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Dopamine transport: pharmacological distinction between the synaptic membrane and the vesicular transporter in rat striatum
European Journal o f Pharmacology, 218 ( 1q92) 175-177 ~(';. 1992 Elsevier Science Publishers B.V. All rights reserved 0014-29q9/92/$05.00
EJP 21O88
Short communication
Dopamine transport: pharmacological distinction between the synapt membrane and the vesicular transporter in rat striatum W i l l i a m R o s t ~ n e ~', J o h n W. Boja b, D a n i e l S c h e r m a n c, F. Ivy C a r r o l l d a n d M i c h a e l J. K u h a r b " I N S E R M U.339, Hopital St. Antoine, 75012 St. Antoine, France, h Neuroscience Branch, NIDA Addiction Research Center, Baltimore. MD 21224, USA, ' CNRS-Rhone-Poulenc Rorer, Vitry-sur-Seine, France and a Research Triangle Institute, Research Triangle Park, N C 27709, USA
Received 30 January 1992, revised MS received 6 May 1992, accepted 19 May 1992
The pharmacological properties of the monoamine transporters in the synaptic vesicles and of the dopamine transportc the synaptic plasma membrane were compared. Tetrabenazine, an inhibitor of the vesicular transporter did not block li binding to the plasma membrane transporter. Various potent cocaine analogues and other compounds active at the pl; membrane transporter did not block ligand binding to the vesicular transix)rter. These data indicate pharmacological differc between the vesicular and synaptic membrane transporters. Dopamine; Dopamine transporters; Synaptic vesicle; Amine uptake
1. Introduction
2. Materials and methods
Cocainc binds to and inhibits monoamine transporters, and some of its behavioral effects have been related to this inhibition, particularly inhibition at plasma m e m b r a n e dopamine transporters (Ritz e t a . , 1987; Bergman et al., 1989; Spealman et al., 1989). Conversely, it has been demonstrated that o~-dihydrotetrabenazine, a derivative of the monoamine depleting agent, tetrabenazine, was able to bind to the vesicular monoamine transporter present in chromaffin granules of the adrcnal medulla and in monoaminergic synaptic vesicles (Henry and Scherman, 1989). Moreover, [3H] a-dihydrotetrabenazine was also recently shown to be a good marker for the visualization of dopaminergic denervation in the striatum (Masuo et al., 1990). In this study, we examined both the effects of potent cocaine analogs (Boja et al., 1990) and other compounds on striatal [3H]tr-dihydrotetrabenazine binding, and the action of a-dihydrotetrabenazine on the striatal binding of a cocaine analog, [3H]W1N 35,428. These experiments were carried out on both homogenized rat striatal m e m b r a n e s and slide-mounted tissue sections.
2.1. [SH]a-dihydrotewabenazine binding 2.1.1. Homogenates Rat striatal homogenates (0.1 r a g / p r o t e i n per were incubated with 1 nM [3H]a-dihydrotetrabena (Dositek, Orsay, France) in the presence of val displacers, for 1 h at room temperature in 20 HEPES-HC1 buffer, pH 8.0, containing 0.3 M suc in 500 txl final volume. Samples were then filtere. Schleicher and Schuell glass fiber filters, washed t with 2 ml of incubation buffer, and counted.
2.1.2. Slide-mounted tissue sectiom" Sections of 20 ~ m through the rat striatum , incubated with 5 nM [3H]a-dihydrotetrabcnazinc 40 min at room temperature in the same buffer a: homogenates. After incubation, they were washed times for 3 min in 40 mM Tris HCI pH 8 at 4°C (M et al., 1990). Sections were wiped off of the slides Schleicher and Schuell filters and counted (Kuhar Unnerstall, 1990). In other experiments, incubated tions were apposed to film to generate autoradiogr
2.2. [~H] WIN ,35,428 binding Correspondence to: M.J. Kuhar, Neuroscience Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 2 1 2 2 4 ,
USA.
[3H]WIN 35,428 binding t o rat striatal membr was conducted according to Madras et al. (198t
17¢)
modified by Boja et al. (1990). Concentrations of 0.5 nM [3H]WIN 35,428 were utilized in a 2 h incubation. RTI-31 (3/3-(4-chlorophenyl)tropane-2-carboxylic acid methyl ester), WIN 35,065-2 (R-3/3-(phenyl)tropan2/3-carboxylic acid methyl ester) and WIN 35,065-3 (S-3/3-(phenyl)tropan-2/3-carboxylic acid methyl ester), were obtained from Research Triangle Institute.
3. Results
Non-radioactive tetrabenazine potently displaced [~H]a-dihydrotetrabenazine binding in homogenates as expected (table 1). The ICs, value of 4 nM is in agreement with its reported K d value (Scherman and Henry, 1983). However, WIN 35,065-2 and RTI 31, potent cocaine analogs that are inhibitors of the cell membrane dopamine transporter (Boja et al., 1990), had no effect of [3H]a-dihydrotetrabcnazine binding up to 30 #M. The inactive compound, WIN 35,065-3. also had no effect. Finally, neither nomifensine nor dand d,l-amphetaminc were able to displace [3H]a-dihydrotetrabenazine binding at micromolar concentrations (table 1). Similar r e s u l t s w e r e obtained in autoradiographic experiments with slide-mounted tissue sections (fig. 1). Conversely, tetrabenazine did not inhibit [3H]WIN 35,428 binding to the plasma membrane dopaminc transporter at 10 -~ M, in contrast to cocaine analogs, suggesting that [3H]a-dihydrotctrabenazinc and [3H] WIN 35,428 labeled different entities,
4. Discussion Cocaine analogs such as WIN 35,065-2 and RTI-31 are potent inhibitors of binding at the synaptic mcmbrane dopamine transporter in rat striatal tissue (Boja
TABLE 1 Inhibition of [311k~-dihydrotetrabenazine and [311]WIN 35,428 binding by N)tent cocaine analogs and other compounds, Average IC50 values were ()btained using various concentrations (10 -~l to 10 5 M) of each tested compound. Each displacement curve has been repeated at least twice. ND. not determined: TBZOH. a-dihydrotctrabenazine, Drug
IC5.(M) [~HJ'IBZOH binding
TBZOIi WIN 35,065-2
WIN35,065-3 RTI-31 d-Amphetamine d.l-Amphetamine Nomifcnsine
4 x 1 0 '~ > 3 x 10 ~ > 3×10 5
> 3× 10 s > 2 x 10- s > 2x 10 5 > 2 × 10 5
[3H]WlN 35.428 binding >3×10 5 2 . 3 x 10 ~
1243x10 ~ 10 " ND
ND ND
~
. , , ~ k
e
I
"~':: "
::"~ii : '::.2 '?"::. . " f -
+ •
Q ' , d ~ i I k
I
Fig. 1. Autoradiographic visualization of [311]a-dihydrotetrabena binding in absence (A) and in presence of inhibitors inclu 2 x 10 ~' unlabeled T B Z (B). Ill ~' M WIN 35,065-2 (C), 10 RTI-31 (D), 2 x 1 0 ~ M d-amphetamine (E). Only tetrabena inhibitedstriatal [~ll]a-dihydrotetrabenazinc binding. Results I typical sections arc shown,
et al., 1990). The fact that the potent cocaine anal were not able to displace [3H]a-dihydrotetrabcnai binding suggests significant pharmacological dif cnces between vesicular transporters compared to p ma membrane transporters. This also has been : gested by uptake studies of Rcith et a. (1989). U now, only indirect evidence has been reported for selectivity of tetrabenazine for the vesicular vel plasma membrane transporter (Henry and Scherrr 1989). It has been suggested (Reith et al., 1989) that cainc concentration in brain could, under certain c ditions, become high enough to affect the vesic~ transporter. WIN 35,065-2 and RTI-31 are two coc~ analogs that are about 4 and 100 times more pol than cocaine in binding studies (Boja et al., 19 Thus, their concentration in brain required to prod dopamine-related activity would be significantly lo than that for cocaine. Yet, no interaction with vesicular transporter could be detected at relati high concentrations. The lack of effect of the coc~ analogs (as well as amphetamine) on vesicular [~Idihydrotetrabenazine binding suggests that s o m e their behavioral effects, and also those of coea result from inhibiting the plasma membrane dopan transporter rather than the vesicular transporter. H ever, while it may not bc likely, it may be possible cocaine analogs could inhibit vesicuhtr amine transl
by some unknown fication of these
mechanism and therefore final vericonclusions may require transport
s t u d i e s in a d d i t i o n t o t h e s e b i n d i n g s t u d i e s . T h e p l a s m a membrane DA transporter has been recently cloned ( S h i m a d a e t al., 1991), a n d o u r d a t a s h o w i n g p h a r m a c o l o g i c a l d i f f e r e n c e s is c o m p a t i b l e w i t h t h e n o t i o n t h a t the vesicular transporter may be derived from a different gene.
Acknowledgements The authors acknowledge the technical support of Ms. T. Kopajtic and clerical support of Ms. B. Cepl.
References Bergman, J., B.K. Madras, S.E. Johnson and RD. Spealman, 1989, Effects of cocaine and related drugs in nonhuman primates Ill self-administration by squirrel monkeys, J. Pharmacol. Exp. Ther. 251, 150. Boja, J.W., F.I. Carroll, M.A. Rahman, A. Philip, A.H. Lewin and M.J. Kuhar, 1990, New, potent cocaine analogs: ligand binding and transport studies in rat striatum, Eur. J. Pharmacol. 184, 329. llenry, J.P. and D. Scherman, 1989, Radioligands of the vesicular
monoamine transporter and their use as markers of monoan storage vesicles, Biochcm. Pharmacol. 38, 2395. Kuhar, M.J. and J.R. Unnerstall, 1990, Receptor autoradiogr~ in: Methods in Neurotransmitter Analysis, ed. |1.1, Yamar (Raven Press)p. 177. Madras, B.K., R.D. Spealman, M.A. Fahey, J.L. Neumeyer, Saha and R.A. Milius, 1989, Cocaine receptors labelec [3H]2/3-carbomethoxs'-3/3-(4-fluorophenyl)tropanc, Mol. Pha col. 36, 518. Masuo, Y., D. Pelaprat, D. Scherman and W. Rost~ne, 1990, [31hydrotetrabenazine, a new marker for the visualizatiou dopaminergic denervation on the rat striatum, Neurosci.
114, 45. Reith, M.E.A., H.K. Kramer, II. Sershen and A. Lajtha, I Cocaine competitively inhibits catecholamine uptake into t synaptic vesicles, Res. Commun. Subst. Abuse 10, 205. Ritz, M.C., R.J. Lamb, S.R. Goldberg and M.J. Kuhar, 1987. caine receptors on dopamine transporters are related to sel ministration of cocaine, Science 237, 1219. Scherman, D. and J. Henry, 1983, The catecholamine carrie bovine chromaffin granules form of the bound amine, Mol. macol. 23, 431. Shimada, S.S. Kitayama, C-L. Lin, A. Patel, E. Nanthakuma Gregor, M. Kuhar and G. Uhl, 1991, Cloning and expression cocaine-sensitive dopamine transporter complementary E Science 254, 576. Spealman, R.D., B.K. Madras and J. Bergman, 1989, Effecl cocaine and related drugs in nonhuman primates. II. Stiml effects on schedule-controlled behavior, J. Pharmacol. Exp. "] 261, 142.
EJP 21O88
Short communication
Dopamine transport: pharmacological distinction between the synapt membrane and the vesicular transporter in rat striatum W i l l i a m R o s t ~ n e ~', J o h n W. Boja b, D a n i e l S c h e r m a n c, F. Ivy C a r r o l l d a n d M i c h a e l J. K u h a r b " I N S E R M U.339, Hopital St. Antoine, 75012 St. Antoine, France, h Neuroscience Branch, NIDA Addiction Research Center, Baltimore. MD 21224, USA, ' CNRS-Rhone-Poulenc Rorer, Vitry-sur-Seine, France and a Research Triangle Institute, Research Triangle Park, N C 27709, USA
Received 30 January 1992, revised MS received 6 May 1992, accepted 19 May 1992
The pharmacological properties of the monoamine transporters in the synaptic vesicles and of the dopamine transportc the synaptic plasma membrane were compared. Tetrabenazine, an inhibitor of the vesicular transporter did not block li binding to the plasma membrane transporter. Various potent cocaine analogues and other compounds active at the pl; membrane transporter did not block ligand binding to the vesicular transix)rter. These data indicate pharmacological differc between the vesicular and synaptic membrane transporters. Dopamine; Dopamine transporters; Synaptic vesicle; Amine uptake
1. Introduction
2. Materials and methods
Cocainc binds to and inhibits monoamine transporters, and some of its behavioral effects have been related to this inhibition, particularly inhibition at plasma m e m b r a n e dopamine transporters (Ritz e t a . , 1987; Bergman et al., 1989; Spealman et al., 1989). Conversely, it has been demonstrated that o~-dihydrotetrabenazine, a derivative of the monoamine depleting agent, tetrabenazine, was able to bind to the vesicular monoamine transporter present in chromaffin granules of the adrcnal medulla and in monoaminergic synaptic vesicles (Henry and Scherman, 1989). Moreover, [3H] a-dihydrotetrabenazine was also recently shown to be a good marker for the visualization of dopaminergic denervation in the striatum (Masuo et al., 1990). In this study, we examined both the effects of potent cocaine analogs (Boja et al., 1990) and other compounds on striatal [3H]tr-dihydrotetrabenazine binding, and the action of a-dihydrotetrabenazine on the striatal binding of a cocaine analog, [3H]W1N 35,428. These experiments were carried out on both homogenized rat striatal m e m b r a n e s and slide-mounted tissue sections.
2.1. [SH]a-dihydrotewabenazine binding 2.1.1. Homogenates Rat striatal homogenates (0.1 r a g / p r o t e i n per were incubated with 1 nM [3H]a-dihydrotetrabena (Dositek, Orsay, France) in the presence of val displacers, for 1 h at room temperature in 20 HEPES-HC1 buffer, pH 8.0, containing 0.3 M suc in 500 txl final volume. Samples were then filtere. Schleicher and Schuell glass fiber filters, washed t with 2 ml of incubation buffer, and counted.
2.1.2. Slide-mounted tissue sectiom" Sections of 20 ~ m through the rat striatum , incubated with 5 nM [3H]a-dihydrotetrabcnazinc 40 min at room temperature in the same buffer a: homogenates. After incubation, they were washed times for 3 min in 40 mM Tris HCI pH 8 at 4°C (M et al., 1990). Sections were wiped off of the slides Schleicher and Schuell filters and counted (Kuhar Unnerstall, 1990). In other experiments, incubated tions were apposed to film to generate autoradiogr
2.2. [~H] WIN ,35,428 binding Correspondence to: M.J. Kuhar, Neuroscience Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 2 1 2 2 4 ,
USA.
[3H]WIN 35,428 binding t o rat striatal membr was conducted according to Madras et al. (198t
17¢)
modified by Boja et al. (1990). Concentrations of 0.5 nM [3H]WIN 35,428 were utilized in a 2 h incubation. RTI-31 (3/3-(4-chlorophenyl)tropane-2-carboxylic acid methyl ester), WIN 35,065-2 (R-3/3-(phenyl)tropan2/3-carboxylic acid methyl ester) and WIN 35,065-3 (S-3/3-(phenyl)tropan-2/3-carboxylic acid methyl ester), were obtained from Research Triangle Institute.
3. Results
Non-radioactive tetrabenazine potently displaced [~H]a-dihydrotetrabenazine binding in homogenates as expected (table 1). The ICs, value of 4 nM is in agreement with its reported K d value (Scherman and Henry, 1983). However, WIN 35,065-2 and RTI 31, potent cocaine analogs that are inhibitors of the cell membrane dopamine transporter (Boja et al., 1990), had no effect of [3H]a-dihydrotetrabcnazine binding up to 30 #M. The inactive compound, WIN 35,065-3. also had no effect. Finally, neither nomifensine nor dand d,l-amphetaminc were able to displace [3H]a-dihydrotetrabenazine binding at micromolar concentrations (table 1). Similar r e s u l t s w e r e obtained in autoradiographic experiments with slide-mounted tissue sections (fig. 1). Conversely, tetrabenazine did not inhibit [3H]WIN 35,428 binding to the plasma membrane dopaminc transporter at 10 -~ M, in contrast to cocaine analogs, suggesting that [3H]a-dihydrotctrabenazinc and [3H] WIN 35,428 labeled different entities,
4. Discussion Cocaine analogs such as WIN 35,065-2 and RTI-31 are potent inhibitors of binding at the synaptic mcmbrane dopamine transporter in rat striatal tissue (Boja
TABLE 1 Inhibition of [311k~-dihydrotetrabenazine and [311]WIN 35,428 binding by N)tent cocaine analogs and other compounds, Average IC50 values were ()btained using various concentrations (10 -~l to 10 5 M) of each tested compound. Each displacement curve has been repeated at least twice. ND. not determined: TBZOH. a-dihydrotctrabenazine, Drug
IC5.(M) [~HJ'IBZOH binding
TBZOIi WIN 35,065-2
WIN35,065-3 RTI-31 d-Amphetamine d.l-Amphetamine Nomifcnsine
4 x 1 0 '~ > 3 x 10 ~ > 3×10 5
> 3× 10 s > 2 x 10- s > 2x 10 5 > 2 × 10 5
[3H]WlN 35.428 binding >3×10 5 2 . 3 x 10 ~
1243x10 ~ 10 " ND
ND ND
~
. , , ~ k
e
I
"~':: "
::"~ii : '::.2 '?"::. . " f -
+ •
Q ' , d ~ i I k
I
Fig. 1. Autoradiographic visualization of [311]a-dihydrotetrabena binding in absence (A) and in presence of inhibitors inclu 2 x 10 ~' unlabeled T B Z (B). Ill ~' M WIN 35,065-2 (C), 10 RTI-31 (D), 2 x 1 0 ~ M d-amphetamine (E). Only tetrabena inhibitedstriatal [~ll]a-dihydrotetrabenazinc binding. Results I typical sections arc shown,
et al., 1990). The fact that the potent cocaine anal were not able to displace [3H]a-dihydrotetrabcnai binding suggests significant pharmacological dif cnces between vesicular transporters compared to p ma membrane transporters. This also has been : gested by uptake studies of Rcith et a. (1989). U now, only indirect evidence has been reported for selectivity of tetrabenazine for the vesicular vel plasma membrane transporter (Henry and Scherrr 1989). It has been suggested (Reith et al., 1989) that cainc concentration in brain could, under certain c ditions, become high enough to affect the vesic~ transporter. WIN 35,065-2 and RTI-31 are two coc~ analogs that are about 4 and 100 times more pol than cocaine in binding studies (Boja et al., 19 Thus, their concentration in brain required to prod dopamine-related activity would be significantly lo than that for cocaine. Yet, no interaction with vesicular transporter could be detected at relati high concentrations. The lack of effect of the coc~ analogs (as well as amphetamine) on vesicular [~Idihydrotetrabenazine binding suggests that s o m e their behavioral effects, and also those of coea result from inhibiting the plasma membrane dopan transporter rather than the vesicular transporter. H ever, while it may not bc likely, it may be possible cocaine analogs could inhibit vesicuhtr amine transl
by some unknown fication of these
mechanism and therefore final vericonclusions may require transport
s t u d i e s in a d d i t i o n t o t h e s e b i n d i n g s t u d i e s . T h e p l a s m a membrane DA transporter has been recently cloned ( S h i m a d a e t al., 1991), a n d o u r d a t a s h o w i n g p h a r m a c o l o g i c a l d i f f e r e n c e s is c o m p a t i b l e w i t h t h e n o t i o n t h a t the vesicular transporter may be derived from a different gene.
Acknowledgements The authors acknowledge the technical support of Ms. T. Kopajtic and clerical support of Ms. B. Cepl.
References Bergman, J., B.K. Madras, S.E. Johnson and RD. Spealman, 1989, Effects of cocaine and related drugs in nonhuman primates Ill self-administration by squirrel monkeys, J. Pharmacol. Exp. Ther. 251, 150. Boja, J.W., F.I. Carroll, M.A. Rahman, A. Philip, A.H. Lewin and M.J. Kuhar, 1990, New, potent cocaine analogs: ligand binding and transport studies in rat striatum, Eur. J. Pharmacol. 184, 329. llenry, J.P. and D. Scherman, 1989, Radioligands of the vesicular
monoamine transporter and their use as markers of monoan storage vesicles, Biochcm. Pharmacol. 38, 2395. Kuhar, M.J. and J.R. Unnerstall, 1990, Receptor autoradiogr~ in: Methods in Neurotransmitter Analysis, ed. |1.1, Yamar (Raven Press)p. 177. Madras, B.K., R.D. Spealman, M.A. Fahey, J.L. Neumeyer, Saha and R.A. Milius, 1989, Cocaine receptors labelec [3H]2/3-carbomethoxs'-3/3-(4-fluorophenyl)tropanc, Mol. Pha col. 36, 518. Masuo, Y., D. Pelaprat, D. Scherman and W. Rost~ne, 1990, [31hydrotetrabenazine, a new marker for the visualizatiou dopaminergic denervation on the rat striatum, Neurosci.
114, 45. Reith, M.E.A., H.K. Kramer, II. Sershen and A. Lajtha, I Cocaine competitively inhibits catecholamine uptake into t synaptic vesicles, Res. Commun. Subst. Abuse 10, 205. Ritz, M.C., R.J. Lamb, S.R. Goldberg and M.J. Kuhar, 1987. caine receptors on dopamine transporters are related to sel ministration of cocaine, Science 237, 1219. Scherman, D. and J. Henry, 1983, The catecholamine carrie bovine chromaffin granules form of the bound amine, Mol. macol. 23, 431. Shimada, S.S. Kitayama, C-L. Lin, A. Patel, E. Nanthakuma Gregor, M. Kuhar and G. Uhl, 1991, Cloning and expression cocaine-sensitive dopamine transporter complementary E Science 254, 576. Spealman, R.D., B.K. Madras and J. Bergman, 1989, Effecl cocaine and related drugs in nonhuman primates. II. Stiml effects on schedule-controlled behavior, J. Pharmacol. Exp. "] 261, 142.