1978) Anal. Biochem. 86, 761-763. 7. Burgen, A. S. V. & Iversen, L. L. (1965) Br. J. Pharmacol. 28. DeLean, A., Munson, P. J. & Rodbard, D. (1978) Am. J. Chemother. 25, 34-49. Physiol. 235, E97-E102. 8. Horn, A. S. (1973) Br. J. Pharmacol. 47, 332-338. 29. Lutz, R. A., Bull, C. & Rodbard, D. (1985) Enzyme 36, 9. Bashford, C. L., Radda, G. K. & Ritchie, G. A. FEBS 197-206. (1975) 30. Rodbard, D., Lennox, R. H., Wray, H. L. & Ramseth, D. Lett. 50, 21-24. (1976) Clin. Chem. 22, 350-358. 10. Bashford, C. L., Radda, G. K. & Ritchie, G. A. (1975) Bio- 31. Draper, N. R. & Smith, H. (1981) Applied Regression Analysis chem. J. 148, 153-156. (Wiley, New York), 2nd Ed., pp. 97-115. 11. Casey, R. P., Njus, D., Radda, G. K. & Sehr, P. A. (1977) 32. Wong, T.-F. J. (1975) Kinetics ofEnzyme Mechanisms (Aca- Biochemistry 16, 972-977. demic, New York), pp. 44-50. 12. Holz, R. N. (1978) Proc. Nati. Acad. Sci. USA 75, 5190-5194. 33. Rehavi, M., Skolnick, P., Brownstein, M. J. & Paul, S. M. 13. Johnson, R. G. & Scarpa, A. (1978) J. Biol. Chem. 254, (1982) J. Neurochem. 38, 889-895. 3750-3760. 34. Johnson, R. G., Carty, S. E., Hayflick, S. & Scarpa, A. (1982) 14. Cidon, S. & Nelson, N. (1983) J. Biol. Chem. 258, 2892-2898. Biochem. Pharmacol. 31, 815-823. Downloaded by guest on September 29, 2021